Team:Kyoto/Secretion/Project

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Revision as of 09:37, 23 September 2012

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 Proteins having torA signal anino acid sequences at N terminal. TatA, TatB, TatC 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.

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"

@@ Line 1: / Line 1: @@
 {{Kyoto/css}}
 {{Kyoto/header}}
-==Introduction==
+==abstruct==
-===How We iGEMer Extract Proteins from Escherichia.coli?===
+======
-<p>Until now, we use only cell death as secretion pathway. It is true that protein in the E.coli emerges when an E.coli die and lysed. However, it is not smart that whenever we need protein synthesized by E.coli we have to kill the E.coli.</p>
+<p>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. </p>
-===Lysis is ...?===
+===Introduction~We need secretion system without cell-death===
-<p>When many many E.coli are exist on environment, it is no problem that we kill a bit E.coli, but when a little E.coli are exist, E.coli might be annihilated if we force them die through introducing lysis genes. When there are little nutriment in the system's environment, E.coli cannot breed. And if there are nutriment in the system's environment, E.coli would consume that and we would have to supply nutriment constantly. In order to maintain your system with E.coli, you must grow E.coli genetically engineered by yourself. We want to save our E.coli when we extract protein, but how ?</p>
+<p> 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） </p>
-===What is TAT Secretion System?===
+===What is TAT Secretion pathway===
-<p>The Twin Arginate Translocation pathway(TAT) is secretion system and E.coli have this system originally. Proteins named tatA, tatB, tatC, and tatD make holes on inner membrane and recognize a signal. Tat ABCD 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 activation of the protein.</p>
+<p>The Twin Arginate Translocation pathway(TAT) is secretion system E.coli originally have. This system can carry proteins Proteins having torA signal anino acid sequences at N terminal. TatA, TatB, TatC 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.</p>
-<p>TAT secretion system make holes on inner membrane. It means protein with torA tag goes to only periplasmic space, not outside of outer membrane. We want to extract proteins from E.coli, not to translocate it into periplasm. We must make a hole on outer membrane in order to extract protein in periplasm.</p>
-===From Periplasm to Outside===
+===Our Tat cassette and kil inducer===
 <p>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.</p>