Abstruct

Even though our fairies can produce FT protein, there remains a big issue: how they can transport proteins to outside their cells? To make it possible, we made Tat cassette and kil protein inducer. This cassette let 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 confirmed and visualized it 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 part 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 are disappeared, 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 carry 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 is make Tat complex on inner membrane and recognize a signal. Tat complex recognize torA signal peptide and then they let protein (with torA) pass. In addition, protein that has passed through TAT pathway, cut off the torA signal. Proteins which secreted by this system have no tag that obstruct the activation of the protein.

Our Tat cassette and kil inducer

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

Detail of Our Secretion System

We constructed a wonderful secretion system contains tatABCD, Kil, and an other gene. PspA(phage-shock protein A) is the gene that E.coli have originally and this gene expressed when their inner membrane is dameged. PspA meintains H+ concentration gradient between periplasm and cytoplasm and membrane potential.

We make many holes on inner and outer membranes. In other words, E.coli which has our secretion system is under the membrane stress conditions. Introducing pspA into our Flower Fairy E.coli, the E.coli come to be able to tolerate that having many holes on the membrane.

Method

Results

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"