From 2012.igem.org

Realize 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. 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!

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.

Easy to use

When you want to use our Flower Fairy E.coli, there is just one thing you must do to get ready. Put it on leaves! The switch is R9 peptide. When you add R9 peptide, FT secreted by Flower Fairy E.coli penetrates cell walls and the plant blooms.

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 has 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.

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

1.EXPRESSION

On the first step; EXPRESSION, E.coli produce florigen inside it.

We wanted E.coli to produce FT protein which is a plants' protein. At first, we needed make E.coli product FT protein witch is a plant protein. There was, however, no guarantee that E.coli can make FT protein. We could not find such research papers as long as we could. Perhaps FT is toxic for E.coli or is delegated in E.coli easily. So we needed to make a new Biobrick of FT protein and to check whether E.coli can produce it. We modified FT gene sequences FT sequence had two cleavage sites of iGEM restriction enzymes and checked the FT expression by Western Blotting.

Modifying FT gene for Biobrick

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

Professor Araki kindly gave us FT cDNA of Arabidopsis thaliana in TOPO blunt end 2(Invitrogen). When we got FT gene, we had 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 mismatch between primer and cDNA.

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.

Inverse PCR is a kind of PCR with the use of retrorse primers. This PCR enables us to mutate FT because tensile direction is reverse. As a result, we could get mutated plasmids, which are not cleaved by iGEM restriction enzymes.

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 purify protein from E.coli. FT and 6 His tagged FT are regulated by T7 promoter, [http://partsregistry.org/Part:BBa_I719005 BBa_I719005] and strong RBS, [http://partsregistry.org/Part:BBa_B0034 BBa_B0034].

Fig.1-3 FT construction. T7 promoter maximizes FT transcriptive activity and 6 His tag enables us to purify FT protein.

Fig.1-4 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.1-3 was transformed into BL21(DE3). Cells were precultured overnight and diluted into fresh SOC medium. IPTG was added when OD600 was approx. 0.5, 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

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. We succeeded in 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? To make it possible, we tried to make 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.

Modified TorA signal

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.

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 Cambrige 2011[http://partsregistry.org/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!

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.

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. 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

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. FT protein from Flower Fairy E.coli needs to enter into plant cells in order to induce plants to bloom.

From an early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems.
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.

Fig.3-1 Image of R9 peptide

the cool system of penetration-R9 peptide

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.
R9 peptide adheres to cell membrane of plants because of hydrophobic character. The cell responds to the stimulus and leads to macropinocytosis. FT protein around an invaginating region of the cell is taken in the cell.

Plants by themselves practice CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.
Then, we determined to cause penetration of FT protein with R9 peptide.

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 macropinocytosis.
3.FT protein around an invaginating region of cell is taken in the cell.

Can E.coli express R9::GFP?

Fig.3-3 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, and then cells were incubated for 4h at 37°C. 100µL of culture was used for SDS-PAGE.
Lane1: Cell lysate 10µL, not induced
Lane2: Cell lysate 10µL, IPTG induced
Lane3: Cell lysate 5µL, not induced
Lane4: Cell lysate 5µL, IPTG induced
Lane5: Cell lysate 2µL, not induced
Lane6: Cell lysate 2µL, IPTG induced

We needed to checked function of R9. Thereby we didn't know whether E.coli expressed R9:GFP, we needed to check it by specific anti GFP monoclonal antibody against GFP. Then we tried to detect specificity of antigen-antibody reaction against GFP and R9:GFP protein to perform Western blotting.

We used the existing GFP generator parts, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].
The parts is consist of T7 promoter 6 his tagged superfolder GFP.
At first, we check the specificity of GFP antibody. (Unfortunately, we used inappropriate molecular marker and couldn't confirm the molecular weights of samples.)

Each samples induced with IPTG showed one main band and one extra band, and not-induced controls showed one bands. The relation of intensity of bands between induced samples and controls is corresponded to the existence of IPTG.
Minor bands of induced samples are considered as the degraded GFP. From this result, we concluded that GFP antibody has enough specificity.

Fig.3-4 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.

After verifying the activity of GFP antibody, We checked the expression of R9::GFP by using the same way(fig) After the 4h of IPTG induction, we noticed that the color of medium withE.coli expressing R9::GFP was different from medium with E.coli express GFP.(fig ) This figure suggests the expression of R9::GFP was growing poorly. The result of Western blotting support the little producing of R9::GFP.(fig ). Even though GFP induced in the same condition was detected,we couldn't get any bands of R9::GFP

Fig.3-6 RT-PCR of R9-GFP
Lane1:100bp ladder Lane2:GAPDH (Negative control) Lane3:GAPDH(R9-GFP) Lane4:GFP (Negative control) Lane5:GFP (R9-GFP)

Fig.3-5 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

We suspected that R9-GFP is not transcribed in mRNA, so we did RT-PCR using GFP specific primers.
GAPDH is used as internal control. The result is shown in fig .There ware the specific band of GFP transcription. As a result, we concluded that R9-GFP mRNA is expressed, but not translated in proteins, perhaps because of its cytotoxicity　of R9 against E.coli.

3. GFP can penetrate membranes with R9 peptide

Fig.3-7 Preparation for sample verifying R9 function 1.Cutting leaves from Arabidopsis thaliana 2.Scratching cuticle

When R9 and GFP were connected, E.coli can't produce R9 fused protein. Then for the purpose of the function of R9, we compared R9+ to R9-. We scratched the cuticle of plant cells and soaked them into a solution of GFP and R9 or only GEP. This GFP protein was purified with 6 His tag. After 5 minutes we washed cells by PBS in order to wash GFP and R9peptide away. Then we succeeded in getting the Figure of GFP fluorescence.

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.

Fig.3-8 Verification of the R9 function with use of GFP.

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) (With confocal microscope Fluoview FU10i OLYMPUS)

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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