From 2012.igem.org

Realizing Flower Fairy in real world

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!!
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.

Putting Flower Fairy E.coli on leaves!

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.

We have to go through four steps in order to achieve our goal――Flower Fairy E.coli.
The four steps are composed of “EXPRESSION”,”SECRETION”, ”PENETRATION”, and ”ACTIVATION”

On each step, we have some problems to be solved.
“EXPRESSION”; It is unclear whether E.coli (prokaryote) could express FT protein, because FT protein is derived from the plant cells (eukaryote).
”SECRETION”; After produced, FT protein has to get out of the E.coli.
”PENETRATION”; FT protein has to penetrate into plant cells.
”ACTIVATION”; 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.

1.EXPRESSION

On the first step; EXPRESSION, We needed to make E.coli produce FT protein.

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.

Modifying FT gene for Biobrick

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.

Fig.1-1 Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes.

Fig.1-2 Inverse PCR Method
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.

We constructed the plasmid shown in the Fig.1-4. One was FT gene with T7 promoter [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.

Fig.1-4 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein.

Fig.1-3 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.

Fig.1-5 Verification of expression of FT protein in E.coli. The result of Western blotting against FT and 6 His:FT proteins.
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.
Lane1: FT Cell lysate, not induced
Lane2: FT Cell lysate, IPTG induced
Lane3: 6 His:FT Cell lysate, not induced
Lane4: 6 His:FT Cell lysate, IPTG induced

Confirming expression of FT

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.

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. As a result, FT and 6 His:FT bands were observed at the expected molecular weight region(Fig.1-5).

We succeeded in mutation and expression of FT and 6 His:FT proteins in E.coli

2.SECRETION

On the second step; ‘’’SECRETION’’’, ‘’E.coli’’ secretes florigen outside of the cell.

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.

Transport FT protein into periplasm

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.
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.
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.

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

Evaluation of kil protein

Using Tat secretion pathway, FT protein is transported into periplasm. 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.

Fig.2-2 The construction used in this assay. The Promoter is Lactose promoter R0010

Fig.2-3 How protein goes through two pathways

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.

Fig.2-4 the result of evaluation of kil protein.
Vertical axis means the value of OD 600. And horizontal axis means incubation time.

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.

Construction of Tat cassette

Tat secretion cassette with constitutive promoter [http://partsregistry.org/Part:BBa_K797004 (BBa_K797004)]
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.

Fig.2-5 Tat secretion cassette with constitutive promoter(BBa_K797004)

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.
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)] ,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 that we performed electrophoresis of this cassette and confirmed the length of our parts.

3.PENETRATION

On the third step; PENETRATION. 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.

Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.

However, normally proteins cannot penetrate cell membranes of plants. Therefore, we needed a method to send FT protein into plant cells. 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. This is a type of Cell Penetrating Peptide (CPP). R9 peptide consists of nine arginine residues. (Fig.3-1)

R9 peptide enables FT protein penetrate membranes by endocytosis

Fig.3-2 Mechanism of endocytosis by R9 peptide
1.R9 peptide adheres to cell membrane of plants because of hydrophobic character.
2.Cells response to the stimulus and cause some kind of endocytosis.
3.FT protein around an invaginating region of cell is taken in the cell.

This is the mechanism of how R9 peptides work.(Fig3-2) Firstly, R9 peptides adhere to a cell membrane of a plant because of hydrophobic character. Secondly, cells response to this stimulus and induce endocytosis. Finally, FT proteins near R9 are taken in the cell.

We connected R9 with protein for a high degree of penetration efficiency

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. 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.

We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915]. perhaps because of its cytotoxicity of R9 against E.coli.

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.

Fig.3-4 Western blotting for checking the expression. No band of R9:::GFP fusion protein.
Lane1: Molecular marker
Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced
Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced
Lane4: R9::GFP Cell lysate 10µL, not induced
Lane5: R9::GFP Cell lysate 10µL, IPTG induced

Fig.3-5 RT-PCR of R9::GFP
Lane1:100bp ladder Lane2:GAPDH (Negative control) Lane3:GAPDH(R9::GFP) Lane4:GFP (Negative control) Lane5:GFP (R9::GFP)

We prepare R9 and GFP individually to visualize the R9 peptides

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

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. To verify it, we performed followed experiment.(Fig3-6)

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. Then we succeeded in getting the Figure of GFP fluorescence.(Fig3-7)

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.

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)

We can make FT protein penetrate cell membrane of plants by R9 peptide function.

4. Activation

On the final step; Activation. We verified whether FT normally worked in plant cells.

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.

How to Verify FT Function

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.

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.
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.

We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.
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 because its molecular weight(27kDa) is relatively similar to that of FT(20kDa.)

Fig.4-2 is the result of RT-PCR.

Fig.4-2 6 His tag fusion FT protein was purified with Ni-NTA agarose column.
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample. FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each. Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.) After incubation, leaves were freezed with liquid nitrogen and glinded immediately.
Total RNA was extracted by phenol-chloroform extraction. cDNA was synthesized by reverse transcription and used as templates of RT-PCR. TUBULIN was used for internal control of mRNA expression.
Lane1: TUBULIN (GFP-R9 treated) amplicon 61bp
Lane2: TUBULIN (FT-R9 treated)
Lane3: FUL (GFP-R9 treated) amplicon 132bp
Lane4: FUL (FT-R9 treated)
Lane5: SEP3 (GFP-R9 treated) amplicon 87bp
Lane6: SEP3 (FT-R9 treated)
Lane7: AP1 (GFP-R9 treated) amplicon 958bp

Fig.4-3 Electrophoresis of RNA. RNA concentration is adjusted.
Lane1: RNA of 16h incubated leaves(GFP-R9 treated)
Lane2: RNA of 16h incubated leaves(FT-R9 treated)
Lane3: RNA of fresh leaves

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.

As shown in the Fig.4-3, RNA samples in this time are degradated.
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)
So, it was required to improve the method of RNA extraction.
We improved following things;
1. Total leaf volume was increased.
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.
3. We centrifuged samples and collected supernatant twice after adding ISOGEN.
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.)
Then we used better quality of RNA for reverse transcription and retried RT-PCR. Fig.4-6 shows the result of this RT-PCR.

Fig.4-4 RNA waveform of long time incubated leaves

Fig.4-5 Improved RNA waveform

Fig.4-6

Fig.4-7
Lane1: 100bp ladder
Lane2: TUBULIN
Lane3: FUL
Lane4: SEP3
Lane5: AP1

From these results, we could not confirm the function of FT, upregulation of FUL, SEP3, and AP1. However, now that we can get good quality of RNA and amplify genes successfully, we are trying to check the function of FT.

Achievement

1.Expression

• Mutate FT sequence
• Standardize FT as an iGEM part
• Confirm expression of FT protein in E.coli

2.Secretion

• Modify TorA signal to be easy to use the signal more
• Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator
• Standardize kil gene
  • Multiply TatABC in order to strengthen Tat secretion system--not yet

3.Penetration

• Keep GFP in or around plant cells using R9 peptide
  • Introduce FT in plant cells using R9 peptide--not yet

4.Activation

• Get high quality of RNA
• Amplify genes successfully
  • Check the function of FT--not yet

Future Works

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.

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.

Biosafety

We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.

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