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.

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.

Expression:FT protein can bloom flowers,but can E.coli make it?

fig.1 There is no guarantee FT protein is

FT, or FLOWER　LOCUS　T, is a protein composed of 175 amino acids. In plants, FT protein is produced in the leaves and transported to shoot apexs. Even at low dose, FT works as a transcription factor and unregulated floral meristem identity genes, thereby making flowers bloom. FT protein can make flowers bloom. Because FT is derived from the eukaryotic cells and we were not sure whether E.coli can synthesize FT protein or not. It might has a harmful effect or be degradated rapidly in E.coli. For these reasons, we try to check the production of FT protein in E.coli

Secretion: Reconstruct and reinforcement Tat system

fig.2 lysis would kill all of Fairies. Flower can't bloom again....

When we determined to make Flower Fairy E.coli, we found that FT protein don’t exit from cytoplasm to surroundings by itself so that we needed to construct a special system for translocate FT protein. In previous iGEM competition, some kinds of parts and devices for protein translocation were already developed. One of the most widely used part is [http://partsregistry.org/Part:BBa_K358004 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 cell-death. Generally speaking, the concentration of E.coli on plant is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies are disappeared, flower wouldn’t bloom.

secretion system

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

1.EXPRESSION

Expression

On the first step; EXPRESSION, E.coli produce florigen inside itself.
When we got FT gene, we had a difficulty in constructing iGEM parts. The first problem is that FT sequence had two cleavage sites of iGEM restriction enzymes. In order to eliminate cleavage sites of iGEM restriction enzymes, we performed Inverse PCR of plasmids with two kinds of primer which have mutation.
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) We constructed the plasmid shown in the Fig.1-3. FT and His tagged FT are regulated by T7 promoter, BBa_I719005 and strong RBS, BBa_B0034.

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

Fig.1-2 Standardization of FT BioBrick. We had mutated and fastened prefix and suffix to FT.

Fig.1-3 FT construction. We had ligated T7 promoter and His tag.

We transformed E.coli and made them express FT gene. In order to confirm the expression of FT protein, we performed western blotting and checked the place of the FT protein band.

fig.1D The result of Western blotting against FT protein.

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2.SECRETION

Secretion

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

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 made 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. Our Secretion team made this protein secretion system and visualized and confirmed its function by using GFP.

What is TAT Secretion pathway?

The Twin Arginate Translocation pathway(TAT) is a secretion system E.coli originally has. This system can carry proteins that have torA signal anino acid sequences at N terminal. TatA, TatB, TatC and TatD compose Tat complex on inner membrane. Tat complex recognizes torA signal peptide and then it transports proteins (with torA) from cytoplasm to periplasm. In addition, proteins that compose TAT pathway cut off the torA signal. Proteins which are secreted by this system don't have tags that obstruct the activation of the protein.

fig.3 without lysis, Fairy product FT protein again and again

Our Tat cassette and kil inducer

TatABC composes a pathway from cytoplasm to periplasm. Kil makes holes in outer membrane and we expect that a protein goes through these holes. Needless to say, the function of outer membrane is essential for E.coli to survive. In other words, overexpression of Kil causes cell death. For this reason, we must find the suitable amount of expression.

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.

Detail of Our Secretion System

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.

Construction

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 week 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(BBa_K797000) and the sequence of pspA (BBa_K797001) individually, and then, we made TAT construction composed of constitutive promoter(BBa_J23107), tatABCD (BBa_K797000),pspA(BBa_K797001) and double terminator (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.

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.

3.PENETRATION

Penetration

1. Cool system for penetration --R9 peptide--

On the third step; PENETRATION. For the purpose of flower formation, FT protein from Flower Fairy E.coli needed to enter into plant cells.

From early stage, we sought various ways to penetrate FT protein into plants, but each way has serious problems. For example, girdling can be used, we think. But, it is difficult to inject FT protein exactly in sieve tube.

fig.3-1 Image of R9 peptide
(powered by winmostar V3.808d, MOPAC2012)

Polyarginine peptide is thought to act on a cell membrane and cause a specific form of endocytosis, that is, macropinocytosis. Macropinocytosis is not caused by an invagination of the cell membrane, but by the growth on the actin mambrane from protrusions into vesicles called macropinosomes, and no receptors are necessary for the process (fig.2). It is reported that plants use CPP to transport biomolecules such as proteins inside the cell, in spite of their cell walls.
Then, we determined to perform penetration by using FT protein with R9 coding region.

fig.3-2 Mechanism of endocytosis by R9 peptide
1.R9 peptides glue to cell membrane of plants because of hydrophobic.
2.Cells response to the stimulus and cause macropinocytosis.
3.FT protein around an invaginating region of cell is taken in the cell.

2. R9 peptide fusion GFP

Pre-experiment

First, we checked the specificity of anti GFP monoclonal antibody.
We characterized the existing GFP generator parts, BBa_I746915.
The parts is consist of T7 promoter 6-his tagged superfolder GFP.

Following figure shows the result of western blotting of [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].
Unfortunately, we used inappropriate molecular marker and could'nt confirm the molecular weights of samples.

fig3-3. Specificity of anti GFP monoclonal antibody. The result of western blotting. Used by GFP monoclonal antibody.
Lane1 : IPTG 0mM, sample 10µL
Lane2 : IPTG 1mM, sample 10µL
Lane3 : IPTG 0mM, sample 5µL
Lane4 : IPTG 1mM, sample 5µL
Lane5 : IPTG 0mM, sample 2µL
Lane6 : IPTG 1mM, sample 2µL

After the 4h of IPTG induction, we noticed that E.coli expressing R9::GFP were growing poorly.

fig3-4.

After the 4h of IPTG induction, we noticed that E.coli expressing R9::GFP were growing poorly

Moreover, we couldn't get any bands of R9::GFP, as shown in the following figure.

Lane1 : Molecular marker
Lane2 : GFP([http://partsregistry.org/Part:BBa_I746915 BBa_I746915]) IPTG 0mM, sample 10µL
Lane3 : GFP([http://partsregistry.org/Part:BBa_I746915 BBa_I746915]) IPTG 1mM, sample 10µL
Lane4 : R9::GFP IPTG 0mM, sample 10µL
Lane5 : R9::GFP IPTG 1mM, sample 10µL

3. Separating R9 peptide and GFP

When R9 and GFP were conected, they didn't work normally. By way of experiment, we separated R9::GFP into two segments and soaked plant cells into a sollution of them. Then we succeeded in penetration by getting the figure of GFP fluoresence.

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.
Fig. Verification of R9 function with use of GFP.

4. Activation

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

FT protein triggers some flowering genes

FT protein increses 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 APETALA 1(AP1), SEPALLATA3(SEP3) and FRUITFULL(FUL). This is because AP1 is the representative protein activated by FT, and SEP3 and FUL activate in leaves. It is difficult for us to handle cells of tips of stems. So we focused on cells of leaves. Leaves' cells are easy to handle for us. We used RT-PCR because of chasing FT protein's function.

This is the result of RT-PCR. The left is control. It is

Achivement

To mutate and standardize FT sequence as a iGEM part.
To confirm expression of FT protein in E.coli.

Future Works

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.

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.

[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
[2]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
[3]Philip A. Wigge et al. "Integration of spatial and temporal information during floral induction in Arabidopsis [4]Sara Trabulo et al.(2010). "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993
[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.
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35
[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
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306
[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
[11]Galán JE, Collmer A.(1999) "Type III secretion machines: bacterial devices for protein delivery into host cells." Science, 284(5418), 1322-8
[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
[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