Abstruct

Even though our fairies can produce FT protein, there remains a big issue: 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 lets E.coli carry proteins with torA signal via Tat protein transportation pathway from cytoplasm to periplasm and Kil protein encourage proteins to move from periplasm to surroundings. We made this protein secretion system and visualized and confirmed its functioning by using GFP.

Introduction

We need secretion system without cell-death

In previous iGEM competition, some kinds of parts and devices for protein translocation were already developed. One of the most widely used parts is lysis cassette(このパーツのページのリンク). This part causes cell lysis and, as a result, makes E.coli scatter materials it includes. This style is, unfortunately, not suitable for our project because of the possibility of accidental all-death. Generally speaking, the concentration of E.coli on flower is not high so that it can’t be ignored that the possibility of occurring all cell-death. Once all our Fairies disappears, flower wouldn’t bloom. In addition to that, lysising E.coli is NOT CUTE.

We tried to seek for a ideal secretion systems, which is not harmful and dangerous for both of our fairies and other livings. Finally, We focus on Twin argenine translocation pathway (Tat pathway) of E.coli. It is not pathogenic and carries various kinds of proteins with torA signal（Tat班古い方のレビュー）

What is TAT Secretion pathway

The Twin Arginate Translocation pathway(TAT) is secretion system E.coli originally have. 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 protein (with torA) from cytoplasm to periplasm. In addition, protein that has passed through TAT pathway cut off the torA signal. Proteins which are secreted by this system have no tag that obstruct the activation of the protein.

Our Tat cassette and kil inducer

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

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 dameged. PspA meintains H+ concentration gradient between periplasm and cytoplasm and membrane potential.

Our secretion system makes many holes on 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 come to be able to maintain the vitality, though they have many holes on the membrane.

Method

Observation with confocal microscope

• PFA
• MilliQ
• Hoechst
• Slide glass

1. Gather fungus body by centrifugal separation
   
2. Add PFA to each microtubes containing fungus body and resuspend them
   

Results

Construction

Tat secretion cassette with constitutive promoter(BBa_K797004)

This cassette allow E.coli to secrete proteins with torA signal. Wild type Tat protein secretion system is too week so that Kyoto 2012 construct Tat cassette to reinforce the ability of transportation of Tat system. This parts include tatA,B,C protein coding region and pspA (phage shock protein A). Tat A,B,C protein is the main component of Tat complex where proteins with torA signal go through and pspA can encourage protein secretion via Tat system. We suggest iGEMers with this new way of secretion and provide them with this cassette regulated by constitutive promoter.

We checked the sequence of tatABCD(BBa_K797000　このページへリンク)and the sequence of pspA(BBa_K797001　このページへリンク) individually, and then, we made TAT construction composed of constitutive promoter(BBa_J23107　このページへリンク), tatABCD(BBa_K797000　このページへリンク), pspA(BBa_K797001　このページへリンク) and double terminator(BBa_B0015　このページへリンク). 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.

Discussion

References

[1]Tracy Palmer and Ben C. Berks "The twin-arginine translocation (Tat) protein export pathway"
[2]J. H. Choi. S. Y. Lee "Secretory and extracellular production of recombinant proteins using Escherichia coli"
[3]G. Miksch · E. Fiedler · P. Dobrowolski · K. Friehs "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"
[4]Brad A. Seibel* and Patrick J. Walsh "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage"