http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Takahiro+Shimosaka&year=&month=2012.igem.org - User contributions [en]2024-03-28T14:55:44ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:45:10Z<p>Takahiro Shimosaka: /* How to use */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
We wanted E.coli to produce FT protein, but E.coli doesn’t have FT plasmid. <br />
So we needed to transform E.coli with FT plasmid and make E.coli express FT. At first step, we decided to construct FT plasmid. <br />
<br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|thumb|right|350px|Fig.2-3 How protein go through two pathways]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-4 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|Fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9. Thereby we didn't know whether ''E.coli'' expressed R9::GFP,and we needed to checked it by specific anti GFP monoclonal antibody against GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9::GFP protein to perform Western blotting. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|Fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|Fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the Figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Tracy Palmer1 and Ben C. Berks2.(2012) "The twin-arginine translocation (Tat)<br />
protein export pathway" Nature 2012 vol 10<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot|]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the "calculate"<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass Get Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:44:16Z<p>Takahiro Shimosaka: /* How to use */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
We wanted E.coli to produce FT protein, but E.coli doesn’t have FT plasmid. <br />
So we needed to transform E.coli with FT plasmid and make E.coli express FT. At first step, we decided to construct FT plasmid. <br />
<br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|thumb|right|350px|Fig.2-3 How protein go through two pathways]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-4 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|Fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9. Thereby we didn't know whether ''E.coli'' expressed R9::GFP,and we needed to checked it by specific anti GFP monoclonal antibody against GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9::GFP protein to perform Western blotting. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|Fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|Fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the Figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Tracy Palmer1 and Ben C. Berks2.(2012) "The twin-arginine translocation (Tat)<br />
protein export pathway" Nature 2012 vol 10<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot|]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass "Golden Pass"]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:40:35Z<p>Takahiro Shimosaka: /* Golden Pass */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|thumb|right|350px|Fig.2-3 How protein go through two pathways]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-4 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|Fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9. Thereby we didn't know whether ''E.coli'' expressed R9::GFP,and we needed to checked it by specific anti GFP monoclonal antibody against GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9::GFP protein to perform Western blotting. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|Fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|Fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the Figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Tracy Palmer1 and Ben C. Berks2.(2012) "The twin-arginine translocation (Tat)<br />
protein export pathway" Nature 2012 vol 10<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot|]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:39:59Z<p>Takahiro Shimosaka: /* Golden Pass */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|thumb|right|350px|Fig.2-3 How protein go through two pathways]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-4 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|Fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9. Thereby we didn't know whether ''E.coli'' expressed R9::GFP,and we needed to checked it by specific anti GFP monoclonal antibody against GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9::GFP protein to perform Western blotting. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|Fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|Fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the Figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Tracy Palmer1 and Ben C. Berks2.(2012) "The twin-arginine translocation (Tat)<br />
protein export pathway" Nature 2012 vol 10<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot| link=http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:38:44Z<p>Takahiro Shimosaka: /* Golden Pass */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|thumb|right|350px|Fig.2-3 How protein go through two pathways]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-4 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|Fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9. Thereby we didn't know whether ''E.coli'' expressed R9::GFP,and we needed to checked it by specific anti GFP monoclonal antibody against GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9::GFP protein to perform Western blotting. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|Fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|Fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the Figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Tracy Palmer1 and Ben C. Berks2.(2012) "The twin-arginine translocation (Tat)<br />
protein export pathway" Nature 2012 vol 10<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:37:27Z<p>Takahiro Shimosaka: /* How to use */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9. Thereby we didn't know whether ''E.coli'' expressed R9::GFP,and we needed to checked it by specific anti GFP monoclonal antibody against GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9::GFP protein to perform Western blotting. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Tracy Palmer1 and Ben C. Berks2.(2012) "The twin-arginine translocation (Tat)<br />
protein export pathway" Nature 2012 vol 10<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:36:56Z<p>Takahiro Shimosaka: /* How to use */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|Fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|Fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP. Thereby we needed to checked it by specific anti GFP monoclonal antibody against R9-GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9-GFP protein. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass | Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:36:08Z<p>Takahiro Shimosaka: /* Golden Pass */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|thumb|left|300px|Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP. Thereby we needed to checked it by specific anti GFP monoclonal antibody against R9-GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9-GFP protein. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br><br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
[http://openwetware.org/wiki/IGEM:Kyoto/GoldenPass| Golden Pass]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:32:09Z<p>Takahiro Shimosaka: /* How to use */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|left|300px]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP. Thereby we needed to checked it by specific anti GFP monoclonal antibody against R9-GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9-GFP protein. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-8. Verification of R9 function with use of GFP.]]<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br><br />
2. set melting temperature<br><br />
3. click the calculate<br><br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:31:32Z<p>Takahiro Shimosaka: /* Golden Pass */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|fig2-1''E.coli'' (left)sample stained with Hoechst under and observed with 352nm wavelength (right)having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
[[File:J2tatpspDT.png|left|300px]]<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP. Thereby we needed to checked it by specific anti GFP monoclonal antibody against R9-GFP. Then We tried to detect specificity of antigen-antibody reaction against GFP and R9-GFP protein. <br><br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. GFP can penetrate membranes with R9 peptide====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡4.jpg|thumb|600px|center|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-5]]<br />
[[File:RT-PCR5.png|thumb|left|300px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[14]Araki, T et al.(2005) “FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex”. Science 309 (5737), 1052–1056<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
====How to use====<br />
1. input the sequences of the parts starting from 5' terminal<br />
2. set melting temperature<br />
3. click the calculate<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:16:11Z<p>Takahiro Shimosaka: /* Software */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP,and then we checked it by specific anti GFP monoclonal antibody against R9-GFP. We couldn't detect antigen-antibody reaction against R9-GFP protein. <br><br />
<br />
<br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fuged protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真3.jpg|thumb|600px|left|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]<br><br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Golden Pass==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|350px|thumb|center|Screen Shot]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:15:08Z<p>Takahiro Shimosaka: /* Software */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP,and then we checked it by specific anti GFP monoclonal antibody against R9-GFP. We couldn't detect antigen-antibody reaction against R9-GFP protein. <br><br />
<br />
<br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fuged protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真3.jpg|thumb|600px|left|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]<br><br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|300px|thumb|center|Screen Shot]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:14:14Z<p>Takahiro Shimosaka: /* Software */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP,and then we checked it by specific anti GFP monoclonal antibody against R9-GFP. We couldn't detect antigen-antibody reaction against R9-GFP protein. <br><br />
<br />
<br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fuged protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真3.jpg|thumb|600px|left|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|300px|thumb|left|Screen Shot]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br><br><br><br><br><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/File:GGA_010.jpgFile:GGA 010.jpg2012-09-27T03:13:17Z<p>Takahiro Shimosaka: </p>
<hr />
<div></div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:12:48Z<p>Takahiro Shimosaka: /* Software */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP,and then we checked it by specific anti GFP monoclonal antibody against R9-GFP. We couldn't detect antigen-antibody reaction against R9-GFP protein. <br><br />
<br />
<br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9-GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9-GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9-GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9-GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9-GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br />
We suspected that R9-GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9-GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity of R9. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fuged protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真3.jpg|thumb|600px|left|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
[[File:GGA_010.jpg|300px|thumb|left|Screen Shot]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T03:10:15Z<p>Takahiro Shimosaka: /* Software */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and 6 His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His-FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His-FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His-FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : 6 His-FT Cell lysate, not induced<br><br />
Lane4 : 6 His-FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
<br />
We made the construct, lacp-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, kil and another gene. Another gene is pspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This Tat secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and cause macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein with R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. Can E.coli express R9-GFP?====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
We needed to checked function of R9-GFP. However, we didn't know whether ''E.coli'' expressed R9-GFP,and then we checked it by specific anti GFP monoclonal antibody against R9::GFP. We couldn't detect antigen-antibody reaction against R9::GFP protein. <br><br />
<br />
<br />
<br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6 his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br />
We suspected that R9:GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9:GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fuged protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真3.jpg|thumb|600px|left|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check FRUITFULL(FUL), SEPALLATA3(SEP3) and APETALA 1(AP1). SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Then we used better quality of RNA for reverese transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
Now we are trying to check the function of FT.<br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Biosafety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers when you input the sequences of the parts and melting temperature.<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:45:49Z<p>Takahiro Shimosaka: /* Software */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : His:FT Cell lysate, not induced<br><br />
Lane4 : His:FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br><br><br><br />
<br />
[[File:wiki intro 4.png|right|350px]] <br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><br />
<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
'''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br />
<br />
Our secretion system is constructed by TatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes TatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of TatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made Tat construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], TatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our ''E.coli'' expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br />
We suspected that R9:GFP is not transcripted in mRNA, so we did RT-PCR using GFP specific primers.<br><br />
GAPDH is used as internal control.<br />
[[File:RT-PCR1R9-GFP.png|thumb|400px|Fig.3-6 RT-PCR of R9:GFP<br><br />
Lane1:100bp ladder<br />
Lane2:GAPDH (Negative control)<br />
Lane3:GAPDH(R9-GFP)<br />
Lane4:GFP (Negative control)<br />
Lane5:GFP (R9-GFP)]]<br />
As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:竹内さんまとめこれで完成1.png|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, ''E.coli'' can't produce R9 fuged protein. Then for the purpose of the function of R9, we compared between R9+ and R9-. We scratched the cuticule of plant cells and soaked them into a sollution of GFP and R9 or only GEP. This GFP protein was purified with 6His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures indicate the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真1.jpg|thumb|center|700px|Fig3-6. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
===Safety===<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.<br><br><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
This software gives you the sequences of primers if you input the sequences of parts and melting temperature.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T02:38:26Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [[Team:Kyoto/Project|'''Golden Gate assembly''']] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg | link=https://2012.igem.org/Team:Kyoto/Project]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T02:37:43Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [[Team:Kyoto/Project|'''Golden Gate assembly''']] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg | link=https://2012.igem.org/Team:Kyoto/Project]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg | link=https://2012.igem.org/Team:Kyoto/Project]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:23:15Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enables us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes. 6 His tag enables us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperate with KAIT-Japan and indicate Bio Safety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : His:FT Cell lysate, not induced<br><br />
Lane4 : His:FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1''E.coli'' sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2''E.coli'' having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes ''E.coli'' scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of ''E.coli'' on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising ''E.coli'' is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must check whether our ''kil''gene is overexpressed or not.<br><br />
[[File:Lacp kil DT.png|thumb|left|350px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
([[File:wiki intro 4.png|left|350px]]; '''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br><br />
<br />
Our secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene and ''E.coli'' has it originally. This gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 peptide.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:scrachScrach.jpg|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真1.jpg|thumb|center|700px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Learn more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:10:32Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Professor Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperate with KAIT-Japan and indicate Bio Safety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : His:FT Cell lysate, not induced<br><br />
Lane4 : His:FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein generater]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from the cytoplasm to the periplasm. The periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to outside[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechst under and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must check whether our ''kil''gene is overexpressed or not.<br><br />
[[File:Lacp kil DT.png|thumb|left|350px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein.<br> Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on survival of ''E.coli''.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to perip<br />
Detail of Our Secretion System<br />
<br />
(たっとコンストラクション図; '''Tat secretion cassette with constitutive promoter'''[[Part:BBa_K797004|'''(BBa_K797004)''']] <br><br><br />
<br />
Our secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene and E.coli has it originally. This gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.<br><br />
Our secretion system makes many holes in inner and outer membranes. In other words, E.coli which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the E.coli comes to be able to maintain the vitality, though they have many holes in the membrane.<br><br><br />
<br />
This cassette allows E.coli to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.<br><br />
We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
<br />
<br />
<br />
[[File:scrachScrach.jpg|thumb|left|320px|Fig.3-5 Preparation for sample verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticline]]<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:顕微鏡写真1.jpg|thumb|center|700px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA in this experiment.<br><br />
To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, RNA samples in this time are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br><br />
3. We centrifuged samples and collectted supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.[2]<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-09-27T01:59:02Z<p>Takahiro Shimosaka: /* Attribution */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-HumanPractice");<br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-HumanPractice'); return false;"></html>[[Image:KyotoTab_HumanPractice.png|link=|HumanPractice]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Safety'); return false;"></html><br />
[[Image:KyotoTab_Safety.png|link=|Safety]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Acknowledgement'); return false;"></html>[[Image:KyotoTab_Acknowledgement.png|link=|Acknowledgement]]<html></a></html></li><br />
</ul><br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-HumanPractice"><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
<br />
This year, we implemented nine plans on "human practice".<br />
<br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br />
<br />
The first project is that we created an iphone app to let many Japanese know synthetic biology. This is because some Japanese people seem to have a bad impression for gene recombination, so we want to modify this bias. In addition, the number of the japanese who know what gene recombination is is still small, so we would like to show how wonderful recombination is to them.<br><br><br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br><br><br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we decided to tell them about the HS division and support students who are interested in it. There will probably be many teams from Japan in the next iGEM HS division.<br />
<br />
<br />
<br />
==Application==<br />
[[File:Kyoto_iColi0.png|left|300px]]<br />
This year we created an iPhone app named "iColi" that is aimed to let many Japanese know synthetic biology and bring it closer to the non-English speakers in Japan by presentation of an example of gene recombination made by iGEMers. This application contains not only the details of gene recombination but also the list of genes and awards the teams won. Furthermore, it includes the explanation about each themes.<br />
<br />
Nowadays most Japanese people don't know about synthetic biology and details of gene recombination, even though there are many genetically-modified crops in Japan, and they often criticize it with poor knowledge. Then we want to make this situation better.<br />
<br />
This is why we create "iColi".<br />
<br />
We think "iColi" will be one of ways to let Japanese people know what synthetic biology is. This app introduces the understandable characters that were presented in the past iGEM competition and "Flower fairy E.coli", furthermore this app contains the description of each characters. <br />
<br />
We already made it complete, and are preparing to release this app in "App Store" this fall.<br />
[[File:i.Colicoli.png|left|250px]]<br />
<br />
<br />
<br />
<br clear="both" /><br />
<br />
==Education==<br />
[[File:KyotoLecture.jpg|link=|right|300px]]<br />
We went to Hibiya high school and Horikawa high school, then we gave a short introduction on synthetic biology and iGEM, including the attractive points of synthetic biology, the ways of changing living things by reconstructing their genes and activities of other undergraduate iGEM teams. <br />
<br />
In Japan, most high school student was not able to learn about synthetic biology and details of gene combination due to a regular curriculum of high school. This is why we decided to let them know how to conduct the experience, what synthetic biology is, and what iGEM is. <br />
<br />
Furthermore we met some eager junior high school students, so we added a brief explanation about biochemistry, especially genes, nucleotide, codon and the basic system of gene expression.<br />
<br />
We used these slides in order to teach synthetic biology.<br />
[[Team:Kyoto/Education]]<br />
<br style="clear: both;" /><br />
<br />
==Walk-in science==<br />
[[File:Walkincampus.jpg|link=|left|300px]]<br />
[[File:BioBrickBlocks.jpg|link=|center|380px]]<br />
The Faculty of Science, Kyoto University, held "Walk-in Science," a science communication event at Kyoto City Hall Station in February 25 & 26.<br />
We iGEM Kyoto also joined it and ran a booth of synthetic biology.<br />
<br />
<br><br>Today, it is a problem in Japan that more young people are moving away from the sciences. Hence, it is important to let people, especially children, know more about sciences.<br />
"Walk-in Science" was an event to show what we study, how interesting sciences are, to people who walk in non-academic place. "Walk-in Science" joiners had booths on the street of the underground mall.<br />
<br />
To walkers of all ages, we told what biobrick is, with "BioBrick Blocks".<br />
Although most of them did not know about biotechnology, everyone listened to our speech and enrich their understanding.<br />
<br style="clear: both;" /><br />
<br />
==Kyoto University Academic Day ==<br />
[[File:Academicday2.jpg|link=|right|300px]]<br />
Kyoto University Academic Day is a place of communication that anyone can notice the attractiveness of learning fun regardless of Citizens and researchers. <br />
<br />
The main purpose of this event is to reflect public opinion in national policy and research activities in the university.<br />
<br />
Actually we used a poster and a screen to tell how to conduct an experiment of gene recombination, what "iGEM" is, and our results of research to children, housewives, teachers, and so on, then we could communicate and explain it with the people who don't know about "iGEM".<br />
<br />
After our presentation many people said "I have had a bad impression on gene recombination and gene modified crops but today I heard the possibility of gene recombination and Synthetic biology, then I change my root thought about it"<br />
<br />
<br />
<br />
<br style="clear: both;" /><br />
<br />
==November Festival and Open Campus at Kyoto University==<br />
[[File:opencampus.jpg|link=|left|300px]]<br />
Kyoto University has some big events for people out of our university.<br />
We iGEM Kyoto joined two of those events: November Festival(NF) and Open Canpus.<br />
<br />
November Festa. is one of the biggest college festivals in West-Japan.<br />
It is held in 4 days, and many people of all ages come not only from Kyoto City but also from far away.<br />
In this festa. we presented what our team does in iGEM 2011 with a poster.<br />
We also explained what synthetic biology and iGEM are.<br />
<br />
Kyoto University's Open Canpus was held in August 9 & 10.<br />
Many high school students visited our univ.<br />
We talk with them about iGEM as one of the activities which college students can join.<br />
We believe that future iGEMER will come from among students we met.<br />
<br style="clear: both;" /><br />
<br />
==Science Agora==<br />
[[File:scienceagora.jpg|link=|right|400px]]<br />
"Science agora" is the biggest event in Japan about science. Last year, we exhibited our results of research that we created "carnivorous E.coli" and "Flower fairy E.coli" as a member of iGEM Japan.<br />
In the science agora, there are university students, office workers, researchers, teachers, artists, housewives… <br />
A variety of people with various backgrounds for example university students, office workers, researchers, teachers, artists, housewives,and so on carry out an event.<br />
<br />
Many children could learn about synthetic biology here, and we were able to communicate with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science Highschool==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
Through we began to communicate with students and teachers of Toyonaka high school.<br />
Toyonaka high school is authorized Super Science High School by Ministry of Education, Culture, Sports, Science and Technology, and conduct a policy that they will increase the number of students who learn university-level education in biology.<br />
As a one of the program, we decided to conduct the lecture that let them get into synthetic biology and experimental technique.<br />
We will be having the lecture about synthetic biology for three days in November.<br />
We thought this activity has several meaning that we are able to encourage regional development of science and technology and prevail synthetic biology .<br />
We would like to continue a communication like this.<br><br />
<br />
==Cooperation -a meeting in iGEM Japan- ==<br />
[[File:igemjapan.jpg|link=|right|300px]]<br />
This summer, we held a meeting in iGEM Japan at Tokyo Metropolitan University.<br />
The main purpose is to strengthen the relations between iGEM teams in Japan and discuss or debate themes that each teams have earnestly with each other.<br />
This meeting made our research project sophisticated , and make a solid contribution to other teams by criticizing their theme, so we can say that we help each other for the point of research project.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Cooperation -an online meeting in iGEM KANSAI- ==<br />
<br />
<br />
In September 28 we will hold an online meeting among iGEM teams which is in kansai area,and we will practice presentations in Skype.<br />
<br />
<br />
<br />
<br />
<br style="clear: both;" /><br />
<br />
<br />
----<br />
</div><br />
<div id="kyoto-tab-Safety"><br />
<br />
=[[File:Kyoto_Safety.png|link=]]=<br />
<br />
'''Q1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety'''? <br />
<br />
We use the following creatures and genes. <br />
<br />
・Creatures<br />
*''Arabidopsis thaliana''<br />
*''Escherichia coli <br />
''<br />
・Genes<br />
*tatABCD operon<br />
*TAMO reductase<br />
*torA signal<br />
*pspA<br />
*Kil <br />
*FLOWERING LOCUS T <br />
*others in iGEM Parts Kit<br />
FLOWERING LOCUS T is derived from ''Arabidopsis thaliana'' and other all genes expect for iGEM Parts Kit are derived from ''E.coli''. All experiments are conducted under the Biosafety level 1 control. All of rubbish containing recombinant E.coli is not carried out of our Lab before being sterilized. We always take care to lock the door of our Lab when there is no person so that the possibility that someone goes into our Lab and takes away recombinant cells is very low.<br />
<br />
We are trained in applicable lab safety to ensure researcher safety and environmental safety. The researchers have also been trained in proper usage of chemicals and equipment. Some of the biological reagents and experiments necessary for the project needed safety measures. They were; Ethidium Bromide (EtBr), phenol, chloroform, 3,5-dinitrosalicylic acid, gas burner, ultra violet LED, polyacrylamide, 2-mercaptoethanol, PFA. Therefore, all researchers keep following rules when we use the chemicals or equipment above.<br />
* EtBr (Ethidium bromide): EtBr is regarded as a mutagen, carcinogen or teratogen. Lab members were briefed on the possible effects of EtBr. During the experiments, we wore gloves. After use, the gloves and gel were separately disposed.<br />
* Phenol and 3,5-Dinitrosalicylic Acid: Phenol and its vapors are corrosive to eyes, skin, and respiratory tract. 3,5-Dinitrosalicylic acid can cause serious irritation to eyes. These were handled under the draft chamber.<br />
* Chloroform: Chloroform is a possible carcinogen. To avoid unnecessary exposure, this was also handled under the draft chamber.<br />
* Gas Burner: We used gas burners and heated the air around the workspace to raise the temperature, thereby reducing contamination. Although the gas burner is a common combustion apparatus we are all familiar with, it can cause a massive disaster if used improperly. We made all-out efforts to keep all the flammable items away from the flame and not to pass behind a person who is using a gas burner.<br />
* Ultra Violet LED: Ultra violet (UVB) is harmful to eyes. Hence, the LEDs were lit only inside the cardboard box and we only look at it to check if it is properly lit to avoid long exposure.<br />
* polyacrylamide,2-mercaptoethanol:We used these reagents for Western Blotting. They are toxic even though they are necessary to check the expression of target proteins so we asked for advice on how we should store them and obeyed the rules of Kyoto University. <br />
* PFA: This reagents are used to microscope E.coli by Confocal laser scanning microscopy. This is ususally stored in Prof.Agata’s Laboratory <br />
<br />
'''Q2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
<br />
* NO. Currently we have no BioBrick which raises any safety issues. The BioBricks we made this year are derived only from lab-safe strains of Arabidopsis thaliana and E.coli. <br />
<br />
<br />
'''Q3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes. We submitted the experiment plan to the Environment, Safety, and Health Organization, Kyoto Univ. (http://www.esho.kyoto-u.ac.jp/index.php) and were allowed to operate genetic modification of the bacteria under the Biosafety level 1(. Experiments were planned following the safety guidelines of the university (http://www.esho.kyoto-u.ac.jp/wp-content /uploads/2008/05/25_01.pdf). These guidelines are based on national laws and this includes several regulations on GMOs (http://www.bch.biodic.go.jp/hourei1.html). We carried out our experiments in the students-laboratory of the Graduate School of Science / Faculty of Science, Kyoto Univ, which has its own department for safety and environment. All of the lab members received training for PCR, culture of cells, miniprep and etc., the minimal genetical operations and use of autoclave. <br />
<br />
'''Q4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
IGEMers should set up a common clear biosafety regulation in iGEM competition and establish a systematic guideline of education about biosafety. This education should include information about biohazard and biotechnology risk assessment and risk analysis.<br />
<br />
Backgrounds of iGEMers have become wider and wider, from high school students to graduate student in biology and from major in physics to major in molecular biology. For some students, iGEM is the first contact to genetic engineering and they have little knowledge of biohazard and importance of biosafety. Each iGEM teams, of course, teaches the knowledge to their members.However, a systematic education way of biosafety will help students to understand biosafety and to conduct their experiments more adequately. <br />
<br />
From these reasons, we recommend that iGEM establish a systematic guideline of education about biosafety. <br />
<br />
<br />
<br />
'''We work together with [[Team:KAIT_Japan|KAIT_Japan]] on issues of safety.'''<br />
<br />
Cooperatively with KAIT_Japan, We iGEM Kyoto placed icons on our Wiki, which immediately enable you to see a risk of parts. The icons are as follows:<br />
<br />
*Bio safety level 1:http://partsregistry.org/Image:Biosafety_Level1.png<br />
*Bio safety level 2:http://partsregistry.org/Image:Biosafety_Level2.png<br />
*Bio safety level 3:http://partsregistry.org/Image:Biosafety_Level3.png<br />
*cases which affect biological community: http://partsregistry.org/Image:Biocenosis.png<br />
*cases which affect the environment :http://partsregistry.org/Image:Environment.png<br />
*cases which affect human bodies:http://partsregistry.org/Image:Humanbody.png<br />
*cases which includes a certain risk because of mutations: http://partsregistry.org/Image:Mutation.png<br />
<br />
*safe: http://partsregistry.org/Image:Safety.png<br />
</div><br />
<div id="kyoto-tab-Acknowledgement"><br />
<br />
=[[File:Kyoto_Acknowledgement.png|link=]]=<br />
<br />
'''iGEM Kyoto 2012 team wouldn't have been able to do much without the lots of support of great advice and attributions. Thank you very much from the bottom of our heart.'''<br><br><br />
<br />
'''Thank you for providing lots of advice on techniques and helping us to solve scientific ploblems.'''<br><br />
<br />
*Knut Woltjen<br><br />
Center for iPS Cell Research and Application(CiRA). Kyoto University<br><br />
*Humihiko Satou<br><br />
Graduate School of Biostudies. Kyoto University<br><br />
*Takayuki Kouchi<br><br />
Graduate School of Biostudies. Kyoto University<br><br />
*Toru Matou & Masaru Kobayashi<br><br />
Faculty/Graduate School of Agriculture<br><br />
*Takashi Endou<br><br />
Faculty/Graduate School of Agriculture<br><br />
*Tan Inoue<br><br />
Graduate School of Biostudies. Kyoto University<br><br />
*Ken Kajita<br />
Faculty of Engineering. Kyoto University<br><br />
*Tatsuya Hirose<br />
Faculty of Science. Kyoto University<br><br><br />
'''Thank you for letting us use laboratory and seminar room over the summer vacation and use freely expensive laboratory equipment.'''<br><br />
<br />
*Kyoto University Committe, Faculty of Science Building #2<br><br><br />
<br />
<br />
== Attribution ==<br />
<br><br />
<br />
'''Thank you for providing of Flower Locus T Plasmid.'''<br><br />
<br />
*Takashi Araki<br><br />
[http://www.lif.kyoto-u.ac.jp/labs/plantdevbio/index.html Laboratory of Plant Deveropmental Biology]<br><br />
2007 Plant Developmental Biology, Graduate School of BIOSTUDIES, Kyoto-Univ.<br><br><br />
<br />
'''Thank you for providing of Arabidopsis thaliana.'''<br><br />
*Tetsuro Okuno<br><br />
Graduate School of Agriculture, Kyoto Univ.<br><br />
*Kojiro Takanashi<br><br />
Research Institute for Sustainable Humanosphere, Kyoto Univ.<br><br />
*Sota Fujii<br><br />
Graduate School of Science, Kyoto Univ.<br><br />
*Toshiharu Shikanai<br><br />
Graduate School of Science, Kyoto Univ.<br><br><br />
<br />
'''Thank you for providing of reagents.'''<br />
*Yasuo Mori<br><br />
Graduate Scholl of Engineering, Kyoto Univ.<br><br><br />
<br />
'''Thank you for providing of MilliQ and EtBr.'''<br><br />
*Tokitaka Oyama<br><br />
Department of Botany, Graduate School of Science, Kyoto Univ.<br><br><br />
<br />
'''Thank you for providing of Arabidopsis thaliana and Syringe. '''<br><br />
*Ikuko Nishimura<br><br />
Department of Botany, Kyoto Univ <br />
*Tomonori Takada<br><br />
Department of Botany, Kyoto Univ<br><br><br />
<br />
'''Thank you for providing of Arabidopsis thaliana. '''<br><br />
*Ikuhiko Nakase<br><br />
Graduate School of Pharmaceutical Sciences, Kyoto-Univ.<br><br><br />
<br />
'''Thank you for your supervising of our using confocal microscope and liquid nitrogen.'''<br><br />
*Makoto Kashima<br><br />
Graduate School of Science, Kyoto Univ<br><br><br />
<br />
'''Thank you for your supervising of our purification of protein.'''<br><br />
*Wataru Shihoya<br><br />
Cellular and Structural Physiology Institute, Nagoya University<br><br><br />
<br />
=[[File:Kyoto_Sponsors.png|link=]]=<br />
'''Kyoto University''' is our school and support us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Intergrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL)''' supported us.<br><br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site]]<br><br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br><br><br><br />
'''CosmoBio''' supported us financially.<br><br />
[[File:CosmoBio.jpg | link=http://www.cosmobio.co.jp/]]<br><br />
</div><br />
<div id="kyoto-tab-Criteria"><br />
<br />
[https://igem.org/2012_Judging_Form?id=797 Criteria]<br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-09-27T01:58:36Z<p>Takahiro Shimosaka: /* Attribution */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-HumanPractice");<br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-HumanPractice'); return false;"></html>[[Image:KyotoTab_HumanPractice.png|link=|HumanPractice]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Safety'); return false;"></html><br />
[[Image:KyotoTab_Safety.png|link=|Safety]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Acknowledgement'); return false;"></html>[[Image:KyotoTab_Acknowledgement.png|link=|Acknowledgement]]<html></a></html></li><br />
</ul><br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-HumanPractice"><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
<br />
This year, we implemented nine plans on "human practice".<br />
<br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br />
<br />
The first project is that we created an iphone app to let many Japanese know synthetic biology. This is because some Japanese people seem to have a bad impression for gene recombination, so we want to modify this bias. In addition, the number of the japanese who know what gene recombination is is still small, so we would like to show how wonderful recombination is to them.<br><br><br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br><br><br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we decided to tell them about the HS division and support students who are interested in it. There will probably be many teams from Japan in the next iGEM HS division.<br />
<br />
<br />
<br />
==Application==<br />
[[File:Kyoto_iColi0.png|left|300px]]<br />
This year we created an iPhone app named "iColi" that is aimed to let many Japanese know synthetic biology and bring it closer to the non-English speakers in Japan by presentation of an example of gene recombination made by iGEMers. This application contains not only the details of gene recombination but also the list of genes and awards the teams won. Furthermore, it includes the explanation about each themes.<br />
<br />
Nowadays most Japanese people don't know about synthetic biology and details of gene recombination, even though there are many genetically-modified crops in Japan, and they often criticize it with poor knowledge. Then we want to make this situation better.<br />
<br />
This is why we create "iColi".<br />
<br />
We think "iColi" will be one of ways to let Japanese people know what synthetic biology is. This app introduces the understandable characters that were presented in the past iGEM competition and "Flower fairy E.coli", furthermore this app contains the description of each characters. <br />
<br />
We already made it complete, and are preparing to release this app in "App Store" this fall.<br />
[[File:i.Colicoli.png|left|250px]]<br />
<br />
<br />
<br />
<br clear="both" /><br />
<br />
==Education==<br />
[[File:KyotoLecture.jpg|link=|right|300px]]<br />
We went to Hibiya high school and Horikawa high school, then we gave a short introduction on synthetic biology and iGEM, including the attractive points of synthetic biology, the ways of changing living things by reconstructing their genes and activities of other undergraduate iGEM teams. <br />
<br />
In Japan, most high school student was not able to learn about synthetic biology and details of gene combination due to a regular curriculum of high school. This is why we decided to let them know how to conduct the experience, what synthetic biology is, and what iGEM is. <br />
<br />
Furthermore we met some eager junior high school students, so we added a brief explanation about biochemistry, especially genes, nucleotide, codon and the basic system of gene expression.<br />
<br />
We used these slides in order to teach synthetic biology.<br />
[[Team:Kyoto/Education]]<br />
<br style="clear: both;" /><br />
<br />
==Walk-in science==<br />
[[File:Walkincampus.jpg|link=|left|300px]]<br />
[[File:BioBrickBlocks.jpg|link=|center|380px]]<br />
The Faculty of Science, Kyoto University, held "Walk-in Science," a science communication event at Kyoto City Hall Station in February 25 & 26.<br />
We iGEM Kyoto also joined it and ran a booth of synthetic biology.<br />
<br />
<br><br>Today, it is a problem in Japan that more young people are moving away from the sciences. Hence, it is important to let people, especially children, know more about sciences.<br />
"Walk-in Science" was an event to show what we study, how interesting sciences are, to people who walk in non-academic place. "Walk-in Science" joiners had booths on the street of the underground mall.<br />
<br />
To walkers of all ages, we told what biobrick is, with "BioBrick Blocks".<br />
Although most of them did not know about biotechnology, everyone listened to our speech and enrich their understanding.<br />
<br style="clear: both;" /><br />
<br />
==Kyoto University Academic Day ==<br />
[[File:Academicday2.jpg|link=|right|300px]]<br />
Kyoto University Academic Day is a place of communication that anyone can notice the attractiveness of learning fun regardless of Citizens and researchers. <br />
<br />
The main purpose of this event is to reflect public opinion in national policy and research activities in the university.<br />
<br />
Actually we used a poster and a screen to tell how to conduct an experiment of gene recombination, what "iGEM" is, and our results of research to children, housewives, teachers, and so on, then we could communicate and explain it with the people who don't know about "iGEM".<br />
<br />
After our presentation many people said "I have had a bad impression on gene recombination and gene modified crops but today I heard the possibility of gene recombination and Synthetic biology, then I change my root thought about it"<br />
<br />
<br />
<br />
<br style="clear: both;" /><br />
<br />
==November Festival and Open Campus at Kyoto University==<br />
[[File:opencampus.jpg|link=|left|300px]]<br />
Kyoto University has some big events for people out of our university.<br />
We iGEM Kyoto joined two of those events: November Festival(NF) and Open Canpus.<br />
<br />
November Festa. is one of the biggest college festivals in West-Japan.<br />
It is held in 4 days, and many people of all ages come not only from Kyoto City but also from far away.<br />
In this festa. we presented what our team does in iGEM 2011 with a poster.<br />
We also explained what synthetic biology and iGEM are.<br />
<br />
Kyoto University's Open Canpus was held in August 9 & 10.<br />
Many high school students visited our univ.<br />
We talk with them about iGEM as one of the activities which college students can join.<br />
We believe that future iGEMER will come from among students we met.<br />
<br style="clear: both;" /><br />
<br />
==Science Agora==<br />
[[File:scienceagora.jpg|link=|right|400px]]<br />
"Science agora" is the biggest event in Japan about science. Last year, we exhibited our results of research that we created "carnivorous E.coli" and "Flower fairy E.coli" as a member of iGEM Japan.<br />
In the science agora, there are university students, office workers, researchers, teachers, artists, housewives… <br />
A variety of people with various backgrounds for example university students, office workers, researchers, teachers, artists, housewives,and so on carry out an event.<br />
<br />
Many children could learn about synthetic biology here, and we were able to communicate with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science Highschool==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
Through we began to communicate with students and teachers of Toyonaka high school.<br />
Toyonaka high school is authorized Super Science High School by Ministry of Education, Culture, Sports, Science and Technology, and conduct a policy that they will increase the number of students who learn university-level education in biology.<br />
As a one of the program, we decided to conduct the lecture that let them get into synthetic biology and experimental technique.<br />
We will be having the lecture about synthetic biology for three days in November.<br />
We thought this activity has several meaning that we are able to encourage regional development of science and technology and prevail synthetic biology .<br />
We would like to continue a communication like this.<br><br />
<br />
==Cooperation -a meeting in iGEM Japan- ==<br />
[[File:igemjapan.jpg|link=|right|300px]]<br />
This summer, we held a meeting in iGEM Japan at Tokyo Metropolitan University.<br />
The main purpose is to strengthen the relations between iGEM teams in Japan and discuss or debate themes that each teams have earnestly with each other.<br />
This meeting made our research project sophisticated , and make a solid contribution to other teams by criticizing their theme, so we can say that we help each other for the point of research project.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Cooperation -an online meeting in iGEM KANSAI- ==<br />
<br />
<br />
In September 28 we will hold an online meeting among iGEM teams which is in kansai area,and we will practice presentations in Skype.<br />
<br />
<br />
<br />
<br />
<br style="clear: both;" /><br />
<br />
<br />
----<br />
</div><br />
<div id="kyoto-tab-Safety"><br />
<br />
=[[File:Kyoto_Safety.png|link=]]=<br />
<br />
'''Q1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety'''? <br />
<br />
We use the following creatures and genes. <br />
<br />
・Creatures<br />
*''Arabidopsis thaliana''<br />
*''Escherichia coli <br />
''<br />
・Genes<br />
*tatABCD operon<br />
*TAMO reductase<br />
*torA signal<br />
*pspA<br />
*Kil <br />
*FLOWERING LOCUS T <br />
*others in iGEM Parts Kit<br />
FLOWERING LOCUS T is derived from ''Arabidopsis thaliana'' and other all genes expect for iGEM Parts Kit are derived from ''E.coli''. All experiments are conducted under the Biosafety level 1 control. All of rubbish containing recombinant E.coli is not carried out of our Lab before being sterilized. We always take care to lock the door of our Lab when there is no person so that the possibility that someone goes into our Lab and takes away recombinant cells is very low.<br />
<br />
We are trained in applicable lab safety to ensure researcher safety and environmental safety. The researchers have also been trained in proper usage of chemicals and equipment. Some of the biological reagents and experiments necessary for the project needed safety measures. They were; Ethidium Bromide (EtBr), phenol, chloroform, 3,5-dinitrosalicylic acid, gas burner, ultra violet LED, polyacrylamide, 2-mercaptoethanol, PFA. Therefore, all researchers keep following rules when we use the chemicals or equipment above.<br />
* EtBr (Ethidium bromide): EtBr is regarded as a mutagen, carcinogen or teratogen. Lab members were briefed on the possible effects of EtBr. During the experiments, we wore gloves. After use, the gloves and gel were separately disposed.<br />
* Phenol and 3,5-Dinitrosalicylic Acid: Phenol and its vapors are corrosive to eyes, skin, and respiratory tract. 3,5-Dinitrosalicylic acid can cause serious irritation to eyes. These were handled under the draft chamber.<br />
* Chloroform: Chloroform is a possible carcinogen. To avoid unnecessary exposure, this was also handled under the draft chamber.<br />
* Gas Burner: We used gas burners and heated the air around the workspace to raise the temperature, thereby reducing contamination. Although the gas burner is a common combustion apparatus we are all familiar with, it can cause a massive disaster if used improperly. We made all-out efforts to keep all the flammable items away from the flame and not to pass behind a person who is using a gas burner.<br />
* Ultra Violet LED: Ultra violet (UVB) is harmful to eyes. Hence, the LEDs were lit only inside the cardboard box and we only look at it to check if it is properly lit to avoid long exposure.<br />
* polyacrylamide,2-mercaptoethanol:We used these reagents for Western Blotting. They are toxic even though they are necessary to check the expression of target proteins so we asked for advice on how we should store them and obeyed the rules of Kyoto University. <br />
* PFA: This reagents are used to microscope E.coli by Confocal laser scanning microscopy. This is ususally stored in Prof.Agata’s Laboratory <br />
<br />
'''Q2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
<br />
* NO. Currently we have no BioBrick which raises any safety issues. The BioBricks we made this year are derived only from lab-safe strains of Arabidopsis thaliana and E.coli. <br />
<br />
<br />
'''Q3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes. We submitted the experiment plan to the Environment, Safety, and Health Organization, Kyoto Univ. (http://www.esho.kyoto-u.ac.jp/index.php) and were allowed to operate genetic modification of the bacteria under the Biosafety level 1(. Experiments were planned following the safety guidelines of the university (http://www.esho.kyoto-u.ac.jp/wp-content /uploads/2008/05/25_01.pdf). These guidelines are based on national laws and this includes several regulations on GMOs (http://www.bch.biodic.go.jp/hourei1.html). We carried out our experiments in the students-laboratory of the Graduate School of Science / Faculty of Science, Kyoto Univ, which has its own department for safety and environment. All of the lab members received training for PCR, culture of cells, miniprep and etc., the minimal genetical operations and use of autoclave. <br />
<br />
'''Q4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
IGEMers should set up a common clear biosafety regulation in iGEM competition and establish a systematic guideline of education about biosafety. This education should include information about biohazard and biotechnology risk assessment and risk analysis.<br />
<br />
Backgrounds of iGEMers have become wider and wider, from high school students to graduate student in biology and from major in physics to major in molecular biology. For some students, iGEM is the first contact to genetic engineering and they have little knowledge of biohazard and importance of biosafety. Each iGEM teams, of course, teaches the knowledge to their members.However, a systematic education way of biosafety will help students to understand biosafety and to conduct their experiments more adequately. <br />
<br />
From these reasons, we recommend that iGEM establish a systematic guideline of education about biosafety. <br />
<br />
<br />
<br />
'''We work together with [[Team:KAIT_Japan|KAIT_Japan]] on issues of safety.'''<br />
<br />
Cooperatively with KAIT_Japan, We iGEM Kyoto placed icons on our Wiki, which immediately enable you to see a risk of parts. The icons are as follows:<br />
<br />
*Bio safety level 1:http://partsregistry.org/Image:Biosafety_Level1.png<br />
*Bio safety level 2:http://partsregistry.org/Image:Biosafety_Level2.png<br />
*Bio safety level 3:http://partsregistry.org/Image:Biosafety_Level3.png<br />
*cases which affect biological community: http://partsregistry.org/Image:Biocenosis.png<br />
*cases which affect the environment :http://partsregistry.org/Image:Environment.png<br />
*cases which affect human bodies:http://partsregistry.org/Image:Humanbody.png<br />
*cases which includes a certain risk because of mutations: http://partsregistry.org/Image:Mutation.png<br />
<br />
*safe: http://partsregistry.org/Image:Safety.png<br />
</div><br />
<div id="kyoto-tab-Acknowledgement"><br />
<br />
=[[File:Kyoto_Acknowledgement.png|link=]]=<br />
<br />
'''iGEM Kyoto 2012 team wouldn't have been able to do much without the lots of support of great advice and attributions. Thank you very much from the bottom of our heart.'''<br><br><br />
<br />
'''Thank you for providing lots of advice on techniques and helping us to solve scientific ploblems.'''<br><br />
<br />
*Knut Woltjen<br><br />
Center for iPS Cell Research and Application(CiRA). Kyoto University<br><br />
*Humihiko Satou<br><br />
Graduate School of Biostudies. Kyoto University<br><br />
*Takayuki Kouchi<br><br />
Graduate School of Biostudies. Kyoto University<br><br />
*Toru Matou & Masaru Kobayashi<br><br />
Faculty/Graduate School of Agriculture<br><br />
*Takashi Endou<br><br />
Faculty/Graduate School of Agriculture<br><br />
*Tan Inoue<br><br />
Graduate School of Biostudies. Kyoto University<br><br />
*Ken Kajita<br />
Faculty of Engineering. Kyoto University<br><br />
*Tatsuya Hirose<br />
Faculty of Science. Kyoto University<br><br><br />
'''Thank you for letting us use laboratory and seminar room over the summer vacation and use freely expensive laboratory equipment.'''<br><br />
<br />
*Kyoto University Committe, Faculty of Science Building #2<br><br><br />
<br />
<br />
== Attribution ==<br />
<br><br />
<br />
'''Thank you for providing of Flower Locus T Plasmid.'''<br><br />
<br />
*Takashi Araki<br><br />
[http://www.lif.kyoto-u.ac.jp/labs/plantdevbio/index.html Laboratory of Plant Deveropmental Biology]<br><br />
2007 Plant Developmental Biology, Graduate School of BIOSTUDIES, Kyoto-Univ.<br><br><br />
<br />
'''Thank you for providing of Arabidopsis thaliana.'''<br><br />
*Tetsuro Okuno<br><br />
Graduate School of Agriculture, Kyoto Univ.<br><br />
*Kojiro Takanashi<br><br />
Research Institute for Sustainable Humanosphere, Kyoto Univ.<br><br />
*Sota Fujii<br><br />
Graduate School of Science, Kyoto Univ.<br><br />
*Toshiharu Shikanai<br><br />
Graduate School of Science, Kyoto Univ.<br><br><br />
<br />
'''Thank you for providing of reagents.'''<br />
*Yasuo Mori*<br><br />
Graduate Scholl of Engineering, Kyoto Univ.<br><br><br />
<br />
'''Thank you for providing of MilliQ and EtBr.'''<br><br />
*Tokitaka Oyama<br><br />
Department of Botany, Graduate School of Science, Kyoto Univ.<br><br><br />
<br />
'''Thank you for providing of Arabidopsis thaliana and Syringe. '''<br><br />
*Ikuko Nishimura<br><br />
Department of Botany, Kyoto Univ <br />
*Tomonori Takada<br><br />
Department of Botany, Kyoto Univ<br><br><br />
<br />
'''Thank you for providing of Arabidopsis thaliana. '''<br><br />
*Ikuhiko Nakase<br><br />
Graduate School of Pharmaceutical Sciences, Kyoto-Univ.<br><br><br />
<br />
'''Thank you for your supervising of our using confocal microscope and liquid nitrogen.'''<br><br />
*Makoto Kashima<br><br />
Graduate School of Science, Kyoto Univ<br><br><br />
<br />
'''Thank you for your supervising of our purification of protein.'''<br><br />
*Wataru Shihoya<br><br />
Cellular and Structural Physiology Institute, Nagoya University<br><br><br />
<br />
=[[File:Kyoto_Sponsors.png|link=]]=<br />
'''Kyoto University''' is our school and support us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Intergrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL)''' supported us.<br><br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site]]<br><br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br><br><br><br />
'''CosmoBio''' supported us financially.<br><br />
[[File:CosmoBio.jpg | link=http://www.cosmobio.co.jp/]]<br><br />
</div><br />
<div id="kyoto-tab-Criteria"><br />
<br />
[https://igem.org/2012_Judging_Form?id=797 Criteria]<br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T01:40:32Z<p>Takahiro Shimosaka: /* Result 2: evaluation of kil protein */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Mr.Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperate with KAIT-Japan and indicate Bio Safety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : His:FT Cell lysate, not induced<br><br />
Lane4 : His:FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competitions, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must find the suitable amount of ''kil'' gene expression.<br><br />
[[File:Lacp kil DT.png|thumb|left|350px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein. Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm.<br />
<br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
[[File:顕微鏡写真1.jpg|thumb|center|700px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. We did freezing with liquid nitrogen more rapidly.<br><br />
3. We centrifuged the samples and collect supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't seem to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013] and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T01:38:40Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [[Team:Kyoto/Project|'''Golden Gate assembly''']] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T01:37:27Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [[https://2012.igem.org/Team:Kyoto/Project| Golden Gate Assembly]] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T01:36:51Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [https://2012.igem.org/Team:Kyoto/Project| Golden Gate Assembly] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T01:35:37Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [https://2012.igem.org/Team:Kyoto/Project|'''Golden Gate Assembly'''] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:KyotoTeam:Kyoto2012-09-27T01:35:05Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:KyotoHeader.png|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<div><br />
=[[File:Kyoto_OurProject.png|link=|180px]]=<br />
==[[File:Kyoto_FlowerFairyEcoli.png|link=]]==<br />
How wonderful would it be, if you can see flowers in full glory throughout the year? If flowers bloom whenever you want, you could eat tasty fruits and see beautiful flowers any time. It is what we have been looking for, the technology to make flowers bloom. There is a plant hormone that enables us to realize this dream. The plant hormone, Florigen, controls and triggers flowering in plants. We will create [[Team:Kyoto/Project|'''Flower Fairy E.coli''']]. This will make Florigen and transfer it into plant cells! This will enable us to make flowers bloom anytime!<br />
<br />
<html><br />
<style type="text/css"><br />
#kyoto-movie {<br />
margin: auto;<br />
width: 650px;<br />
height: 404px;<br />
overflow: hidden;<br />
position: relative;<br />
}<br />
#kyoto-movie > img {<br />
margin-left: 43px;<br />
height: 404px;<br />
width: 568px;<br />
position: absolute;<br />
z-index: 100;<br />
}<br />
#kyoto-movie-base {<br />
width: 1850px;<br />
height: 404px;<br />
padding: 0px 135px;<br />
background-image: url(https://static.igem.org/mediawiki/2012/5/5e/Kyoto_Nega.png);<br />
position: absolute;<br />
z-index: 50;<br />
}<br />
#kyoto-movie-base > img {<br />
margin: 60px -2px;<br />
padding: 0px;<br />
width: 400px;<br />
height: 284px;<br />
}<br />
</style><br />
<script type="text/javascript"><br />
function moveRight() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left - 404;<br />
if (left < -1212) left =-1212;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
function moveLeft() {<br />
var base = $("#kyoto-movie-base");<br />
var left = base.position().left + 404;<br />
if (left > 0) left = 0;<br />
base.animate({<br />
"left" : left+"px"<br />
},500);<br />
}<br />
$(function() {<br />
$("#kyoto-movie").click(function(event){<br />
var x = event.pageX-$(this).offset().left;<br />
if (x < 133) { return; }<br />
if (x > 520) { return; }<br />
if (x < 320) { moveLeft(); }<br />
else if (x > 320) { moveRight(); }<br />
});<br />
});<br />
</script><br />
<div id="kyoto-movie"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1f/Kyoto_Frame.png"></img><br />
<div id="kyoto-movie-base"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e1/Kyoto_Movie1.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Kyoto_Movie2.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/3/33/Kyoto_Movie3.png"></img><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Kyoto_Movie4.png"></img><br />
</div><br />
</div><br />
</html><br />
<br />
==[[File:Kyoto_GoldenGateAssembly.png|link=]]==<br />
<br />
Have you ever hoped for a faster experiment? We guess, you have. In order to achieve a successful study, we have to experiment day by day. This is the case for iGEM, too. iGEMers tend to experiment on ''Escherichia.coli'', and almost all experiments take a lot of time. Unless our guess is wrong, all scholars and iGEMers undergo hardships that make it seem impossible to complete their work by a deadline. [[https://2012.igem.org/Team:Kyoto/Project|'''Golden Gate Assembly''']] made up by Carola Engler, Romy Kandzia, Sylvestre Marillonnet enables us to construct genes faster than any other assembly methods. We will confirm this assembly method and will so some experiments to check how the number of promoters influences the strength of transcription.<br />
<br />
[[File:Kyoto_GoldenGateAssembly.jpg]]<br />
[[File:Kyoto_GoldenPrimerDesigner.jpg]]<br />
<br clear="both" /><br />
<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
This year, we implemented <font size="3">nine projects</font> on [[Team:Kyoto/Consideration|"'''human practice'''"]]. <br><br />
<br />
Especially, we gave high priority to two programs, "Making iPhone app" and "Educational program" <br><br />
We created an iPhone app named "iColi" which is a way to bring Japanese closer to synthetic biology.<br />
In addition, we carried out a lecture of synthetic biology and biochemistry at many places, Hibiya high school, Horikawa high school, Kyoto university, and so on.<br />
Our activities which we conduct this year actually help many people know synthetic biology and gene recombination.<br />
<br />
<font size="5">Application</font><br><br />
----<br />
[[File:Kyoto_iColi0.png|right|300px]]<br />
One of the main projects is to creat an iphone apps to help many Japanese know synthetic biology. <br />
<br />
We set the project because some Japanese people seem to have a bad impression for gene recombination, and we want to modify this bias. <br />
<br />
In addition, few Japanese people know what gene recombination is all about, so we would like to show them how wonderful recombination is.<br />
<br />
<br />
<font size="5">Education</font><br><br />
----<br />
The second project is to introduce the synthetic biology to high school students, as we would like many high school students to participate in iGEM. <br />
This summer, we carried out lectures about gene recombination and biochemistry in "Seiryo festival" at Hibiya high school, furthermore we held a poster session in open campus of Kyoto university.<br />
<br />
Due to the curriculum in our country, it is rather difficult for high school students to take part in iGEM undergraduate category, so we tell them about the HS division and support students who are interested in it. We hope that many japanese teams take part in the next iGEM HS division.<br><br />
<br />
<font size="5">Other activities</font><br><br />
----<br />
*Kyoto university Academic day<br><br />
*November Festival and Open Campus at Kyoto University<br><br />
*Science agora<br><br />
*Tie-up with Super Science High school<br><br />
*Cooperation<br><br />
<br />
<br style="clear: both;" /><br />
<br />
=[[File:Kyoto_Criteria.png|link=]]=<br />
You can see it at [https://igem.org/2012_Judging_Form?id=797 here].<br />
<br />
=[[File:Kyoto_Sponsors.png]]=<br />
<div style="background-color: #ffffff;"><br />
[[File:KUlogo.png | link=http://www.kyoto-u.ac.jp/|250px]]<br />
[[File:CosmoBio.png | link=http://www.cosmobio.co.jp/|250px]]<br />
[[File:IDTLogo2010.png | link=http://www.idtdna.com/site|250px]] <br />
[[File:MBLlogo.gif | link=http://www.mbl.co.jp/e/index.html]]<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T01:31:00Z<p>Takahiro Shimosaka: /* Result 1: Modified TorA signal */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Mr.Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperate with KAIT-Japan and indicate Bio Safety Level of our parts.<br><br><br><br />
[[File:westernFT3.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : FT Cell lysate, not induced<br><br />
Lane2 : FT Cell lysate, IPTG induced <br> <br />
Lane3 : His:FT Cell lysate, not induced<br><br />
Lane4 : His:FT Cell lysate, IPTG induced <br> ]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed stop codon doesn't appear when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competition, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must find the suitable amount of ''kil'' gene expression.<br><br />
[[File:Lacp kil DT.png|thumb|left|350px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein. Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm.<br />
<br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:顕微鏡写真1.jpg|thumb|right|750px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. We did freezing with liquid nitrogen more rapidly.<br><br />
3. We centrifuged the samples and collect supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us to introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T01:25:12Z<p>Takahiro Shimosaka: /* Result 2: evaluation of kil protein */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Mr.Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperate with KAIT-Japan and indicate Bio Safety Level of our parts.<br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal doesn't appear stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competition, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on a flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, a flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must find the suitable amount of ''kil'' gene expression.<br><br />
[[File:Lacp kil DT.png|thumb|left|350px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein. Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm.<br />
<br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:顕微鏡写真.jpg|thumb|right|600px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. We did freezing with liquid nitrogen more rapidly.<br><br />
3. We centrifuged the samples and collect supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T01:21:42Z<p>Takahiro Shimosaka: /* Result 1: Modified TorA signal */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Mr.Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br><br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice|left|BioSafetyLevel1]]<br />
<br>We cooperate with KAIT-Japan and indicate Bio Safety Level of our parts.<br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal doesn't appear stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competition, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must find the suitable amount of ''kil'' gene expression.<br><br />
[[File:Lacp kil DT.png|thumb|left|350px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|left|350px|Fig.2-2 the result of evaluation of kil protein. Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
====Result 3: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm.<br />
<br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:顕微鏡写真.jpg|thumb|right|600px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step; '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. We did freezing with liquid nitrogen more rapidly.<br><br />
3. We centrifuged the samples and collect supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T01:08:55Z<p>Takahiro Shimosaka: /* Result 1: Modified TorA signal */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside it.<br><br><br />
Mr.Araki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
Of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
<br />
[[File:FT1.png|thumb|left|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
<br />
[[File:FT2.png|thumb|left|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br><br><br><br />
<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br><br><br><br><br><br><br><br><br><br><br><br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design an applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't appear stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: evaluation of kil protein====<br />
In previous iGEM competition, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette. This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappear, flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.<br><br />
To be sure, Tat pathway can carry proteins from cytoplasm into periplasm. There is,however,still a obstacle for proteins to effect materials outside ''E.coli'' the outer membrane. To encourage protein to get outside, we expressed ''kil'' gene. ''Kil'' gene is the Colding site of Colicin E1 lysis protein(kil protein.)<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Larger hole area means larger amount of proteins can go out. On the other hand, the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''Kil'',threfore, causes cell death. For this reason, we must find the suitable amount of ''kil'' gene expression.<br><br />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-1 The construction used in this assay. the Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|right|350px|Fig.2-2 the result of evaluation of kil protein. Vertical axis means the value of OD 600.<br />
and horizontal axis means incuvation time.]]<br><br />
<br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of ''Arabidopsis thaliana'' leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step, '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
We improved following things;<br><br />
1. Total leaf volume was increased.<br><br />
2. We did freezing with liquid nitrogen more rapidly.<br><br />
3. We centrifuged the samples and collect supernatant twice after adding ISOGEN.<br><br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we are trying to check the function of FT.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T00:28:21Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br><br />
Mr.Arzki kindly gave us FT cDNA of ''Arabidopsis thaliana'' in TOPO blunt end 2(Invitrogen). When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primers which contain one base mis-match between primer and cDNA. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't appear stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構改.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane to penetrate GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step, '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we can characterize FT more precisely.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
We create the segments which have complementary ligation sites so that we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T00:19:56Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't appear stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1E.coli sample stained with Hoechstunder and observed with 352nm wavelength<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and observed with 489nm wavelength<br />
</gallery><br />
<br><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane penetrating GFP in order to confirm R9 peptide system by fluorecence. This is because we didn't know whether our E.coli expressed R9 peptide fusioned GFP properly. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step, '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we can characterize FT more precisely.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, when we create the segments so that ligation sites become complementary, we can introduce plural DNA segments into one plasmid at the same time and arrange them in the way we like . (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T00:14:23Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid appearing a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't appear stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1the same sample under UV<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide adheres to cell membrane of plants because of hydrophobic character.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody against R9::GFP. We wanted membrane penetrating GFP in order to confirm R9 peptide system by fluorecence. because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br><br />
Minor bands of induced samples are considered as the degradated GFP. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were connected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step, '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we can characterize FT more precisely.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
Most restrict enzyme cuts its own recognition sites. <br />
But BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts downstream of the recognition site as shown in the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time when we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T00:00:40Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<html><a id="FFEResults"></a></html><br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011[[Part:BBa_K233307|'''(BBa_K233307)''']] cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid raising a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:青色.jpg|fig2-1the same sample under UV<br />
File:緑色.jpg|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(fig.3-1).<br><br><br>[[File:R9.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br>]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis,a specific form of endocytosis. The use of CPP in plant cells is already verified.<br><br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:R9取り込み機構.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody(Wako, 012-22541). The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step, '''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we can characterize FT more precisely.<br><br><br><br><br />
<div><br />
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
<br />
[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBricks are useful for us because we can look for required BioBrick parts from their registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly takes advantage of the characteristic restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four bp for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams to use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:31:17Z<p>Takahiro Shimosaka: /* Plasmid backbone (BBa_K797013) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br><br />
Now we can characterize FT more precisely. <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone [http://partsregistry.org/Part:BBa_K797013 BBa_K797013]==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].(See the part of Golden Gate Assembly)<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:30:05Z<p>Takahiro Shimosaka: /* Plasmid backbone (BBa_K797013) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3), a breed of competent cell. Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE. <br><br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it was required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (BBa_K797013)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
[[File:電気泳動.PNG|250px|center|]]<br />
After we confirmed this part has the restriction cites and restriction enzyme cutting sites of BsaI. After this, we mixed DpnI with enzyme-treated plasmid and conducted ligation.<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:26:44Z<p>Takahiro Shimosaka: /* Plasmid backbone (psB1K3) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT and 6 His::FT proteins in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925-01.png|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it is required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (BBa_K797013)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:電気泳動.PNG|250px|center|]]<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:23:48Z<p>Takahiro Shimosaka: /* Plasmid backbone (psB1K3) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT protein in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it is required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (psB1K3)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:23:27Z<p>Takahiro Shimosaka: /* Plasmid backbone (psB1K3) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT protein in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it is required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (psB1K3)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:22:59Z<p>Takahiro Shimosaka: /* Plasmid backbone (psB1K3) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT protein in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it is required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (psB1K3)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|center|]]<br />
<br><br><br><br><br><br><br />
<br />
<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:21:33Z<p>Takahiro Shimosaka: /* Plasmid backbone (psB1K3) */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT protein in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, the waveform of it has a law peak at 260nm(Fig.4-4.)<br><br />
So, it is required to improve the method of RNA exraction.<br><br />
<br />
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br />
[[File:RNA0926.png|thumb|350px|Fig.4-4 Improved RNA waveform]]<br />
[[File:TotalRNA20120926.jpeg|thumb|400px|Fig.4-5]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (psB1K3)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
[[File:GGA_003.jpg|300px|left|]]<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:18:06Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes .6 His tag enable us to protein purification from ''E.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using anti-FT goat antibody and checked the place of the FT protein band.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region. We succeeded in expression of FT protein in ''E.coli''.<br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT and 6 His::FT proteins. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, long-time incubated RNA samples are degradated.<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
Moreover, RNA waveform of long time incubated has a law peak at 260nm(Fig.4-4.)<br><br />
So, it is required to improve the method of RNA exraction.<br><br />
<br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
==Plasmid backbone (psB1K3)==<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<br />
<br />
==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:09:09Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR with the use of restrose primers. This PCR enable us to mutate FT because tensile direction is reverse.<br />
<br />
<br />
of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes . His tag enable us to protein purification from ''e.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using FT specific antibody and checked the place of the FT protein band.<br><br />
As a result, FT and His:FT bands were observed at the expected molecular weight region. <br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT protein. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated.<br><br />
Moreover, RNA waveform of long time incubated has a law peak at 260nm(Fig.4-4.)<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
So, it is required to improve the method of RNA exraction.<br><br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated<br><br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
[[File:RNA0925.png|300px]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:Golden⑤_syuusei.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC"(Figure 1) and cuts DNA like the Figure 2.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 4) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:04:52Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR, with the primers of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes before completing our secretion system. His tag enable us to protein purification from ''e.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using FT specific antibody and checked the place of the FT protein band.<br><br />
As a result, FT and His:FT bands were observed at the expected molecular weight region. <br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT protein. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated.<br><br />
Moreover, RNA waveform of long time incubated has a law peak at 260nm(Fig.4-4.)<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
So, it is required to improve the method of RNA exraction.<br><br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated<br><br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
[[File:RNA0925.png|300px]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:GGA_002.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC" and cuts DNA like the figure 3.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 2) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T23:04:03Z<p>Takahiro Shimosaka: /* What's Golden Gate Assembly */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR, with the primers of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to purify FT protein in order to take an experiment of FT penetration into plant cell membranes before completing our secretion system. His tag enable us to protein purification from ''e.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to purify FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using FT specific antibody and checked the place of the FT protein band.<br><br />
As a result, FT and His:FT bands were observed at the expected molecular weight region. <br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT protein. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated.<br><br />
Moreover, RNA waveform of long time incubated has a law peak at 260nm(Fig.4-4.)<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
So, it is required to improve the method of RNA exraction.<br><br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated<br><br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
[[File:RNA0925.png|300px]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.1 BsaI recognition sites.]][[File:GGA_002.jpg|300px|thumb|center|Fig.2 irreversibe ligation]]<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.3 conventional method.]][[File:GGA_004.jpg|300px|thumb|center|Fig.4 Golden Gate Assembly.]]<br />
<br />
<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC" and cuts DNA like the figure 3.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 2) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T22:58:56Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR, with the primers of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to refine FT protein in order to take an experiment of FT introduction into plant cells before completing our secretion system. His tag enable us to protein purification from ''e.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to refine FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using FT specific antibody and checked the place of the FT protein band.<br><br />
As a result, FT and His:FT bands were observed at the expected molecular weight region. <br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT protein. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated.<br><br />
Moreover, RNA waveform of long time incubated has a law peak at 260nm(Fig.4-4.)<br><br />
[[File:RNA0925.png|thumb|300px|Fig.4-4 RNA waveform of long time incubated leaves]]<br />
So, it is required to improve the method of RNA exraction.<br><br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated<br><br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
[[File:RNA0925.png|300px]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"] to make it easier to use Golden Gate assembly.<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.1 conventional method.]][[File:GGA_002.jpg|300px|thumb|center|Fig.2 Golden Gate Assembly.]]<br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.3 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.4 irreversibe ligation]]<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC" and cuts DNA like the figure 3.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 2) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T22:54:53Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR, with the primers of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3. We wanted to maximize the expression level of FT. T7 promoter is the strongest promoter in terms of FT expression, so we use T7 promoter. And we wanted to refine FT protein in order to take an experiment of FT introduction into plant cells before completing our secretion system. His tag enable us to protein purification from ''e.coli''.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to refine FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using FT specific antibody and checked the place of the FT protein band.<br><br />
As a result, FT and His:FT bands were observed at the expected molecular weight region. <br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT protein. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with TorA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. We made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have TorA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes TorA signal peptide and then it transports proteins (with TorA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active.<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old TorA signal cause a stop codon between signal peptide and target coding sequence(CDS) when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between TorA signal and a target protein CDS, we made Frameshift mutation twice on TorA signal. As a result, you can make TorA-fusion target protein by standard or 3A assembly. In addition to that, our TorA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified TorA signal and confirmed our TorA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells(Fig.1-1,1-2). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-TorA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with TorA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with TorA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We performed RT-PCR in order to investigate FT protein's function. <br><br />
<br><br />
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as control.<br><br />
GFP was used as a control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|400px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated<br><br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
[[File:RNA0925.png|300px]]<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. When we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we tried to make it easier to use this assembly. And we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"].<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.1 conventional method.]][[File:GGA_002.jpg|300px|thumb|center|Fig.2 Golden Gate Assembly.]]<br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.3 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.4 irreversibe ligation]]<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC" and cuts DNA like the figure 3.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 2) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosakahttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-26T22:36:33Z<p>Takahiro Shimosaka: /* link= */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
showTab("kyoto-tab-Florigen"); <br />
});<br />
</script><br />
</html><br />
<br />
<ul id="kyoto-tabs"><br />
<li><html><a href="" onclick="showTab('kyoto-tab-Florigen'); return false;"></html><br />
[[Image:KyotoTab_FlowerFairyE.png|link=|Florigen]]<html></a></html></li><br />
<li><html><a href="" onclick="showTab('kyoto-tab-GoldenGate'); return false;"></html><br />
[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
</ul><br />
<br />
<div id="kyoto-tab-contents"><br />
<div id="kyoto-tab-Florigen" class="displayOn"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#FFEIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#FFEResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#FFEDiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#FFEReferences]]</li><br />
</ul><br />
<br />
<html><a id="FFEIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=|Introduction]]=<br />
Have you ever seen flower fairies? <br />
Probably no(some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. What if we can live with flower fairies? Their lovely powers to make flowers bloom would be profitable for us, including application to agriculture. That’s why we set our project to realize it with synthetic biology, Flower Fairy E.coli!<br />
<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focus on FT protein, the identity of Florigen.'''<br><br><br />
<div style="background-color: #ffffff;" width="700"><br />
[[File:Kyoto_wiki_intro_1_Expression.png|frameless|266px|link=#Expression]]<br />
[[File:Kyoto_wiki_intro_1_Activation.png|frameless|373px|link=#Activation]]<br><br />
[[File:Kyoto_wiki_intro_1_Secretion.png|frameless|266px|link=#Secretion]]<br />
[[File:Kyoto_wiki_intro_1_Penetration.png|frameless|373px|link=#Penetration]]<br><br />
</div><br />
There are four issues in order to create Flower Fairy E.coli. It is unclear whether ''E.coli''(prokaryote) could produce functional FT properly because FT is usually produced in the plant cells(eukaryote). After produced, FT have to go through four walls, inner and outer membranes, a cell wall of the plant cells and a cell membrane of the plant. Even though FT could get inside of the cells, it is unknown whether FT protein transcribed in ''E.coli'' can activate shoot apex cells and bloom flowers.<br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<html>We have to go through four steps for purpose of obtaining our goal-Flowering Fairy E.coli-<br><br />
The four steps are composed of <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,<br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html><br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|250px|left|Expression]]<br />
On the first step; '''EXPRESSION''', ''E.coli'' produce florigen inside itself.<br><br />
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation. Inverse PCR is a kind of PCR, with the primers of which 3’ ends get longer in the direction of the outside of reproduction domain in contradiction to nomal PCR. We succeeded in mutating FT plansmid by this PCR method.<br><br />
As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. In this way, we made FT gene available(Fig.1-2)<br />
We constructed the plasmid shown in the Fig.1-3.<br />
FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.<br><br />
<br><br><br />
[[File:FT1.png|thumb|300px|Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:FT2.png|thumb|300px|Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
[[File:FTconstruction.png|thumb|300px|Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and His tag enables us to refine FT protein. ]]<br />
<br />
In order to confirm the expression of FT protein, we performed western blotting using FT specific antibody and checked the place of the FT protein band.<br><br />
As a result, FT and His:FT bands were observed at the expected molecular weight region. <br />
<br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br />
[[File:Western-FT.png|thumb|600px|left|fig.1-4 Verification of expression of FT protein in ''E.coli''. The result of Western blotting against FT protein. <br><br />
Each plasmid shown in Fig.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, then cells were incubated for 4h or overnight at 20°C. 100µL of culture was used for SDS-PAGE.<br />
Lane1 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane2 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane3 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane4 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
Lane5 : Cell lysate diluted into 100µL of sample buffer, not induced<br><br />
Lane6 : Cell lysate diluted into 100µL of sample buffer, IPTG induced 4h.<br> <br />
Lane7 : Cell lysate diluted into 100µL of sample buffer, IPTG induced overnight<br><br />
Lane8 : Cell lysate diluted into 50µL of sample buffer, IPTG induced overnight<br><br />
]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left| Secretion ]]<br />
On the second step; '''SECRETION''', ''E.coli'' secretes florigen outside of the cell.<br><br><br />
Even though our ''E.coli'' can produce FT protein, a big issue remains: how they can transport proteins to the outside of the cells? To make it possible, we tried to make [[#Secretion Group|Tat cassette and kil protein inducer]]. This cassette makes ''E.coli'' carry proteins with torA signal via Tat protein transportation pathway from cytoplasm to periplasm. Periplasm is a space between inner and outer membrane. Kil protein encourages proteins to move from periplasm to surroundings[8]. Our Secretion team made this protein secretion system and visualized and confirmed its function by using GFP. <br><br />
<br />
=====Result 1: Modified TorA signal=====<br />
The Twin Arginate Translocation pathway(Tat) is one of the secretion systems ''E.coli'' originally has. This system can carry proteins that have torA signal anino acid sequences at N terminal. TatA, TatB and TatC compose Tat complex on inner membrane. Tat complex recognizes torA signal peptide and then it transports proteins (with torA) from cytoplasm to periplasm with maintaining their folding. In short, proteins secreted via Tat pathway can keep active[].<br />
<br />
In this experiment, we wanted to design a applicable TorA signal device to meet various needs and to check the function of signal sequence. TorA signal was, actually,submitted by Canbrige 2011(BBa_K233307). These signals, however, doesn't contain RBS so that you need to make RBS by yourself. In addition, old torA signal cause a stop codon between signal peptide and target CDS when you assemble them by standard or 3A assembly. For these reasons, all of other teams make an effort to combine TorA signal to targets, such as using Gibson assembly. That's too trouble! <br />
<br />
To avoid to raise a stop codon in scar sequence between torA signal and a target protein CDS, we made Frameshift mutation twice on torA signal. As a result, you can make torA-fusion target protein by standard or 3A assembly. In addition to that, our torA signal has RBS. This can be an easy-to-use biobrick part. It can shorten the time of constructions because it only needs a promoter and a target protein. <br />
<br />
We read the sequence data of our modified torA signal and confirmed our torA signal don't raise stop codon when it used in Standard or 3A assembly. <br />
Using green fluorescent protein (GFP) as a target protein, we observed the torA-GFP fusion-expressing cells(Fig.1-1,1-2). The torA-GFP fusion was successfully expressed. This means RBS in our torA signal worked.<br />
<br />
<gallery widths=300px heights=300px><br />
File:顕微鏡1.jpg|fig2-1the same sample under UV<br />
File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|fig2-2E.coli having Lacp-torA-GFP-DT and stained with Hoechst<br />
</gallery><br />
<br />
====Result 2: Construction of Tat cassette====<br />
TatABC composes a pathway from cytoplasm to periplasm. <br />
<br />
====Result 3: Assay of Kil====<br />
Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for ''E.coli'' to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.<br><br><br />
We made the construct, lacp-RBS-Kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600. The table below shows the results. This result indicates that the expression of LacP-RBS-Kil-DT(pSB3C5) makes no effect on suvival of E.coli.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! scope="col"| IPTG<br />
! scope="col"| 0<br />
! scope="col"| 0.001mM<br />
! scope="col"| 0.01mM<br />
! scope="col"| 0.1mM<br />
! scope="col"| 1mM<br />
|-<br />
! scope="row"| 0.5h<br />
|0.202<br />
|0.207<br />
|0.201<br />
|0.207<br />
|0.200<br />
|-<br />
! scope="row"| 1h<br />
|0.406<br />
|0.428<br />
|0.424<br />
|0.402<br />
|0.421<br />
|-<br />
! scope="row"| 1.5h<br />
|0.796<br />
|0.813<br />
|0.779<br />
|0.751<br />
|0.802<br />
|-<br />
! scope="row"| 2h<br />
|1.107<br />
|1.129<br />
|1.141<br />
|1.092<br />
|1.124<br />
|-<br />
! scope="row"| 2.5h<br />
|1.546<br />
|1.565<br />
|1.541<br />
|1.532<br />
|1.578<br />
|-<br />
! scope="row"| 3h<br />
|1.912<br />
|1.933<br />
|1.890<br />
|1.883<br />
|1.940<br />
|-<br />
! scope="row"| 3.5h<br />
|2.300<br />
|2.295<br />
|2.238<br />
|2.247<br />
|2.259<br />
|-<br />
! scope="row"| 4h<br />
|2.546<br />
|2.545<br />
|2.528<br />
|2.490<br />
|2.520<br />
|-<br />
! scope="row"| 4.5h<br />
|2.688<br />
|2.663<br />
|2.603<br />
|2.633<br />
|2.666<br />
|-<br />
! scope="row"| 5h<br />
|2.699<br />
|2.826<br />
|2.787<br />
|2.593<br />
|2.673<br />
|-<br />
! scope="row"| 5.5h<br />
|2.863<br />
|2.742<br />
|2.754<br />
|2.741<br />
|2.756<br />
|-<br />
! scope="row"| 6h<br />
|2.831<br />
|2.876<br />
|2.758<br />
|2.759<br />
|2.728<br />
|-<br />
! scope="row"| 20h<br />
|2.671<br />
|2.706<br />
|2.680<br />
|2.606<br />
|2.619<br />
|-<br />
|}<br />
<br />
====Detail of Our Secretion System====<br />
Our wonderful secretion system is constructed by tatABCD, Kil and another gene. Another gene is PspA (phage-shock protein A) gene. ''E.coli'' has it originally and this gene is expressed when their inner membrane is damaged. PspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm.Our secretion system makes many holes in inner and outer membranes. In other words, ''E.coli'' which has our secretion system is under the membrane stress conditions. But by introducing pspA into our Flower Fairy E.coli, the ''E.coli'' comes to be able to maintain the vitality, though they have many holes in the membrane.<br />
<br />
===Construction===<br />
<p>'''Tat secretion cassette with constitutive promoter(BBa_K797004)'''</p><br />
<p>This cassette allows ''E.coli'' to secrete proteins with torA signal. Wild type Tat protein secretion system is so weak that Kyoto 2012 constructs Tat cassette to reinforce the ability of transportation of Tat system. This part includes tatA, B and C proteins coding region and pspA (phage shock protein A). Tat A, B and C proteins are the main components of Tat complex where proteins with torA signal go through, and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggests this new way of secretion and provides iGEMers with this cassette regulated by constitutive promoter.</p><br />
<p>We checked the sequence of tatABCD[[Part:BBa_K797000|'''(BBa_K797000)''']] and the sequence of pspA [[Part:BBa_K797001|'''(BBa_K797001)''']] individually, and then, we made TAT construction composed of constitutive promoter[[Part:BBa_J23107|'''(BBa_J23107)''']], tatABCD<br />
[[Part:BBa_K797000|'''(BBa_K797000)''']],pspA[[Part:BBa_K797001|'''(BBa_K797001)''']] and double terminator<br />
[[Part:BBa_B0015|'''(BBa_B0015)''']]. This TAT secretion cassette is too long device to sequence, so that we performed electrophoresis of this cassette and confirmed the length of our parts. <br><br><br />
Considering that the sequences of tatABCD and pspA are correct,and the length of TAT secretion casssette is correct, we declare that this construction of TAT secretion cassette has been completed.</p><br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
<br />
On the third step; '''PENETRATION'''.<br />
FT protein from Flower Fairy ''E.coli'' needs to enter into plant cells in order to induce plants to bloom.<br><br><br />
From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. <br><br />
In such a situation, we found the way of penetration into cells using polyarginine. This is called R9 peptide, a type of Cell Penetrating Peptide (CPP). R9 peptide comprises a sequence conjugated to nine arginine residues. (fig.1).<br><br><br>[[File:R9peptide.png|thumb|right|300px|fig.3-1 Image of R9 peptide <br> (powered by winmostar V3.808d, MOPAC2012)]]<br />
<br />
R9 peptide is thought to act on a cell membrane and causes macropinocytosis, a specific form of endocytosis.<br />
R9 peptide glues to cell membrane of plants because of hydrophobic.The cell responses to the stimulus and cause macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.<br />
<br />
Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.<br><br />
Then, we determined to cause penetration of FT protein by R9 coding region.<br><br><br />
<br />
[[File:Macropinocytosis.png|thumb|left|450px|fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides glue to cell membrane of plants because of hydrophobic.<br> 2.Cells response to the stimulus and cause macropinocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br><br><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br><br><br><br />
<br />
====2. GFP antibody specificity check====<br />
<br />
[[File:Western-GFPgenerator.png|thumb|right|300px|fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. BBa_I746915 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.7, then cells were incubated for 4h at 37°C. 100&micro;L of culture was used for SDS-PAGE.<br><br />
Lane1 : Cell lysate 10µL, not induced<br><br />
Lane2 : Cell lysate 10µL, IPTG induced<br><br />
Lane3 : Cell lysate 5µL, not induced<br><br />
Lane4 : Cell lysate 5µL, IPTG induced<br><br />
Lane5 : Cell lysate 2µL, not induced<br><br />
Lane6 : Cell lysate 2µL, IPTG induced<br>]]<br><br />
<br />
First, we checked the specificity of anti GFP monoclonal antibody, because we must confirm if R9 peptide fusioned GFP is expressed in ''E.coli''. <br><br />
We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].<br><br />
The parts is consist of T7 promoter 6-his tagged superfolder GFP.<br><br />
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples. <br><br />
<br />
Each samples induced with IPTG showed one main band and one extra band, and uninduced controls showed one bands. <br />
The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG. <br />
From this result, we concluded that GFP antibody has enough specificity. <br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
After the 4h of IPTG induction, we noticed that ''E.coli'' expressing R9::GFP were growing poorly.<br><br />
Moreover, we couldn't get any bands of R9::GFP, as shown in the Fig.3-4.<br><br />
[[File:culture1.png|thumb|left|300px|fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.<br />
]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|450px|left|fig.3-5 Lane1 : Molecular marker<br><br />
Lane2 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, not induced<br><br />
Lane3 : GFP([Part:BBa_I746915|BBa_I746915]) Cell lysate 10µL, IPTG induced<br><br />
Lane4 : R9::GFP Cell lysate 10µL, not induced<br><br />
Lane5 : R9::GFP Cell lysate 10µL, IPTG induced<br>]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br />
<br />
====3. Separating R9 peptide and GFP====<br />
[[File:Nc_10_merge.jpg|thumb|right|300px|Fig3-. Verification of R9 function with use of GFP.<br><br />
These pictures shows cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst (10 times magnification), GFP fluorescence (10 times), nuclei by Hoechst (60 times), GFP fluorescence (60 times)<br />
(With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
<br />
When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. After 5 minutes we washed cells by PBS in order to wash GFP away. Then we succeeded in penetration by getting the figure of GFP fluoresence.<br><br><br />
The controls on the left were soaked in only GFP, and the samples on the right-hand side were soaked in GFP and R9. These two pictures show the action of R9 peptide. R9 peptide kept GFP in or around plant cells. This figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP.<br><br />
Fig. Verification of R9 function with use of GFP.<br />
<br clear="both" /><br />
<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|Activation]]<br />
On the final step,'''Activation'''. We verified whether FT normally worked in plant cells.<br><br> <br />
FT protein is derived from plant cells and it is capable of post-translational modification. ''E.coli'' cannot do post-translational modification, so FT protein derived from Flower Fairy E.coli may not work normally. As a final step, we tried to confirm whether FT protein by our ''E.coli'' led to flower formation.<br><br><br><br><br><br><br><br />
===How to Verify FT Function===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom.FT protein upregulates some flowering genes and they induce plants to bloom. Verification of upreguration of flowering genes. ]]<br />
<br />
<br />
FT protein increases transcriptive activity of several proteins which lead to flower formation. For that reason it can be said that we have verified FT function when we have found rises of activities of the proteins. <br><br />
Although such proteins activated by FT are various, we check APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We used RT-PCR because of chasing FT protein's function. <br><br />
<br><br />
We did RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
Two types of samples were prepared, one is treated with FT and the other is treated with GFP as control.<br><br />
GFP was used as control because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br><br />
<br><br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR5.png|thumb|left|500px|Fig.4-2.His tag fusion FT protein was purified with Ni-NTA agarose column.<br><br />
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample.<br />
FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each.<br />
Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.)<br />
After incubation, leaves were freezed with liquid nitrogen and glinded immediately.<br><br />
Total RNA was extracted by phenol-chloroform extraction. <br />
cDNA was synthesized by reverse transcription and used as templates of RT-PCR. <br />
TUBULIN was used for internal control of mRNA expression.<br><br />
Lane1:TUBULIN (GFP-R9 treated) amplicon 61bp<br><br />
Lane2:TUBULIN (FT-R9 treated) <br><br />
Lane3:FUL (GFP-R9 treated) amplicon 132bp<br><br />
Lane4:FUL (FT-R9 treated) <br><br />
Lane5:SEP3 (GFP-R9 treated) amplicon 87bp<br><br />
Lane6:SEP3 (FT-R9 treated) <br><br />
Lane7:AP1 (GFP-R9 treated) amplicon 958bp<br>]]<br />
<br />
From this results, though we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1 <br />
but we could amplify each gene successfully.<br><br />
One possible reason of this failure is the poor quality of extracted RNA, resulted from 16h incubation <br />
after harvesting leaves.<br><br />
We compared the total RNA by electrophoresis, shown in fig.4-3.<br><br />
[[File:TotalRNA20120925.jpeg|thumb|400px|Fig.4-3 Electrophoresis of RNA.<br />
RNA concentration is adjusted.<br><br />
Lane1:RNA of 16h incubated leaves(GFP-R9 treated)<br><br />
Lane2:RNA of 16h incubated leaves(FT-R9 treated)<br><br />
Lane3:RNA of fresh leaves]]<br />
As shown in the Fig.4-3, RNA of long time incubated samples are degradated<br />
<br />
[[File:RT-PCR1R9-GFP.png|300px]]<br />
[[File:RNA0926.png|300px]]<br />
RNA0925.png<br />
<br />
==Achivement==<br />
<br />
To mutate and standardize FT sequence as a iGEM part.<br><br />
To confirm expression of FT protein in ''E.coli''.<br><br />
<br />
<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
<p>We noticed only flowering and florigen in this time but there are many many other plant hormones. We made translocation pathway from ''E.coli'' into plant cells, so we will be able to introduce plant hormones into plant cells if ''E.coli'' can make them. It means we can control plant growth in any stage through genetically engineered ''E.coli''. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.</p><br />
<p>Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to controll all living cells using this technology.</p><br />
<br />
<html><a id="FFEReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
<br />
[1]Microsugar Chang et al. (2005)"Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488<br><br />
[2]Paula Teper-Bamnolker and Alon Samach1 (2005) "The flowering integrator FT regulates SEPALLATA3 and<br />
FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675<br><br />
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis<br><br />
[4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery <br />
systems" Pharmaceuticals, 3, 961-993<br><br />
[5]Unnamalai N, Kang BG, Lee. (2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells." FEBS Lett 21;566(1-3):307-10.<br />
<br><br />
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96<br><br />
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35<br><br />
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli cntrolled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50<br><br />
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306<br><br />
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Mol Microbiol, 39(1), 47-53<br><br />
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8<br><br />
[12]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death." J Bacteriol, 170(10), 4963-6<br><br />
[13]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway." J Bacteriol, 186(2), 366-73<br />
[[Image:KyotoBSL1.png|link=https://2012.igem.org/Team:KAIT_Japan/Human_Practice]]<br />
</div><br />
<br />
<div id="kyoto-tab-GoldenGate"><br />
<br />
<ul class="kyoto-post-it"><br />
<li>[[File:Kyoto_Introduction.png|30px|link=#GGAIntroduction]]</li><br />
<li>[[File:Kyoto_Experiment.png|30px|link=#GGAResults]]</li><br />
<li>[[File:Kyoto_Discussion.png|30px|link=#GGADiscussion]]</li><br />
<li>[[File:Kyoto_Reference.png|30px|link=#GGAReferences]]</li><br />
</ul><br />
<br />
<html><a id="GGAIntroduction"></a></html><br />
<br />
=[[File:Kyoto_IntroductionHeader.png|link=]]=<br />
<p>BioBrick is useful for us because we can look for required BioBrick parts from its registory and recombine genes easily. If we want to introduce many parts into one plasmid, however, we have to repeat the process; restrict enzyme digestion and ligation. It takes us too much time and sometimes we lose enough time for other experiments.</p><br />
<p>We want to reduce the time required for the recombination of genes and get enough time for verification of the expression and the effect of genes.</p><br />
<p>Golden Gate assembly is the one of the ways to make it possible.</p><br />
[[File:GGA_001.jpg|600px|center|]]<br />
<p>Some teams like 2011 WHU-China have used this assembly. But they didn't intend to spread Golden Gate Assembly through other iGEM teams. So, we tried to make it easier to use this assembly. And we created plasmid backbone parts [http://partsregistry.org/Part:BBa_K797013 "BBa_K797013"].<br />
</p><br />
<p>We also created a software which designs primers for Golden Gate assembly.</p><br />
<br />
<br />
==What's Golden Gate Assembly==<br />
Golden Gate Assembly is developed by Carola Engler, Ramona Gruetzner, Romy Kandzia and Sylvestre Marillonnet.<br> <br />
[[File:GGA_001.jpg|300px|thumb|left|Fig.1 conventional method.]][[File:GGA_002.jpg|300px|thumb|center|Fig.2 Golden Gate Assembly.]]<br />
This method enables us introduce plural gene segments into one plasmid all at once.<br />
[[File:GGA_003.jpg|300px|thumb|left|Fig.3 BsaI recognition sites.]][[File:GGA_004.jpg|300px|thumb|center|Fig.4 irreversibe ligation]]<br />
Golden Gate Assembly uses the feature of restrict enzyme "BsaI".<br><br />
BsaI recognizes the sequence "GGTCTC" and cuts DNA like the figure 3.<br />
And BsaI activity is independent of the sequences of the downstream of the<br />
recognition site.<br><br />
<br />
Restrict enzyme digestion and ligation are completed by just one PCR because once DNA is cut and ligated irreversibly, the recognition site of BsaI disappears.<br />
<br />
So, we can introduce plural DNA segments into one plasmid at the same time if we create the segments so that ligation sites become complementary. (Figure 2) <br />
<br><br><br />
<br />
Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
<html><a id="GGAResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]] =<br />
Before we started assembly, we had to make proper gene segments for Golden Gate assembly.<br />
The DNA segments we created had four base pair for ligation, one base pair spacer, <br />
BsaI recognition sites and four base pair at the both ends.<br />
<br />
We amplified plasmid backbone(psB1K3).<br />
<br />
After amplification, we assembled these DNA segments referring to [https://2012.igem.org/Team:Kyoto/MethodAndMaterial the protocol].<br />
<br />
Other iGEM teams can use this backbone plasmid for their Golden Gate assembly.<br />
[[File:GGA_005.jpg|250px|center|]]<br />
<html><a id="GGADiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png|link=]]=<br />
We created the backbone plasmide psB1K3 and a software to design primers for Golden Gate assembly.<br />
They make it easier to use Golden Gate assembly. <br />
We want other teams use this parts and software to use precious time efficiently. <br />
<html><a id="GGAReferences"></a></html><br />
<br />
=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
[1]Carola Engler, Romy Kandzia, Sylvestre Marillonnet "A One Pot, One Step, Precision Cloning Method with High Throughput Capability"PLoS ONE 3(11): e3647.<br><br />
[2]Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet"Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes" PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 doi:10.1371/journal.pone.0003647<br><br />
<br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Takahiro Shimosaka