http://2012.igem.org/wiki/index.php?title=Special:Contributions/TJR&feed=atom&limit=50&target=TJR&year=&month=2012.igem.org - User contributions [en]2024-03-29T06:36:11ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:59:10Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO", (though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. This is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First of all, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with TorA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT protein around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT protein made by '' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT protein. <br><br><br><br />
<br />
===The detail function of FT===<br />
FT protein increases transcript activities of flowering factors which lead to flower formation in a plant cell. <br />
let us introduce the function mechanism of FT protein. Actually, FT function is to up-regulate genes relating to bloom flowers. at the stage of flowering of plants, the expressions are incresed in these genes, FUL, SEP3, and so on. These genes work on shoot apex and change the form of shoot apex in order to start flowering. We focused on the amount of FUL and SEP3 because they are the most popular genes among them.<br />
<br />
<br />
<br />
[[File:Flowering factors1.png|thumb|center|610px|Fig.4-1 FT protein triggers some flowering genes and they induce flower formation. FT protein up-regulates some flowering genes and they induce plants to bloom. ]]<br />
<br />
<br><br />
<br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our ''E.coli'', we tried to check the amount of flowering factors' mRNA upregulated by FT. Experimental procedure is as follows.<br><br />
1. We prepared solutions. One is composed of R9 peptides and GFP, and the other is composed of R9 peptides and FT. GFP protein is used as a control group. 2. We injected these solutions into each leaf. 3.After incubation, we tried to perform RT-PCR in order to check up-regulation of flowering factors.<br> <br />
<br />
We used leaf cells of Arabidopsis thaliana , insted of cells of a shoot apex. This is because shoot apex' cells are too small to observe. FT protein upregulates FUL and SEP3 genes in leaf cells, too.<br />
<br />
<br />
<br />
This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT protein by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:jyoge.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:ABC.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: Electrophoresis of RNA. RNA concentration is adjusted. These band showed all sample were purified correctly <br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig4-5 The result of RT-PCR<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]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control.<br />
<br><br><br><br><br><br><br><br />
<br />
===Checking FT protein purification by Western Blotting===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig The result of Western Blotting the results of against 6his::FT using anti-FT antibody There was no band showing FT protein]]<br />
We could not confirm FT function though enough quality of RNA was extracted. Therefore, we questioned about FT protein quality, and performed western blotting to confirm FT protein is successfully purified.<br><br />
Unfortunately, the band of FT was not detected. Because of the time shortage, we could not conduct further experiment. We have to reconsider about FT purification and now we are trying.<br><br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABC, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the relative RNA expression of FUL and SEP3 by qPCR. However, there was not any significant difference between experimental group and negative control.<br />
We found that FT was not successfully purified and there is the room for improvement in that.<br />
In addition of this, several reasons can be considered. <br />
The cause of failure may be that FT protein was not active or decomposed in the cell or even that FT protein didn't enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:42:22Z<p>TJR: /* Improvement of secretion system */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO", (though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. This is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First of all, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made by '' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. <br />
let us introduce the function mechanism of FT protein. Actually, FT function is to up-regulate genes relating to bloom flowers. at the stage of flowering of plants, the expressions are incresed in these genes, FUL, SEP3, and so on. These genes work on shoot apex and change the form of shoot apex in order to start flowering. <br />
<br />
<br />
<br />
[[File:Flowering factors1.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
<br><br />
<br><br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:jyoge.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:ABC.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig The result of RT-PCR]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking FT protein purification by Western Blotting===<br />
We could not confirm FT function though enough quality of RNA was extracted. Therefore, we questioned about FT protein quality, and performed western blotting to confirm FT protein is successfully purified.<br><br />
Unfortunately, the band of FT was not detected. Because of the time shortage, we could not conduct further experiment. We have to reconsider about FT purification and now we are trying.<br><br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:41:12Z<p>TJR: /* Establishing an effective way of RNA purification */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO", (though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. This is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First of all, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=350px heights=152px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made by '' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. <br />
let us introduce the function mechanism of FT protein. Actually, FT function is to up-regulate genes relating to bloom flowers. at the stage of flowering of plants, the expressions are incresed in these genes, FUL, SEP3, and so on. These genes work on shoot apex and change the form of shoot apex in order to start flowering. <br />
<br />
<br />
<br />
[[File:Flowering factors1.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
<br><br />
<br><br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:jyoge.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:ABC.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig The result of RT-PCR]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. Its cause might be that FT protein was not active or decomposed in the cell, or that FT proteins did not enter plant cells in the first place. If injection process does not work well, or FT does not get into the cell, it is natural that the activity of FT does not be observed. To clarify the cause, we conducted Western Blotting on Plant leaves we had injected FT proteins.<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:40:45Z<p>TJR: /* Injecting FT and verifying its function by RT-PCR */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO", (though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. This is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First of all, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=350px heights=152px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made by '' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. <br />
let us introduce the function mechanism of FT protein. Actually, FT function is to up-regulate genes relating to bloom flowers. at the stage of flowering of plants, the expressions are incresed in these genes, FUL, SEP3, and so on. These genes work on shoot apex and change the form of shoot apex in order to start flowering. <br />
<br />
<br />
<br />
[[File:Flowering factors1.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
<br><br />
<br><br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:jyoge.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig The result of RT-PCR]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. Its cause might be that FT protein was not active or decomposed in the cell, or that FT proteins did not enter plant cells in the first place. If injection process does not work well, or FT does not get into the cell, it is natural that the activity of FT does not be observed. To clarify the cause, we conducted Western Blotting on Plant leaves we had injected FT proteins.<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/File:ABC.pngFile:ABC.png2012-10-27T03:39:48Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/File:Jyoge.pngFile:Jyoge.png2012-10-27T03:39:19Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:31:56Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO", though some of you might have come across them in your childhood, because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=350px heights=152px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors1.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig The result of RT-PCR]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. Its cause might be that FT protein was not active or decomposed in the cell, or that FT proteins did not enter plant cells in the first place. If injection process does not work well, or FT does not get into the cell, it is natural that the activity of FT does not be observed. To clarify the cause, we conducted Western Blotting on Plant leaves we had injected FT proteins.<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/File:Flowering_factors1.pngFile:Flowering factors1.png2012-10-27T03:31:20Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:28:58Z<p>TJR: /* Improvement of secretion system */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO", though some of you might have come across them in your childhood, because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=350px heights=152px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. Its cause might be that FT protein was not active or decomposed in the cell, or that FT proteins did not enter plant cells in the first place. If injection process does not work well, or FT does not get into the cell, it is natural that the activity of FT does not be observed. To clarify the cause, we conducted Western Blotting on Plant leaves we had injected FT proteins.<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:25:19Z<p>TJR: /* Improvement of secretion system */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=400px heights=173px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:24:00Z<p>TJR: /* Improvement of secretion system */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/File:Detail_explanation_of_TAT_system_improvement.pngFile:Detail explanation of TAT system improvement.png2012-10-27T03:23:31Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:16:31Z<p>TJR: /* Kil protein had no significant effect on E.coli's growth */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/File:Detail_explanation_of_TAT_system.pngFile:Detail explanation of TAT system.png2012-10-27T03:15:28Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:11:14Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conduct qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:10:37Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conduct qPCR about and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:09:49Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conduct qPCR about and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:09:01Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
[[File:Flowering factors.png|thumb|center|610px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.<br />
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conduct qPCR about and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig. <br />
<br><br />
<br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:07:40Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
[[File:Flowering factors.png|thumb|right|300px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL.<br />
The result is shown in fig.<br />
We were able to get sufficiently purified RNA but it is suspicious that the band of tublin,internal control,<br />
we conduct qPCR about the same and compared the relative RNA expression of each gene. <br />
However, there was not significant difference between experimental group.<br />
The result is shown in fig.<br />
<br><br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:06:47Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
[[File:Flowering factors.png|thumb|center|300px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<br />
<br />
As shown in the Fig.4-3, this time, RNA samples were degradated.<br><br />
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br><br />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL.<br />
The result is shown in fig.<br />
We were able to get sufficiently purified RNA but it is suspicious that the band of tublin,internal control,<br />
we conduct qPCR about the same and compared the relative RNA expression of each gene. <br />
However, there was not significant difference between experimental group.<br />
The result is shown in fig.<br />
<br><br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:05:43Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
[[File:Flowering factors.png|thumb|middle|300px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL.<br />
The result is shown in fig.<br />
We were able to get sufficiently purified RNA but it is suspicious that the band of tublin,internal control,<br />
we conduct qPCR about the same and compared the relative RNA expression of each gene. <br />
However, there was not significant difference between experimental group.<br />
The result is shown in fig.<br />
<br><br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:04:41Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
[[File:Flowering factors.png|right|300px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL.<br />
The result is shown in fig.<br />
We were able to get sufficiently purified RNA but it is suspicious that the band of tublin,internal control,<br />
we conduct qPCR about the same and compared the relative RNA expression of each gene. <br />
However, there was not significant difference between experimental group.<br />
The result is shown in fig.<br />
<br><br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-27T03:01:28Z<p>TJR: /* The detail function of FT */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is "NO",(though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell are taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. <br />
So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.<br />
<br><br />
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br><br />
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <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|link=|Activation]]<br />
<br>The final step is; '''Activation'''. We verified whether FT proteins made in'' E.coli'' work normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT===<br />
[[File:Flowering factors.png|thumb|right|300px|Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT produced by our Fairies, we used leaf cells of ''Arabidopsis thaliana '' , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.<br><br />
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<br />
<br><br><br />
[[File:4つ.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/><br />
B: Improved RNA waveform<br/><br />
C: <br/><br />
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]<br />
<br clear="both" /><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL.<br />
The result is shown in fig.<br />
We were able to get sufficiently purified RNA but it is suspicious that the band of tublin,internal control,<br />
we conduct qPCR about the same and compared the relative RNA expression of each gene. <br />
However, there was not significant difference between experimental group.<br />
The result is shown in fig.<br />
<br><br />
===Checking whether FT protein really introduced or not by Western Blotting===<br />
At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If injection process was something wrong and FT didn't get the inside of the cell, the activity of FT,of course, wouldn't be observed. To clarify the point, we conducted Western Blotting to Plant leaves injected FT protei<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
At '''PENETRATION''', GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.<br />
It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).<br />
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.<br />
<br />
<br />
At''' Activation''', in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/File:Flowering_factors.pngFile:Flowering factors.png2012-10-27T03:00:52Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-26T21:37:22Z<p>TJR: /* We used R9 and GFP separately and to check the R9 peptides function */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is, "NO" (though some of you might have come across them in your childhood), because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition to the happy feelings, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!<br />
In order to realize our dream, we focused on FT protein, known as Florigen.This protein is a kind of plant hormone. First, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy ''E.coli'' on leaves!===<br />
When you want to use our Flower Fairy ''E.coli'', all you have to do is just put them on plant leaves! When you spread Flower Fairy ''E.coli'', FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy <i>E.coli</i>.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> (prokaryote) can express FT protein, because FT protein is derived from plant cells (eukaryote).<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, FT protein has to get out of the <i>E.coli</i>.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is not clear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We had to make ''E.coli'' produce FT protein.<br />
<br />
However, in the natural environment, wild''E.coli'' don't have ''FT'' gene. Therefore we tried to make a new BioBrick part including ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
<br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of ''E.coli'', the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' using affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).<br><br />
So we successfuly confirmed the FT expression!<br />
<br />
[[File:Exp-plac.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT protein<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in confirming the mutation and the expression of ''FT'' and 6 His:FT protein in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. ''We want'' E.coli ''to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via Tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/><br />
<br />
====We improved usability of TorA signal BioBrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' to secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can enable'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have. <br />
To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.<br />
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) <br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <br />
<br />
<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br clear="both"/><br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell is taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively regardless of whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT protein around an invaginating region of the cell is taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken by endocytosis. <br />
In other words, it can be said that it induces endocytosis regardless of whether R9 peptides and target proteins are fused.<br />
<br><br />
We wonder whether we should fuse R9 and target proteins. <br><br />
From this system, we thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we considered that fusing R9 peptide with a target protein may leads to higher efficiency.'''<br />
<br />
'''So, we tried to fuse R9 peptides with target proteins to increase penetration efficiency'''<br />
<br />
We transformed ''E.coli'', arranging R9's sequence and target protein's gene in line. And after that we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was poor in growth(Fig3-3). Then, we checked R9's effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we did not find the protein (Fig3-5). These results indicated poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
<br clear="both"/><br />
<br />
====We used R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
We wondered whether R9 peptides work properly and put FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, need to verify the function of R9 peptides. For this reason, we performed following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticles of ''Arabidopsis thaliana'''s leaves by scratching them. Second, we soaked the leaves into a solution of only GFP, or GFP and R9 for each. After 5 minutes we washed cells by PBS in order to put GFP and R9 peptides away from leaves. After that, we compared leaves having soaked in only GFP and ones having soaked R9 and GFP. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or around plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <br />
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<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|link=|Activation]]<br />
<br>On the final step; '''Activation'''. We verified whether FT protein made in'' E.coli'' worked normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor so that we can check the ability of FT protein by observing the transcript levels of genes FT protein upregulate. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT as a transcriptional factor===<br />
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom. FT protein up-regulates some flowering genes and they induce plants to bloom. ]]<br />
<br />
FT protein increases transcript activity of several genes which leads to flower formation. The protein can . <br><br />
<br><br />
<br><br />
<br />
<br><br><br><br><br><br><br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
To evaluate the function of FT which our Fairy made, we used leaf cells of ''Arabidopsis thaliana '' , not soot apex. Plants produce FT protein in their leaves and carry it to soot apex. For these reason, we assumed that our Flower Fairy can make flowers bloom only if it can introduce active FT protein in leaves. In addition to that, we can get lots of sample easily. Previous research said that FT gene can also up-regulate some gene,such as FUL and SEP3, in leaves so that We injected FT protein into leaves and conducted RT-PCR to value the amount of expressed RNA.<br><br />
FT protein is injected by a syringe. Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control. GFP was used as a control to confirm the result of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 />
<br />
===Establishing an effective way of RNA purification===<br />
We found that RNA purification method should be improved for performing RT-PCR successfully .<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<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 />
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<br />
<br />
<br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-6]]<br />
[[File:RT-PCR7.jpeg|thumb|300px|Fig.4-7 <br><br />
Lane1: 100bp ladder<br><br />
Lane2: TUBULIN <br><br />
Lane3: FUL<br><br />
Lane4: SEP3<br><br />
Lane5: AP1]]<br />
<br clear="both" /><br />
<br><br />
<br />
===Conducting RT-PCR and qPCR again by using new RNA purification===<br />
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL<br />
<br />
===Checking whether FT protein really introduced or not===<br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
In order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.<br />
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.<br />
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.<br />
<br />
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. <br />
In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.<br />
<br />
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.<br />
<br />
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.<br />
<br />
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. <br />
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.<br />
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein. <br />
<br />
<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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 />
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<br />
[1][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T18:21:27Z<p>TJR: /* November Festival and Open Campus at Kyoto University */</p>
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<br />
===This year, we implemented nine plans on "human practice".===<br />
<br />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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 />
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<br />
==Education==<br />
[[File:KyotoLecture.jpg|link=|right|300px]]<br />
We went to Hibiya high school and Horikawa high school, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
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<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<br />
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<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 who live in Kyoto, 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 />
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<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 />
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<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 Campus.<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 Campus was held in August 9 & 10.<br />
Many high school students visited our univ.<br />
We talked with them about iGEM as one of the activities which college students can join.<br />
We believe that some of them will join iGEM in the future.<br />
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<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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
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<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
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<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
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==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which could make our research safer, we got their proposal about safety. They performed "Safety Icon Project".In this project, they designed icons in safety, which you could tell whether the parts in registry were safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]].<br />
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'''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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-26T17:59:18Z<p>TJR: /* Realizing Flower Fairy in real world */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
[[File:FFT_kyoto.png |left|300px]]<br />
Have you ever seen flower fairies? Probably the answer is, "NO" (though some of you might have come across them in your childhood), because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition to the happy feelings, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy ''E.coli'' with synthetic biology!!<br />
'''Our goal is to produce ''E.coli'' which can make flowers bloom as Flower Fairies. To make it possible, we focused on FT protein, known as Florigen.'''This protein is a kind of plant hormone. First, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy ''E.coli'' on leaves!===<br />
When you want to use our Flower Fairy ''E.coli'', all you have to do is just put them on plant leaves! When you spread Flower Fairy ''E.coli'', FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy <i>E.coli</i>.<br><br />
These four steps are <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 />
<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 />
<br><br />
<html>In each step, we have some problems to be attacked.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> (prokaryote) can express FT protein, because FT protein is derived from plant cells (eukaryote).<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, FT protein has to get out of the <i>E.coli</i>.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is not clear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
The first step is '''EXPRESSION'''.<br> We needed to enable ''E.coli'' to produce FT protein.<br />
<br />
As a matter of course, ''E.coli'' doesn’t have ''FT'' gene. Therefore we had to make a new BioBrick part of ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new biobrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]<br />
<br><br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7polymerase. In our strain of ''E.coli'', the expression of T7 pol can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from ''E.coli'' with affinity chromatography.<br />
<br />
We needed to confirm that E. coli can express FT protein, because there was a possibility that ''E.coli'' can not translate FT or FT is unstable or toxic in ''E.coli''. <br><br />
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.<br><br />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region(Fig.1-2).<br />
<br />
[[File:PolP.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT proteins<br><br />
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br>''' We succeeded in making mutation and confirming the expression of ''FT'' and 6 His:FT proteins in ''E.coli!'''''<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
The second step is '''SECRETION'''''. We want ''E.coli'' to secrete FT protein to outside of the cell.''<br><br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables ''E.coli'' to transport FT protein continuously. <br clear="both"/><br />
====Proteins are required to go through two membranes====<br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT proteins have two hurdles in order to get out of the cell]]<br />
''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', therefore, FT protein has to pass through the two membranes. So we searched for secretion systems to make FT proteins go through these membranes. <br />
<br />
<br clear="both"/><br />
<br />
====TorA signal enables proteins to go through the inner membrane via tat pathway====<br />
[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]<br />
Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation(Tat) pathway. Tat pathway is more suitable than other secretion systems for us, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and transports into the periplasm only proteins that have a TorA signal at their N terminals.<br />
<br clear="both"/><br />
<br />
====We improved usability of TorA signal biobrick====<br />
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS, which regulate additional processing time for iGEMers who use the parts. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, therefore, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, but TorA would be expressed. <br><br />
<br />
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of LacP-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal biobricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]<br />
[[File:光る大腸菌.jpg|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having Lacp-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br />
====Kil protein enables proteins to go through outer membrane====<br />
[[File:Kil make holes.png|thumb|right|250px|Fig.2-5 Kil protein makes holes on outer membrane.]]<br />
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of ''E.coli''. So in our project, we introduced ''kil'' gene into ''E.coli''. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <br clear="both"/><br />
<br />
====Kil protein had no significant effect on ''E.coli'''s growth====<br />
We made the construction lacP-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of LacP-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''. <br />
<br />
'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' to secrete FT protein without cell lysis!!(Fig.2-7)'''<br />
<br />
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.<br />
<br> Vertical axis means the value of OD 600.<br />
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]<br />
<br />
[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.<br />
<br><br />
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]<br />
<br clear="both"/><br />
<br />
====Improvement of secretion system====<br />
<br />
Now we can make ''E.coli'' secrete FT proteins outside of them by using Tat pathway and kil protein. But E. coli secrete only a small amount of FT proteins when they use Tat transporters they originally have. To make ''E.coli'' secrete enough amount of FT proteins, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which makes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H+ concentration gradient between periplasm and cytoplasm. On our project, we expected that ''E.coli'' would secrete more proteins if those two genes are expressed more. It is because in our plan inner membrane and outer membrane have many holes and'' E.coli'' are exposed to the membrane stress. We, therefore, constructed Tat secretion cassette with constitutive promoter [http://partsregistry.org/Part:BBa_K797004 (BBa_K797004)].(Fig.2-8) <br><br />
<br />
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is increased and pspA diminishes stress of membranes. In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9) <<br />
<br />
<gallery widths=300px heights=130px> <br />
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)<br />
File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.<br />
</gallery> <br />
<br><br />
Kyoto 2012 suggests this new way of secretion, that is using Tat pathway and kil protein, and provides iGEMers with this cassette regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. This Tat secretion cassette is too long device to sequence, so we performed electrophoresis of this cassette and confirmed the length of our parts.<br />
<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
The third step is '''PENETRATION'''.<br />
In order to induce flower formation, FT proteins from ''E.coli'' must enter into plant cells. This is because FT proteins upregulate other proteins which lead to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.<br><br />
<br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br />
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.<br />
<br />
This is the mechanism of how R9 peptides work(Fig3-2).<br />
<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br><br />
Secondly, the cell responds to this stimulus and starts to endocytose.<br />
<br><br />
Finally, proteins around the endocytosing region of the cell is taken into the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work effectively regardless of whether or not R9 peptides are fused with target proteins. <br />
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br><br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2 Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]<br />
<br clear="both"/><br />
<br />
==== From the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====<br />
Considering this system, target proteins near the R9 peptides are taken by endocytosis. <br />
In other words, it can be said that it induces endocytosis regardless of whichever R9 peptides and target proteins are fused.<br />
<br><br />
We tried to wonder whether we should fuse R9 and target proteins. <br><br />
From this system, we thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we considered that fusing R9 peptide with a target protein may leads to higher efficiency.'''<br />
<br />
====So, we tried to fuse R9 peptides with target proteins to increase penetration efficiency ====<br />
<br />
We transformed ''E.coli'', arranging R9's sequence and target protein's gene in line. And after that we tried to make ''E.coli'' express R9::GFP fusion protein.<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was poor in growth(Fig3-3). Then, we checked R9's effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we did not find the protein (Fig3-5). These results indicated poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<gallery widths=190px heights=210px> <br />
File:culture1N.png|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.<br />
File:Western-R9-GFP2N.png|Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.<br> Lane1: Molecular marker<br> Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced<br> Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced<br> Lane4: R9::GFP Cell lysate 10µL, not induced<br> Lane5: R9::GFP Cell lysate 10µL, IPTG induced<br />
File:RT-PCR1R9-GFPN.png|Fig.3-5 RT-PCR of R9::GFP<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><br />
</gallery> <br />
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<br />
====We prepared R9 and GFP separately and to check the R9 peptides function====<br />
<br />
<br />
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
We wondered whether R9 system really works properly and puts FT proteins into plant cells. Although animal cells often endocytose, there are few examples of plant cell’s endocytosis. Therefore, we need to verify the system.<br />
To verify it, we performed following experiment(Fig3-6). <br />
<br />
<br />
First, we removed the cuticle of ''Arabidopsis thaliana'''s leaves by scratching them. Arabidopsis thaliana is a model plant. Second, we soaked the leaves into a solution of only GFP, or GFP and R9 for each. These GFP proteins were purified with 6 His tag inserted in our 1st Step, '''Expression'''. After 5 minutes we washed cells by PBS in order to wash GFP and R9 peptide away from leaves. After that, we compared leaves including only GFP and leaves including R9 and GFP for each. <br />
Then we succeeded in getting the Figure of GFP fluorescence(Fig3-7).<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides work properly kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function!'''. <br />
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<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|link=|Activation]]<br />
<br>On the final step; '''Activation'''. We verified whether FT protein made in'' E.coli'' worked normally in plant cells.<br><br> <br />
Actually, FT protein is a transcriptional factor so that we can check the ability of FT protein by observing the transcript levels of genes FT protein upregulate. <br><br><br><br />
<br />
<br />
<br><br />
<br><br />
===The detail function of FT as a transcriptional factor===<br />
[[File:FULSPE3AP1.jpg|thumb|right|200px|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. ]]<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) and SEPALLATA3(SEP3). This is because SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tops 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 />
<br />
<br><br><br><br />
===leave is suitable for checking the function of FT ===<br />
<br />
===Injecting FT and verifying its function by RT-PCR===<br />
FT protein is injected by a syringe. Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control. GFP was used as a control to confirm the result of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)<br />
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]<br />
<br>We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.<br><br />
<br />
<br />
Fig.4-2 is the result of RT-PCR. <br />
[[File:RT-PCR6.jpeg|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 extraction.<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<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 clear="both" /><br />
<br />
<br />
=====improving the way of RNA extraction=====<br />
<br />
=====conducting qPCR=====<br />
<br />
=====Whether FT protein went into plant cell?=====<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-6]]<br />
[[File:RT-PCR7.jpeg|thumb|300px|Fig.4-7 <br><br />
Lane1: 100bp ladder<br><br />
Lane2: TUBULIN <br><br />
Lane3: FUL<br><br />
Lane4: SEP3<br><br />
Lane5: AP1]]<br />
<br clear="both" /><br />
From these results, we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1. <br />
However, now that we can get good quality of RNA and amplify genes successfully, we are trying to check the function of FT.<br><br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T17:56:45Z<p>TJR: /* Cooperation with KAIT_Japan on issues of safety. */</p>
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<br />
===This year, we implemented nine plans on "human practice".===<br />
<br />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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 />
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<br />
==Education==<br />
[[File:KyotoLecture.jpg|link=|right|300px]]<br />
We went to Hibiya high school and Horikawa high school, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
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<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<br />
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<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 who live in Kyoto, 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 />
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<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 />
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<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 Campus.<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 Campus 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 />
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<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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
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<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
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<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
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<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which could make our research safer, we got their proposal about safety. They performed "Safety Icon Project".In this project, they designed icons in safety, which you could tell whether the parts in registry were safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]].<br />
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</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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T17:56:11Z<p>TJR: /* Cooperation with KAIT_Japan on issues of safety. */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
<html><br />
<script type="text/javascript"><br />
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<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 />
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[[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 />
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<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 />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
<br style="clear: both;" /><br />
<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<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 who live in Kyoto, 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 Campus.<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 Campus 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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which could make our research safer, we got their proposal about safety. They performed "Safety Icon Project".In this project, they designed icons in safety, which you can tell whether the parts in registry were safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]].<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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T17:55:04Z<p>TJR: /* Cooperation with KAIT_Japan on issues of safety. */</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 />
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<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 />
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<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 />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
<br style="clear: both;" /><br />
<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<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 who live in Kyoto, 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 Campus.<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 Campus 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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which could make our research safer, we got their proposal about safety. They performed "Safety Icon Project".In this project they designed icons in safety, which you can tell whether the parts in registry were safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]].<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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T17:52:31Z<p>TJR: /* Cooperation with KAIT_Japan on issues of safety. */</p>
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<br />
=[[File:Kyoto_HumanPractice.png|link=]]=<br />
<br />
===This year, we implemented nine plans on "human practice".===<br />
<br />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
<br style="clear: both;" /><br />
<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<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 who live in Kyoto, 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 Campus.<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 Campus 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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which could make our research safer, we got their proposal about safety. They did "Safety Icon Project". They designed icons in safety, which you can tell whether the parts in registry are safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]].<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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T17:44:35Z<p>TJR: /* Cooperation with KAIT_Japan on issues of safety. */</p>
<hr />
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<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 />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
<br style="clear: both;" /><br />
<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<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 who live in Kyoto, 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 Campus.<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 Campus 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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which can make our research safer, we got their proposal about safety. They did "Safety Icon Project". They designed icons in safety, which you can tell whether the parts in registry are safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]].<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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-26T17:40:11Z<p>TJR: /* Science Agora */</p>
<hr />
<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
{{Kyoto/header}}<br />
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<script type="text/javascript"><br />
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[[Image:KyotoTab_Safety.png|link=|Safety]]<html></a></html></li><br />
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<div id="kyoto-tab-HumanPractice"><br />
<br />
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<br />
===This year, we implemented nine plans on "human practice".===<br />
<br />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
<br style="clear: both;" /><br />
<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
read more:[[File:Japanese.pdf|link=|right|300px]]<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 who live in Kyoto, 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 Campus.<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 Campus 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 Science agora, a variety of people with various backgrounds were there. <br />
Many children learned about synthetic biology here, and we communicated with many people.<br />
<br />
<br style="clear: both;" /><br />
<br />
==Tie-up with Super Science High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which can make our research safer, we got their proposal about safety. They did "Safety Icon Project". They designed icons in safety, which you can tell whether the parts in registry are safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]]<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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-26T04:05:11Z<p>TJR: /* We prepared R9 and GFP separately */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
Have you ever seen flower fairies? Probably the answer is no (though some of you might have come across them in your childhood), because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition to the happy feelings, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli<br> with synthetic biology!!<br />
'''Our goal is to produce E.coli which can make flowers bloom as Flower Fairies. To make it possible, we focused on FT protein, known as Florigen.'''This protein is a kind of plant hormone. First, FT proteins are produced in leaves. then move to the shoot apex and bloom flowers. Therefore, FT proteins were the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli with R9 peptides, FT proteins are secreted by them and penetrate cell membranes of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These 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 />
<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 />
<br><br />
<html>On each step, we had some problems to attack.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether ''E.coli'' (prokaryote) can express FT proteins, because FT proteins are derived from plant cells (eukaryote).<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, FT proteins have to get out of the ''E.coli''.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT proteins have to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT proteins could get into the cells, it is not clear whether FT from ''E.coli'' can activate genes in shoot apex cells and lead it to flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
On the first step; '''EXPRESSION''', We needed to make ''E.coli'' produce FT protein.<br />
<br />
As a matter of course, ''E.coli'' doesn’t have ''FT'' gene. Therefore we had to make a new BioBrick part of ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' sequence had two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). This PCR enables us to mutate ''FT'' gene. As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes, and by fastening prefix and suffix to ''FT'' ,we could make new biobrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Changing the transcription’s direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' BioBrick. We had mutated and fastened prefix and suffix to ''FT''.]]<br />
<br><br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promoter and IPTG switches the transcription of T7 RNA polymerase. 6His tag, which is used in later steps, enabled us to purify protein from ''E.coli'' with affinity chromatography.<br />
<br />
We needed to confirm that E. coli had correctly expressed FT, because there was a possibility that ''E.coli'' could not translate FT or, the FT was unstable or toxic. <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(Fig.1-2).<br />
<br />
<br>''' We succeeded in making a mutation and confirming the expression of ''FT'' and 6 His:FT proteins in ''E.coli'''''<br />
<br />
<br />
[[File:Exp-FTwestern.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT proteins<br><br />
In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
On the second step; '''SECRETION''''' E.coli'' secretes florigen outside of the cell.<br />
<br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But on our project, lysis is not so good. It is because lysis possibly causes all Fairies death. <br />
Then, the effect of Flower Fairy E.coli won't continue. <br />
So, we adopted a new method by which ''E.coli'' can transport FT protein without lysis. <br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|150px|Fig.2-1.5 FT protein has two hurdles in order to get out of the cell]]<br />
<br clear="both"/><br />
<br />
=====Transport FT protein into periplasm=====<br />
<br />
[[File:Sec-inner-pathway1.png|thumb|right|150px|Fig.2-2 FT protein with torA signal go through inner membrane.]]<br />
<br />
Wild ''E.coli'' has many secretion systems. So we use one of these secretion systems, called Twin Arginate Translocation(Tat) pathway. Tat pathway is superior to others secretion systems because on Tat pathway ''E.coli'' secretes proteins into periplasm keeping proteins’ conformation and their functions. The mechanism of Tat pathway is that Tat transporter recognizes TorA signal and transports proteins that have TorA signal at N terminal into periplasm. <br />
<br />
<br clear="both"/><br />
<br />
====Our modified torA signal====<br />
We tried to combine TorA signal with FT protein. Actually, we searched previous iGEM projects and found some teams submitted TorA signal as iGEM parts (for example, [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]). There were, however, two big problems. One problem is that these parts don’t have RBS, so when iGEMers use these parts, they have to spend additional processing time. The other is that stop codons appear between signal region and target coding sequence when iGEMers assemble these parts and some parts by standard or 3A assembly. So if iGEMers use these parts, TorA-fusion protein is not expressed or only TorA is expressed. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-2 Old torA signal and our modified torA signal]]<br><br />
So we made new applicable TorA signal (BBa_). Our part has two advantages. BBa_ contains RBS and doesn’t have stop codon between signal region and target cording sequence. We read the sequence data of Bba_ and confirmed stop codon doesn't appear when it is used in Standard or 3A assembly. Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells (Fig.2-3). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked. <br><br />
<br />
[[File:光る大腸菌.jpg|thumb|center|600px|Fig.2-3 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. ''E.coli'' (right) sample having Lacp-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br><br />
<br />
====Transport FT protein out of ''E.coli''====<br />
[[File:Sec-outer-hole.png|thumb|right|200px|Fig.2-4 Kil protein makes holes on outer membrane.]]<br />
Using Tat secretion pathway, FT protein is transported into periplasm. Next, FT protein needs to be transported out of E.coli. In order to transport, we used kil protein, which is derived from λ phage. Kil protein makes holes in outer membrane of ''E.coli''. So on our project, we introduce ''kil'' gene into ''E.coli'' and want FT protein to go through these holes. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <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 />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. The Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|right|300px|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 incubation time.]]<br />
[[File:wiki intro 4.png|thumb|left|300px|Fig.2-3 How protein goes through two pathways]]<br><br><br><br><br />
[[File:Sec-integration.png|thumb|left|600px|Fig.2-5 How protein get out of the cell.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
====Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter''' [http://partsregistry.org/Part:BBa_K797004 (BBa_K797004)]<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 />
[[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 [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABCD [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)]<br />
,pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]<br />
. 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 />
[[File:Sec-futurework-construction.png|thumb|center|400px|Fig.2-6 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br><br />
[[File:Sec-futurework-pspA.png|thumb|center|600px|Fig.2-7 pspA enables TAT pathway to be improved more.]]<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
On the third step; '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins leading to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate cell membranes of plants. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found the method for the penetration of cell membrane with R9 peptides.<br />
<br />
<br />
<br clear="both"/><br />
<br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br><br />
R9 peptide consists of nine arginine residues. (Fig.3-1) It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a kind of endocytosis, so R9 peptide is suited to our purpose.<br />
<br />
This is the mechanism of how R9 peptides work.(Fig3-2)<br />
<br />
Firstly, R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br><br />
Secondly, Cells response to the stimulus and induced endocytosis.<br><br />
Finally, FT protein around an endocytosing region of cell is taken in the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work regardless of whether or not R9 peptides are connected with target proteins. <br />
There are few examples about endocytosis of plants, so '''we needed to check the function of R9 against plants'''.<br><br />
<br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|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 some kind of endocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br />
<br />
<br><br />
<br clear="both"/><br />
<br />
----<br />
<br />
====The above R9 system indicates that connecting R9 peptide with a target protein may lead to higher efficiency ====<br />
Considering the R9 system, target proteins near the R9 peptides are taken by endocytosis. <br />
In other words, it can be said that the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. Therefore, we thought connecting R9 with a target protein is more efficient than disconnecting R9 peptides from target proteins. And it is easy to connect them by amide bond when we transform E.coli with a plasmid arranging R9's sequence and target protein's gene in line.<br />
<br />
====So, We tried to connect R9 with target proteins for penetration efficiency by amide bond====<br />
<br />
we transformed E.coli with a plasmid arranging R9's sequence and target protein's gene in line.And after that we tried to make E.coli express R9::GFP fusion protein.<br />
<br />
<br />
<br />
<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was poor in growth.(Fig3-3) Then, we checked R9 effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we didn’t find the protein (Fig3-5). These results insist poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<br />
[[File:culture1N.png|thumb|left|190px|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.]]<br />
[[File:Western-R9-GFP2N.png|thumb|190px|left|Fig.3-4 Western blotting for checking the expression. No band of R9:::GFP fusion protein.<br> 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 />
[[File:RT-PCR1R9-GFPN.png|thumb|190px|left|Fig.3-5 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 />
<br />
<br clear="both"/><br />
<br />
----<br />
<br />
====We prepared R9 and GFP separately====<br />
<br />
<br />
[[File:R9とGFPでやるやつ.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticle 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
We wondered whether R9 system really work properly and put FT protein into plant cells? Although animal cells often endocytose, there are few examples of plant cell’s endocytosis. Therefore, We need to verify the system.<br />
To verify it, we performed followed experiment.(Fig3-6) <br />
<br />
<br />
First, we scratched the cuticle of ''Arabidopsis thaliana'', a model plant. Second, we soaked them into a solution of only GFP, or GFP and R9 for each. These GFP proteins were purified with 6 His tag inserted in our 1st Step, "Expression". After 5 minutes we washed cells by PBS in order to wash GFP and R9 peptide away from leaves. After that, we compared leaves including only GFP, and leaves including R9 and GFP for each. <br />
Then we succeeded in getting the Figure of GFP fluorescence.(Fig3-7)<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides work properly kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function'''. <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|link=|Activation]]<br />
<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 />
===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 />
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) and SEPALLATA3(SEP3). This is because SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tops 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|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 extraction.<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<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 clear="both" /><br />
<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-6]]<br />
[[File:RT-PCR7.jpeg|thumb|300px|Fig.4-7 <br><br />
Lane1: 100bp ladder<br><br />
Lane2: TUBULIN <br><br />
Lane3: FUL<br><br />
Lane4: SEP3<br><br />
Lane5: AP1]]<br />
<br clear="both" /><br />
From these results, we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1. <br />
However, now that we can get good quality of RNA and amplify genes successfully, we are trying to check the function of FT.<br><br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-26T03:57:18Z<p>TJR: /* We prepared R9 and GFP separately */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
Have you ever seen flower fairies? Probably the answer is no (though some of you might have come across them in your childhood), because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition to the happy feelings, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli<br> with synthetic biology!!<br />
'''Our goal is to produce E.coli which can make flowers bloom as Flower Fairies. To make it possible, we focused on FT protein, known as Florigen.'''This protein is a kind of plant hormone. First, FT proteins are produced in leaves. then move to the shoot apex and bloom flowers. Therefore, FT proteins were the key to our project.<br />
<br><br />
<br />
===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli with R9 peptides, FT proteins are secreted by them and penetrate cell membranes of a plant, and the plant starts blooming.<br><br><br />
<br />
<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br><br />
These 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 />
<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 />
<br><br />
<html>On each step, we had some problems to attack.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether ''E.coli'' (prokaryote) can express FT proteins, because FT proteins are derived from plant cells (eukaryote).<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, FT proteins have to get out of the ''E.coli''.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT proteins have to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT proteins could get into the cells, it is not clear whether FT from ''E.coli'' can activate genes in shoot apex cells and lead it to flower formation.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
On the first step; '''EXPRESSION''', We needed to make ''E.coli'' produce FT protein.<br />
<br />
As a matter of course, ''E.coli'' doesn’t have ''FT'' gene. Therefore we had to make a new BioBrick part of ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' sequence had two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). This PCR enables us to mutate ''FT'' gene. As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes, and by fastening prefix and suffix to ''FT'' ,we could make new biobrick parts of ''FT'' (Fig.1-1 C). <br />
<br />
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Changing the transcription’s direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' BioBrick. We had mutated and fastened prefix and suffix to ''FT''.]]<br />
<br><br />
=== Confirming expression of FT ===<br />
<br />
We constructed the plasmid shown in the Fig.1-2. One was ''FT'' gene with T7 promoter<br />
([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was ''FT'' gene only with T7 promoter. T7 promoter is a strong promoter and IPTG switches the transcription of T7 RNA polymerase. 6His tag, which is used in later steps, enabled us to purify protein from ''E.coli'' with affinity chromatography.<br />
<br />
We needed to confirm that E. coli had correctly expressed FT, because there was a possibility that ''E.coli'' could not translate FT or, the FT was unstable or toxic. <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(Fig.1-2).<br />
<br />
<br>''' We succeeded in making a mutation and confirming the expression of ''FT'' and 6 His:FT proteins in ''E.coli'''''<br />
<br />
<br />
[[File:Exp-FTwestern.png|thumb|left|650px|Fig.1-2 Construction of ''FT'' generator and the results of Western blotting against FT and 6 His:FT proteins<br><br />
In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.]]<br />
<br />
<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
On the second step; '''SECRETION''''' E.coli'' secretes florigen outside of the cell.<br />
<br />
Now our ''E.coli'' can produce FT protein, but a big issue still remains: <br />
how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. <br />
But on our project, lysis is not so good. It is because lysis possibly causes all Fairies death. <br />
Then, the effect of Flower Fairy E.coli won't continue. <br />
So, we adopted a new method by which ''E.coli'' can transport FT protein without lysis. <br />
<br />
[[File:Sec-intro-hurdles.png|thumb|right|150px|Fig.2-1.5 FT protein has two hurdles in order to get out of the cell]]<br />
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<br />
=====Transport FT protein into periplasm=====<br />
<br />
[[File:Sec-inner-pathway1.png|thumb|right|150px|Fig.2-2 FT protein with torA signal go through inner membrane.]]<br />
<br />
Wild ''E.coli'' has many secretion systems. So we use one of these secretion systems, called Twin Arginate Translocation(Tat) pathway. Tat pathway is superior to others secretion systems because on Tat pathway ''E.coli'' secretes proteins into periplasm keeping proteins’ conformation and their functions. The mechanism of Tat pathway is that Tat transporter recognizes TorA signal and transports proteins that have TorA signal at N terminal into periplasm. <br />
<br />
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<br />
====Our modified torA signal====<br />
We tried to combine TorA signal with FT protein. Actually, we searched previous iGEM projects and found some teams submitted TorA signal as iGEM parts (for example, [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]). There were, however, two big problems. One problem is that these parts don’t have RBS, so when iGEMers use these parts, they have to spend additional processing time. The other is that stop codons appear between signal region and target coding sequence when iGEMers assemble these parts and some parts by standard or 3A assembly. So if iGEMers use these parts, TorA-fusion protein is not expressed or only TorA is expressed. <br><br />
[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-2 Old torA signal and our modified torA signal]]<br><br />
So we made new applicable TorA signal (BBa_). Our part has two advantages. BBa_ contains RBS and doesn’t have stop codon between signal region and target cording sequence. We read the sequence data of Bba_ and confirmed stop codon doesn't appear when it is used in Standard or 3A assembly. Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells (Fig.2-3). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked. <br><br />
<br />
[[File:光る大腸菌.jpg|thumb|center|600px|Fig.2-3 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. ''E.coli'' (right) sample having Lacp-TorA-GFP-DT and observed with 489nm wavelength.]]<br />
<br><br />
<br />
====Transport FT protein out of ''E.coli''====<br />
[[File:Sec-outer-hole.png|thumb|right|200px|Fig.2-4 Kil protein makes holes on outer membrane.]]<br />
Using Tat secretion pathway, FT protein is transported into periplasm. Next, FT protein needs to be transported out of E.coli. In order to transport, we used kil protein, which is derived from λ phage. Kil protein makes holes in outer membrane of ''E.coli''. So on our project, we introduce ''kil'' gene into ''E.coli'' and want FT protein to go through these holes. But the function of outer membrane is essential for ''E.coli'' to survive. Overexpression of ''kil'' gene, therefore, causes cell death. For this reason, we must check whether ''kil'' gene is harmful or not under our condition. <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 />
[[File:Lacp kil DT.png|thumb|left|300px|Fig.2-2 The construction used in this assay. The Promoter is Lactose promoter R0010]]<br />
[[File:Kill assay.png|thumb|right|300px|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 incubation time.]]<br />
[[File:wiki intro 4.png|thumb|left|300px|Fig.2-3 How protein goes through two pathways]]<br><br><br><br><br />
[[File:Sec-integration.png|thumb|left|600px|Fig.2-5 How protein get out of the cell.]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
====Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter''' [http://partsregistry.org/Part:BBa_K797004 (BBa_K797004)]<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 />
[[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 [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABCD [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)]<br />
,pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]<br />
. 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 />
[[File:Sec-futurework-construction.png|thumb|center|400px|Fig.2-6 Tat secretion cassette with constitutive promoter(BBa_K797004) ]]<br><br />
[[File:Sec-futurework-pspA.png|thumb|center|600px|Fig.2-7 pspA enables TAT pathway to be improved more.]]<br />
<br />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
<br />
On the third step; '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins leading to flower formation in plant cells.<br />
<br />
However, normally proteins cannot penetrate cell membranes of plants. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found the method for the penetration of cell membrane with R9 peptides.<br />
<br />
<br />
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<br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<br />
[[File:R9.png|thumb|right|250px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
<br><br />
R9 peptide consists of nine arginine residues. (Fig.3-1) It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a kind of endocytosis, so R9 peptide is suited to our purpose.<br />
<br />
This is the mechanism of how R9 peptides work.(Fig3-2)<br />
<br />
Firstly, R9 peptide adheres to cell membrane of plants because of hydrophobic character.<br><br />
Secondly, Cells response to the stimulus and induced endocytosis.<br><br />
Finally, FT protein around an endocytosing region of cell is taken in the cell.<br><br />
<br />
<br />
<br />
<br />
R9 peptides seem to work regardless of whether or not R9 peptides are connected with target proteins. <br />
There are few examples about endocytosis of plants, so '''we needed to check the function of R9 against plants'''.<br><br />
<br />
[[File:Pene-R9-mechanism.png|thumb|left|650px|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 some kind of endocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br />
<br />
<br><br />
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<br />
----<br />
<br />
====The above R9 system indicates that connecting R9 peptide with a target protein may lead to higher efficiency ====<br />
Considering the R9 system, target proteins near the R9 peptides are taken by endocytosis. <br />
In other words, it can be said that the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. Therefore, we thought connecting R9 with a target protein is more efficient than disconnecting R9 peptides from target proteins. And it is easy to connect them by amide bond when we transform E.coli with a plasmid arranging R9's sequence and target protein's gene in line.<br />
<br />
====So, We tried to connect R9 with target proteins for penetration efficiency by amide bond====<br />
<br />
we transformed E.coli with a plasmid arranging R9's sequence and target protein's gene in line.And after that we tried to make E.coli express R9::GFP fusion protein.<br />
<br />
<br />
<br />
<br />
<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was poor in growth.(Fig3-3) Then, we checked R9 effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we didn’t find the protein (Fig3-5). These results insist poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
<br />
<br />
[[File:culture1N.png|thumb|left|190px|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of ''E.coli'' with R9::GFP fusion protein on the right hand is cloudier than that on the left.]]<br />
[[File:Western-R9-GFP2N.png|thumb|190px|left|Fig.3-4 Western blotting for checking the expression. No band of R9:::GFP fusion protein.<br> 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 />
[[File:RT-PCR1R9-GFPN.png|thumb|190px|left|Fig.3-5 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 />
<br />
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<br />
----<br />
<br />
====We prepared R9 and GFP separately====<br />
<br />
<br />
[[File:R9とGFPでやるやつ.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticle 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
We wondered whether R9 system really work properly and put FT protein into plant cells? Although animal cells often endocytose, there are few examples of plant cell’s endocytosis. Therefore, We need to verify the system.<br />
To verify it, we performed followed experiment.(Fig3-6) <br />
<br />
<br />
First, we scratched the cuticle of Arabidopsis thaliana, a model plant. Second, we soaked them into a solution of only GFP, or GFP and R9 for each. This GFP protein was purified with 6 His tag inserted in our 1st Step, "Expression". After 5 minutes we washed cells by PBS in order to wash GFP and R9 peptide away from leaves. After that, we compared leaves including only GFP, and leaves including R9 and GFP for each. <br />
Then we succeeded in getting the Figure of GFP fluorescence.(Fig3-7)<br />
<br />
<br />
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides work properly kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP, because this was taken by a confocal microscopy and seen as cross sections.<br />
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<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function'''. <br />
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<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|link=|Activation]]<br />
<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 />
===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 />
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) and SEPALLATA3(SEP3). This is because SEP3 and FUL are activated in leaves. It is difficult for us to handle cells of tops 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|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 extraction.<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<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 />
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<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-6]]<br />
[[File:RT-PCR7.jpeg|thumb|300px|Fig.4-7 <br><br />
Lane1: 100bp ladder<br><br />
Lane2: TUBULIN <br><br />
Lane3: FUL<br><br />
Lane4: SEP3<br><br />
Lane5: AP1]]<br />
<br clear="both" /><br />
From these results, we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1. <br />
However, now that we can get good quality of RNA and amplify genes successfully, we are trying to check the function of FT.<br><br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
<br />
=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/File:%EF%BC%B2%EF%BC%99%E3%81%A8%EF%BC%A7%EF%BC%A6%EF%BC%B0%E3%81%A7%E3%82%84%E3%82%8B%E3%82%84%E3%81%A4.pngFile:R9とGFPでやるやつ.png2012-10-26T03:55:34Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Team:Kyoto/TeamTeam:Kyoto/Team2012-10-26T03:12:45Z<p>TJR: /* Students */</p>
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<div id="kyoto-tab-Member"><br />
=[[File:Kyoto_Members.png|link=]]=<br />
=== '''Students''' ===<br />
<div id="kyoto-members"><br />
<div><br />
[[Image:Kyoto_TOMOHIRO_NOBEYAMA.jpg|link=|center|150px|]]<br />
'''TOMOHIRO NOBEYAMA'''<br />
Team Leader<br>Secretion group<br />
----<br />
Tomohiro is 2nd year student and belongs to the faculty of science. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_AKANE_SATO.jpg|link=|center|150px|]]<br />
'''AKANE SATO'''<br />
Experiment Leader<br>Florigen group<br />
----<br />
Akane is a second year student and belongs to the faculty of pharmaceutical sciences.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_FUMITAKA_HASHIYA.jpg|link=|center|150px|]]<br />
'''FUMITAKA HASHIYA'''<br />
----<br />
Fumitaka is 3rd year student and belongs to the faculty of science. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_HATSUHO_KANO.jpg|link=|center|150px|]]<br />
'''HATSUHO KANO'''<br />
Florigen group<br />
----<br />
Hatsuho is 1st year student and he belongs to the faculty of science. He is interested in Synthetic Biology.<br />
</div><br />
<br />
<div><br />
[[Image:Hyungcheol_Chae.jpg|link=|center|150px|]]<br />
'''HYUNGCHEOL CHAE'''<br />
Florigen group<br />
----<br />
Hello~ I'm a sophomore studying applied life science at Kyoto University. I hope to have a lot of fun iGEM. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_KANJI_NAKAGAWA.jpg|link=|center|150px|]]<br />
'''KANJI NAKAGAWA'''<br />
Florigen group<br />
-----<br />
Kanji is 1st year student and belongs to the faculty of science. He is interested in science widely, especially in micro biology. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_KAORU_RICHARD_KOMATSU.jpg|link=|center|150px|]]<br />
'''KAORU R. KOMATSU'''<br />
Human practice Leader<br><br />
----<br />
He's absorbed in apple products, so he often says that "nothing more apple, nothing more apple!"<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_KEN_TAJIRI.jpg|link=|center|150px|]]<br />
'''KEN TAJIRI'''Florigen group<br>Illustrator, Designer<br />
----<br />
Ken is crazy. He always runs, always sleeps on the floor. He loves wearing skirts.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_KENJI_OKUMURA.jpg|link=|center|150px|]]<br />
'''KENJI OKUMURA'''<br />
Golden Gate group<br />
----<br />
Kenji is 2nd year student and belongs to the faculty of agriculture.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_KENYA_UENo.jpg|link=|center|150px|]]<br />
'''KENYA UENO'''<br />
----<br />
Kenya is 1st year student and he belongs to the faculty of science.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_MASAKO_TERASAKA.jpg|link=|center|150px|]]<br />
'''MASAKO TERASAKA'''<br />
Secretion group<br />
----<br />
Masako is 1st year student and belongs to the faculty of medicine.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_MITSUAKI_YOSHIDA.jpg|link=|center|150px|]]<br />
'''MITSUAKI YOSHIDA'''<br />
Secretion group<br />
----<br />
Mitsuaki is 1st year student and he belongs to the faculty of science. He is interested in mathematics, physics, biology, and so on.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_MOE_YANAGI.jpg|link=|center|150px|]]<br />
'''MOE YANAGI'''<br />
Florigen group<br>Designer<br />
----<br />
Moe is 1st year student and she belongs to the faculty of agriculture. She is interested in applied life science. She has a full of curiosity. Yeah!<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_RYOSUKE_KIDA.jpg|link=|center|150px|]]<br />
'''RYOSUKE KIDA'''<br />
Design Leader<br>Golden Gate Leader<br />
----<br />
Ryosuke is 3rd year student, and he belongs to the faculty of agriculture. He has been interested in food science since he was 1st year. His other interests are movies, books and music. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_RYOSUKE_TAKEUCHI.jpg|link=|center|150px|]]<br />
'''RYOSUKE TAKEUCHI'''<br />
Florigen Leader<br />
----<br />
Ryosuke is 2nd year student. He is interested in DIYbio, and wants to realize the harmony between iGEM and DIYbio. If you are interested, please tell to him.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_SANAE_IZUMI.jpg|link=|center|150px|]]<br />
'''SANAE IZUMI'''<br />
Secretion Leader<br />
----<br />
Sanae is second year student and belongs to the faculty of pharmaceutical sciences. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_SATOSHI_KUSABA.jpg|link=|center|150px|]]<br />
'''SATOSHI KUSABA'''<br />
Human practice<br>Illustrator, Designer<br />
----<br />
Satoshi is 3rd year student and belongs to the faculty of science (physics).<br />
He also does kendo, a modern Japanese martial art of sword-fighting.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_SAYAKA_DANTSURI.jpg|link=|center|150px|]]<br />
'''SAYAKA DANTSUZI'''<br />
Florigen group<br />
----<br />
Sayaka is 1st year student and she belongs to the faculty of science.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_SUGURU_KATO.jpg|link=|center|150px|]]<br />
'''SUGURU KATO'''<br />
Programer<br>Wiki Edit Leader<br>Golden Gate group<br />
----<br />
Suguru is 2nd year student and belongs to the faculty of science.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_TAKAHIRO_SHIMOSAKA.jpg|link=|center|150px|]]<br />
'''TAKAHIRO SHIMOSAKA'''<br />
Golden Gate group<br />
----<br />
He is sophomore and he majors in industrial chemistry.<br><br />
He is interested in microorganisms and fermented food.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_TETSUYA_KAWATA.jpg|link=|center|150px|]]<br />
'''TETSUYA KAWATA'''<br />
----<br />
Tetsuya is 4th year student and belongs to the faculty of science.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_TORU_NIINA.jpg|link=|center|150px|]]<br />
'''TORU NIINA'''<br />
Florigen group<br>Wiki Editor<br>Designer<br />
----<br />
Toru is 1st year student and belongs to the faculty of science. He is interested in whole science, especially in biology and chemistry. <br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_YOSHITAKA_HIRANO.jpg|link=|center|150px|]]<br />
'''YOSHITAKA HIRANO'''<br />
Secretion group<br />
----<br />
Yoshitaka is the 1st year student and belongs to the faculty of agriculture. He is interested in biomass, especially in cellulose.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_YOSHIYUKI_OHTA.jpg|link=|center|150px|]]<br />
'''YOSHIYUKI OHTA'''<br />
Florigen group<br />
----<br />
Yoshiyuki is 2nd year student and belongs to the faculty of agriculture.<br />
</div><br />
<br />
<div><br />
[[Image:Kyoto_YUSUKE_KOMOTO.jpg|link=|center|150px|]]<br />
'''YUSUKE KOMOTO'''<br />
Secretion group<br />
----<br />
I'm an elite.<br />
</div><br />
</div><br />
<br />
==Group Photograph==<br />
[[File:579556_342527215818961_125847331_n.jpg|600px|center|link=]]<br />
</div><br />
<div id="kyoto-tab-Advisor"><br />
<br />
=[[File:Kyoto_Advisors.png|link=]]=<br />
;Akira Nabetani<br />
:Graduate School of Biostudies, Kyoto University<br />
;Humihiko Satou<br />
:Graduate School of Biostudies, Kyoto University<br />
;Ikuhiko Nakase<br />
:Graduate School of Pharmaceutical Sciences<br />
;Ikuko Nishimura<br />
:Department of Botany, Kyoto University <br />
;Ken Kajita<br />
:Faculty of engineering, Kyoto University<br />
;Knut Woltjen<br />
:Center for iPS Cell Research and Apprication(CiRA), Kyoto University<br />
;Kojirou Takanashi<br />
:Research Institute for Sustainable Humanosphere, Kyoto University<br />
;Makoto Kashima<br />
:Graduate School of Science, Kyoto University<br />
;Masaru Kobayashi<br />
:Graduate School of Agriculture, Kyoto University<br />
;Miki Imanishi<br />
:Institute for Chemical Reserch, Kyoto University<br />
;Shin Yonehara<br />
:Laboratory of Molecular and Cellular Biology, Graduate School of Biostudies, Kyoto University<br />
;Takayuki Kouchi<br />
:Graduate School of Biostudies, Kyoto University<br />
;Tan Inoue<br />
:Graduate School of Biostudies, Kyoto University<br />
;Takashi Araki<br />
:Plant Developmental Biology, Graduate School of BIOSTUDIES, Kyoto University.<br />
;Takashi Endou<br />
:Graduate School of Agriculture, Kyoto University<br />
;Tetsurou Okuno<br />
:Graduate School of Agriculture, Kyoto University<br />
;Tokitaka Oyama<br />
:Department of Botany, Graduate School of Science, Kyoto University<br />
;Tomo Murayama<br />
:Graduate School of Pharmacy, Kyoto University<br />
;Tomonori Takada<br />
:Department of Botany, Kyoto University <br />
;Toru Matou<br />
:Graduate School of Agriculture, Kyoto University<br />
;Toshiharu Shikanai<br />
:Graduate School of Science, Kyoto University<br />
;Wataru Shihoya<br />
:Cellular and Structural Physiology Institute, Nagoya University<br />
;Yoshihiko Fujita<br />
:Graduate School of Biostudies, Kyoto University<br />
</div><br />
<div id="kyoto-tab-ContactUs"><br />
<br />
<div><br />
<br />
=[[File:Kyoto_Contact.png|link=]]=<br />
Contact us if you need more information. Our contact details are the following!<br /><br />
{| style="background-color:transparent"<br />
|[[File:Email icon.jpg|100px]]<br />
|Mail to '''igem.kyoto(a)gmail.com'''.<br />
|-<br />
|[[File:KyotoLogo.png|100px|link=http://openwetware.org/wiki/IGEM:Kyoto/home]]<br />
|Visit our [http://openwetware.org/wiki/IGEM:Kyoto/home website].<br />
|-<br />
|[[File:twitter-bird.png|100px|link=http://twitter.com/iGEMkyoto]]<br />
|Follow [http://twitter.com/iGEMkyoto @iGEMkyoto].<br />
|-<br />
|[[File:fblogo.jpg|100px|link=http://www.facebook.com/IgemKyoto iGEM Kyoto facebook page]]<br />
|Like [http://www.facebook.com/IgemKyoto iGEM Kyoto facebook page].<br />
|}<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>TJRhttp://2012.igem.org/Template:Kyoto/Project/FlowerFairyTemplate:Kyoto/Project/FlowerFairy2012-10-25T02:26:17Z<p>TJR: /* Putting Flower Fairy E.coli on leaves! */</p>
<hr />
<div><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 />
===Realizing Flower Fairy in real world===<br />
Have you ever seen flower fairies? Probably no (some of you might come across them in your childhood), because they are imaginary creatures existing only in fairy tales. Don’t you think it is happy if you can live with flower fairies? In addition to feeling happiness, 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, known as Florigen.'''This protein is a kind of plant hormone. At first, FT protein is produced in leaves. After that, it moves to shoot apex and bloom flowers. Therefore, FT protein is a key to our project.<br />
<br><br />
<br />
===Putting Flower Fairy E.coli on leaves!===<br />
When you want to use our Flower Fairy E.coli, there is just one thing you must do to get ready. Only putting them on leaves! When you spread Flower Fairy E.coli adding R9 peptide, FT protein secreted by them penetrates into cell walls and the plant blooms.<br><br><br />
<br />
<html>We have to go through four steps in order to achieve our goal――Flower 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 />
<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 />
<br><br />
<html>On each step, we have some problems to be solved.<br><br />
<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether E.coli (prokaryote) could express FT protein, because FT protein is derived from plant cells (eukaryote).<br><br />
<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, FT protein has to get out of the E.coli.<br><br />
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br><br />
<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even though FT protein could get inside of the cells, it is unknown whether FT transcribed in E.coli can activate shoot apex cells and bloom flowers.</html><br />
<br />
<html><a id="FFEResults"></a></html><br />
<br />
=[[File:Kyoto_Experiments&ResultsHeader.png|link=]]=<br />
<br />
<br />
<html><a name="Expression"></a></html><br />
==1.EXPRESSION==<br />
<br />
[[File:アイコン1.png|200px|left|link=|Expression]]<br />
<br />
On the first step; '''EXPRESSION''', We needed to make ''E.coli'' produce FT protein.<br />
<br />
As a matter of course, ''E.coli'' doesn’t have ''FT'' gene. Therefore we had to make a new BioBrick part of ''FT'' gene and introduce it into ''E.coli''.<br />
<br />
<br><br><br><br />
<br><br />
=== Modifying ''FT'' gene for Biobrick ===<br />
<br />
''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' sequence had two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1), therefore we modified ''FT'' gene sequences by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-2). This PCR enables us to mutate ''FT'' gene. As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes. <br />
<br />
<br />
<br />
[[File:FT1.png|thumb|left|200px|Fig.1-1 Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes. ]]<br />
[[File:InversePCRMethod.jpg|thumb|right|200px|Fig.1-2 Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Changing the transcription’s direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.]]<br />
[[File:FT2.png|thumb|left|200px|Fig.1-3 Standardization of ''FT'' BioBrick. We had mutated and fastened prefix and suffix to FT.]]<br />
We constructed the plasmid shown in the Fig.1-4. One was FT gene with T7 promoter<br />
[http://partsregistry.org/Part:BBa_I719005 BBa_I719005] and 6His tag, and the other was FT gene only with T7 promoter. T7 promoter is strong promoter and IPTG switches transcription of T7 RNA polymerase. 6His tag, which is used at later steps, enables us to purify protein from ''E.coli'' with affinity chromatography.<br><br><br />
<br />
[[File:FTconstruction.png|thumb|left|200px|Fig.1-4 ''FT'' construction. T7 promoter maximizes ''FT'' transcriptive activity and 6 His tag enables us to purify FT protein. ]]<br clear="both"><br />
[[File:westernFT3.png|thumb|300px|left|Fig.1-5 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.1-4 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, and 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 />
=== Confirming expression of FT ===<br />
<br />
<br />
Then we confirmed whether E. coli was correctly expressing FT by Western blotting, because there is possibility that ''E.coli'' cannot translate FT or that FT is not stable or is toxic. <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 />
As a result, FT and 6 His:FT bands were observed at the expected molecular weight region(Fig.1-5).<br />
<br />
''' We succeeded in mutation and expression of ''FT'' and 6 His:FT proteins in ''E.coli'''''<br />
<br />
<br clear="both" /><br />
<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
<br />
[[File:アイコン2.png|250px|left|link=|Secretion ]]<br />
On the second step; ‘’’SECRETION’’’, ‘’E.coli’’ secretes florigen outside of the cell.<br />
<br />
Now our ‘’E.coli’’ can produce FT protein, but a big issue still remains: how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. But on our project, lysis is not so good. It is because lysis possibly causes all Fairies death. Then, the effect of Flower Fairy E.coli won't continue. So, we adopted a new method by which ‘’E.coli’’ can transport FT protein without lysis. <br><br><br><br />
<br />
=====Transport FT protein into periplasm=====<br />
We chose transport system of wild ‘’E.coli’’, called The Twin Arginate Translocation(Tat) pathway. The points that Tat pathway is better than the other secretion system is that ‘’E.coli’’ secretes proteins without lysis and keeping proteins’ conformation. The mechanism of Tat pathway is that Tat transporter recognizes TorA signal and transports proteins that have TorA signal at N terminal into periplasm. <br> <br />
We tried to combine TorA signal with FT protein. Actually, we searched previous iGEM projects and found some teams submitted TorA signal as iGEM parts (for example, [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]). There were, however, two big problems. One problem is that these parts don’t have RBS, so when iGEMers use these parts, they have to spend additional processing time. The other is that stop codons appear between signal region and target coding sequence when iGEMers assemble these parts and some parts by standard or 3A assembly. So if iGEMers use these parts, TorA-fusion protein is not expressed or only TorA is expressed. <br><br />
So we made new applicable TorA signal (BBa_). Our part has two advantages. BBa_ contains RBS and doesn’t have stop codon between signal region and target cording sequence. We read the sequence data of Bba_ and confirmed stop codon doesn't appear when it is used in Standard or 3A assembly. Using green fluorescent protein (GFP) as a target protein, we observed the TorA-GFP fusion-expressing cells (Fig.2-1). The TorA-GFP fusion was successfully expressed. This means RBS in our TorA signal worked. <br><br />
<br />
<gallery widths=600px heights=300px><br />
File:光る大腸菌.jpg|Fig.2-1 ''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 />
====Evaluation of kil protein====<br />
Using Tat secretion pathway, FT protein is transported into periplasm.<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, lysis of ''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 an 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 coding 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 goes 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'', therefore, causes cell death. For this reason, we must check whether our ''kil'' gene is overexpressed or not.<br><br><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 incubation 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 />
====Construction of Tat cassette====<br />
'''Tat secretion cassette with constitutive promoter''' [http://partsregistry.org/Part:BBa_K797004 (BBa_K797004)]<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 />
[[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 [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABCD [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)]<br />
,pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]<br />
. 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 />
<html><a name="Penetration"></a></html><br />
<br />
==3.PENETRATION==<br />
<br />
[[File:アイコン3改.png|250px|left|link=|Penetration ]]<br />
On the third step; '''PENETRATION'''.<br />
In order to induce flower formation, FT protein from E.coli must enter into plant cells. This is because FT protein upregulates other proteins leading to flower formation in plant cells.<br />
<br><br><br><br><br><br><br><br><br><br />
<br />
[[File:R9.png|thumb|right|200px|Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.]]<br />
However, normally proteins cannot penetrate cell membranes of plants. Therefore, we needed a method to send FT protein into plant cells.<br />
Thanks to the advice from Doctor Washida, we found the method for the cell membrane penetration. A polyarginine ,called '''R9 peptides''', makes FT protein penetrate a cell membrane in this method.<br />
This is a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. <br />
(Fig.3-1)<br />
<br />
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<br />
=====R9 peptide enables FT protein penetrate membranes by endocytosis=====<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 some kind of endocytosis.<br> 3.FT protein around an invaginating region of cell is taken in the cell. ]]<br />
<br />
This is the mechanism of how R9 peptides work.(Fig3-2)<br />
Firstly, R9 peptides adhere to a cell membrane of a plant because of hydrophobic character.<br />
Secondly, cells response to this stimulus and induce endocytosis.<br />
Finally, FT proteins near R9 are taken in the cell.<br />
<br />
<br />
<br />
<br />
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<br />
----<br />
<br />
====We connected R9 with protein for a high degree of penetration efficiency====<br />
<br />
<br />
We thought shorting the distance between R9 and protein led to a high degree of penetration efficiency, because R9 induces endocytosis with R9 itself bonded to membranes. Therefore we connected R9 with protein.<br />
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, E.coli expressing the R9::GFP fusion protein was poor in growth.(Fig3-3) Then, we checked R9 effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we didn’t find the protein (Fig3-5). These results insists poor translation or quick breakdown of the protein. <br />
<br />
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. <br />
perhaps because of its cytotoxicity of R9 against ''E.coli''. <br />
<br />
[[File:culture1.png|thumb|left|190px|Fig.3-3 Poor growth of ''E.coli'' expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of E.coli with R9::GFP fusion protein on the right hand is cloudier than that on the left.]]<br />
<br />
[[File:Western-R9-GFP2.png|thumb|190px|left|Fig.3-4 Western blotting for checking the expression. No band of R9:::GFP fusion protein.<br> 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 />
[[File:RT-PCR1R9-GFP.png|thumb|190px|left|Fig.3-5 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 />
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----<br />
<br />
====We prepare R9 and GFP individually to visualize the R9 peptides====<br />
<br />
<br />
[[File:竹内さんまとめlast.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Scratching cuticle 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]<br />
<br />
<br />
we wondered whether R9 system really work properly and put FT protein into plant cells? Although animal cells often endocytose, there are few examples of plant cell’s endocytosis. Therefore, We need to verify the system.<br />
To verify it, we performed followed experiment.(Fig3-6) <br />
<br />
<br />
<br />
First,we scratched the cuticle of Arabidopsis thaliana, a model plant. Second,we soaked them into a solution of only GEP, or GFP and R9 for each. This GFP protein was purified with 6 His tag inserted in our 1st Step,"Expression". After 5 minutes we washed cells by PBS in order to wash GFP and R9 peptide away from leaves. After that, we compared leaves including only GFP, and leaves including R9 and GFP for each. <br />
Then we succeeded in getting the Figure of GFP fluorescence.(Fig3-7)<br />
<br />
The control group on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptide work properly kept GFP in or around plant cells. This Figure strongly suggests that R9 peptide works successfully and penetrates cell membrane with GFP, because this was taken by a confocal microscopy and seen as cross sections.<br />
<br clear="both"/><br />
<br />
[[File:Final.jpg|thumb|600px|center|Fig.3-7 Verification of the R9 peptide function with use of GFP.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 , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)]]<br />
<br />
'''We can make FT protein penetrate cell membrane of plants by R9 peptide function'''. <br />
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<html><a name="Activation"></a></html><br />
<br />
==4. Activation==<br />
[[File:アイコン4.png|250px|left|link=|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 />
===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 />
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|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 />
[[File:TotalRNA20120925-01.png|thumb|300px|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 />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.<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 extraction.<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 collected 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 reverse transcription and retried RT-PCR.<br />
Fig.4-6 shows the result of this RT-PCR.<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 />
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<br />
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-6]]<br />
[[File:RT-PCR7.jpeg|thumb|300px|Fig.4-7 <br><br />
Lane1: 100bp ladder<br><br />
Lane2: TUBULIN <br><br />
Lane3: FUL<br><br />
Lane4: SEP3<br><br />
Lane5: AP1]]<br />
<br clear="both" /><br />
From these results, we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1. <br />
However, now that we can get good quality of RNA and amplify genes successfully, we are trying to check the function of FT.<br><br />
<br />
==Achievement==<br />
1.Expression<br><br />
* Mutate FT sequence<br />
* Standardize FT as an iGEM part<br />
* Confirm expression of FT protein in ''E.coli''<br />
2.Secretion<br><br />
*Modify TorA signal to be easy to use the signal more<br />
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator<br />
*Standardize kil gene<br />
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''<br />
3.Penetration<br><br />
*Keep GFP in or around plant cells using R9 peptide<br />
**Introduce FT in plant cells using R9 peptide--'''not yet'''<br />
4.Activation<br><br />
*Get high quality of RNA<br />
*Amplify genes successfully<br />
**Check the function of FT--'''not yet'''<br />
<html><a id="FFEDiscussion"></a></html><br />
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=[[File:Kyoto_DiscussionHeader.png]]=<br />
<br />
===Future Works===<br />
<br />
We noticed only flowering and florigen in this time but there are 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.<br />
<br />
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 control all living cells using this technology.<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 />
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[1][http://www.ncbi.nlm.nih.gov/pubmed/15695452 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][http://www.ncbi.nlm.nih.gov/pubmed/16155177 Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/16099980 Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059]<br><br />
[4][http://www.mdpi.com/1424-8247/3/4/961/htm Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993]<br><br />
[5][http://www.ncbi.nlm.nih.gov/pubmed/15147914 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 />
[6][http://www.ncbi.nlm.nih.gov/pubmed/22683878 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][http://www.ncbi.nlm.nih.gov/pubmed/14966662 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][http://www.ncbi.nlm.nih.gov/pubmed/9042754 Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled 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][http://www.ncbi.nlm.nih.gov/pubmed/11854367 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][http://www.ncbi.nlm.nih.gov/pubmed/11123687 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][http://www.ncbi.nlm.nih.gov/pubmed/3139642 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), 4963-4966]<br><br />
[12][http://www.ncbi.nlm.nih.gov/pubmed/14702305 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), 366-373]<br><br />
[13][http://www.ncbi.nlm.nih.gov/pubmed/16099979 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]</div>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-24T16:40:33Z<p>TJR: /* Attribution */</p>
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<br />
===This year, we implemented nine plans on "human practice".===<br />
<br />
Our purpose of these projects is that we let many Japanese people know iGEM and participate in it.<br />
This is why we implemented nine projects.<br />
In order to achieve our purpose, we gave many people lessons the correct knowledge about Genetic Engineering and eliminate the bias toward it at many places.<br />
Besides, we attempted to communicate with local people who live in Kyoto through teaching science, synthetic biology, and "iGEM".<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 still small, so we would like to show how wonderful recombination is to them.<br><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 />
==Application==<br />
[[File:Kyoto_iColi0_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 theme.<br><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 />
<br />
<font size="6">This is why we create "iColi".</font><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 />
<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 />
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<br />
==Education==<br />
[[File:KyotoLecture.jpg|link=|right|300px]]<br />
We went to Hibiya high school and Horikawa high school, and 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<br />
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<br />
==The 3rd international symposium on liberal arts and general education==<br />
[[File:article2.jpg|link=|right|300px]]<br />
<font size="5">We wrote an article about "Japanese Attitude toward Genetic Engineering" </font><br />
<br />
and presented it in the 2nd International Symposium on Liberal Arts and General Education which took place at Clock Tower Centennial Hall in Kyoto University, on November 23, 2011.<br />
Many people starve to death because they are unable to grow enough crops in their impoverished countries. Food shortage has become one of the most serious problems in the world. Some people expect that genetic engineering can solve this problem because genetically modified plants can grow more easily in barren land .<br />
However, some people worry that genetically modified foods may do harm to our health and the environment. It is often reported that Japanese people tend to avoid genetically modified foods. Sure enough, previous surveys of attitudes toward genetic engineering showed that, in Japan, more people had “negative” or “neutral” opinions regarding genetically modified foods than people in other nations.<br />
These findings piqued our interest in the Japanese public’s views on genetic engineering and made clear to us the importance of active discussion on the subject of genetic engineering. In conjunction with other university students in Japan, we designed questionnaires asking for subjects’ impressions of genetic engineering and carried out a nationwide survey in order to clarify the reasons for Japanese people’s attitudes toward the subject .<br />
In this paper, we suggest that educational differences have created a gap between the attitudes of students and adults of their parents generation.<br />
<br />
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<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 who live in Kyoto, 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 />
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<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 Campus.<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 Campus 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 High school==<br />
<br />
[[File:highschool.jpg|link=|left|300px]]<br />
<br style="clear: both;" /><br />
<font size ="4">We began to communicate with students and teachers of Toyonaka high school.</font><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 diffuse 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=|left|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 team have earnestly with each other.<br />
This meeting made our research project sophisticated, and makes 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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which can make our research safer, we got their proposal about safety. They did "Safety Icon Project". They designed icons in safety, which you can tell whether the parts in registry are safety or not at a glance. You can see the explanation of the icons and their meanings [[Team:Kyoto/Cooperation Program|here]]<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: These reagents are used to microscope ''E.coli'' by Confocal laser scanning microscopy. This is usually 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 problem.'''<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 Committee, 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 us 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 supports us.<br><br />
[[File:KUlogo.jpg | link=http://www.kyoto-u.ac.jp/|]]<br><br><br><br />
'''Integrated DNA Technologies (IDT), Japanese branch, Medical & Biological Laboratories Company (MBL), Leave a Nest''' 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 />
[[File:Leave_a_Nest.jpg | link=http://www.cactus.co.jp/aboutus/partners.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>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-10-20T14:31:17Z<p>TJR: /* 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-HumanPractice");<br />
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<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 />
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<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_7.png|left|250px]]<br />
[[File:Kyoto_iColi0_1.png|80px]]<br />
[[File:Kyoto_iColi0_2.png|80px]]<br />
[[File:Kyoto_iColi0_5.png|80px]]<br />
[[File:Kyoto_iColi0_6.png|80px]]<br />
[[File:Kyoto_iColi0_3.png|80px]]<br />
<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. "iColi" is an encyclopedia of E. coli made in iGEM. 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 [[Team:Kyoto/Education|these slides]] in order to teach synthetic biology.<br />
<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 />
Moreover we suggested holding an online meeting among iGEM teams which is in kansai area, and we will practice presentations via Skype.<br />
<br style="clear: both;" /><br />
<br />
==Cooperation with KAIT_Japan on issues of safety. ==<br />
[[Team:KAIT_Japan|KAIT_Japan]] has an issue of safety. When we were trying some program which can make our research safer, we got their proposal about safety. They did " Safety Icon Project". They designed icons in safety, which you can tell whether the parts in registry are safety or not at a glance. You can see the explanation of the icons and their meanigs [[Team:Kyoto/Cooperation Program|here]]<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 problem.'''<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>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:43:54Z<p>TJR: /* 3. Separating R9 peptide and GFP */</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 />
[[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 />
<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>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:42:47Z<p>TJR: /* 3. Separating R9 peptide and GFP */</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 />
[[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|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 Bio Safety Level of our parts.<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 />
<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>TJRhttp://2012.igem.org/File:%E7%AB%B9%E5%86%85%E3%81%95%E3%82%93%E3%81%BE%E3%81%A8%E3%82%81%E3%81%93%E3%82%8C%E3%81%A7%E5%AE%8C%E6%88%90%EF%BC%91.pngFile:竹内さんまとめこれで完成1.png2012-09-27T02:41:17Z<p>TJR: </p>
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<div></div>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:21:50Z<p>TJR: /* Result 3: Construction of Tat cassette */</p>
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<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 />
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<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 />
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[[Image:KyotoTab_Golden.png|link=|GoldenGate]]<html></a></html></li><br />
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<div id="kyoto-tab-Florigen" class="displayOn"><br />
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<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 />
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[[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 />
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<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
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[[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 />
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====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 />
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====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 />
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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 />
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====3. Separating R9 peptide and GFP====<br />
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[[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 />
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[[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 />
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<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 />
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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 />
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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 />
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[[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>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:21:27Z<p>TJR: /* Result 3: Construction of Tat cassette */</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|thumb|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 />
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>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:20:21Z<p>TJR: /* Result 3: Construction of Tat cassette */</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|thumb|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 />
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>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T02:19:14Z<p>TJR: /* Result 3: Construction of Tat cassette */</p>
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<div>[[Image:Header_Kyoto_not_home.jpg|975px|link=Team:Kyoto]]<br />
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<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 />
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<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 />
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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 />
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[[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 />
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[[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 />
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[[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 />
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<html><a name="Secretion"></a></html><br />
<br />
==2.SECRETION==<br />
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[[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 />
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====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]]; '''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 />
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[[File:アイコン3改.png|250px|left| Penetration ]]<br />
====1. Cool system for penetration --R9 peptide-- ====<br />
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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 />
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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 />
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====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 />
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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 />
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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 />
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[[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 />
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====3. Separating R9 peptide and GFP====<br />
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[[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 />
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[[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 />
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<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 />
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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 />
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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 />
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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 />
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[[File:RNA0926.png|thumb|320px|Fig.4-4 Improved RNA waveform]]<br><br />
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[[File:TotalRNA20120926.jpeg|thumb|400px|left|Fig.4-5]]</div><br />
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==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 />
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<html><a id="FFEDiscussion"></a></html><br />
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=[[File:Kyoto_DiscussionHeader.png]]=<br />
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===Future Works===<br />
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<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 />
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<html><a id="FFEReferences"></a></html><br />
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=[[File:Kyoto_ReferencesHeader.png|link=]]=<br />
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[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>TJRhttp://2012.igem.org/File:Wiki_intro_4.pngFile:Wiki intro 4.png2012-09-27T02:16:38Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Team:Kyoto/ConsiderationTeam:Kyoto/Consideration2012-09-27T01:02:07Z<p>TJR: /* Application */</p>
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<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 />
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</html><br />
<br />
=Consideration=<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 />
*Okuno Tetsuro<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 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 />
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</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>TJRhttp://2012.igem.org/File:I.Colicoli.pngFile:I.Colicoli.png2012-09-27T01:00:34Z<p>TJR: </p>
<hr />
<div></div>TJRhttp://2012.igem.org/Team:Kyoto/ProjectTeam:Kyoto/Project2012-09-27T00:55:07Z<p>TJR: /* 1.EXPRESSION */</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 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: evaluation of kil protein====<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><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 />
<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 />
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[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 />
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<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 />
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<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 />
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<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 />
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<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 />
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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 />
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Read more about [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005553 Golden Gate Assembly].<br />
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==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 />
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We amplified plasmid backbone(psB1K3).<br />
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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 />
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[[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 />
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==Software==<br />
We created a software which design primers to create DNA segments for Golden Gate assembly.<br />
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<html><a id="GGADiscussion"></a></html><br />
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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 />
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[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 />
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