Team:Paris/Production overview strategy

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<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Production_overview#top | Production]] > [[Team:Paris/Production_overview#bottom | Overview]] > [[Team:Paris/Production_overview_strategy#overview | Our Strategy]]
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<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Production_overview#top | Vesicle production system]] > [[Team:Paris/Production_overview_strategy#overview | Our Strategy]]
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===B. Our strategy===
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==B. Our strategy==
We want to destabilize the outer membrane to create outer membrane vesicles (OMVs).   
We want to destabilize the outer membrane to create outer membrane vesicles (OMVs).   
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The first strategy is the knock out of tol/pal gene witch destabilizes the membrane. It's an active system, whereas the use of conditional mutant is a passive system. A previous study [2] focused on the development of a gene expression system able to induce production of large amounts of OMVs. They used different domains of two protein of Tol-Pal system. This team [2] sends us their plasmid and we were able to begin our work quickly.
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The first strategy is the knock out of tol/pal gene witch destabilizes the membrane. It's an active system, whereas the use of conditional mutant is a passive system. <font color=yellow>Que veux tu dire par actif et passif? Il faut expliquer</font>
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A previous study<sup><span id="1">[[Team:Paris/Production_overview_strategy#References|[1]]]</span></sup> focused on the development of a gene expression system able to induce production of large amounts of OMVs. They used different domains of two protein of Tol-Pal system. This team <sup><span id="1">[[Team:Paris/Production_overview_strategy#References|[1]]]</span></sup> send us their plasmid and we were able to begin our work quickly.
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To achieve our goal, we decided to take advantage of the second domain of TolR (TolRII). <font color=yellow> Tu ne peux pas dire ca sans expliquer d'abord qu'il ya plusieurs domaines dans TolR et quelles sont leur fonctions... Il faut expliquer pourquoi ca peut être intéressant d'exprimer uniquement des domaines de TolR ou TolA; dire qui a déjà fait ca et quels ont été leur résultats... Il faut aussi expliquer pourquoi dans les constructions on utilise ompAS</font> We don’t use the third domain of TolA because, it doesn’t work so well and TolAIII have lot of PstI domain in his sequence. In order to achieve our project, we will over express specifically designed biobricks containing TolRII fused with OmpA signal which allows it to migrate in the periplasm<sup><span id="1">[[Team:Paris/Production_overview_strategy#References|[2]]]</span></sup>. So Tol-Pal system will become bad and the membrane integrity will be destabilised. Lot of vesicle could be creating.  
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To achieve our goal, we decided to take advantage of the second domain of TolR (TolRII). We don’t use the third domain of TolA because, it doesn’t work so well and TolAIII have lot of PstI domain in his sequence. In order to achieve our project, we will over express specifically designed biobricks containing TolRII fused with OmpA signal which allows it to migrate in the periplasm[3]. So Tol-Pal system will become bad and the membrane integrity will be destabilised. Lot of vesicle could be creating.
 
If bacteria stay in this conformation, there is lysis. We try to create an ON/OFF system to stop the vesicles creation.
If bacteria stay in this conformation, there is lysis. We try to create an ON/OFF system to stop the vesicles creation.
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In the same framework, we could also over express various Tol ligand (like colicin) to destabilize the membrane. But it doesn’t work.
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In the same framework, we could also over express various Tol ligand (like colicin) to destabilize the membrane. But it doesn’t work. <font color=yellow> tu ne peux pas faire une affirmation comme ca sans aucune explication ni référence...</font>
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====References====
====References====
<ol class="references">
<ol class="references">
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<li> [[Team:Paris/Production_overview_strategy#2 | ^]]The Tol-Pal proteins of the Escherichia coli cell envelope an energized system required for outer membrane integrity. Lloubès & Journet 2001 - [http://www.ncbi.nlm.nih.gov/pubmed/11501670 11501670]</li>
 
<li> [[Team:Paris/Production_overview_strategy#3 | ^]]Improved methods for producing outer membrane vesicles in gram-negative bacteria. Henry & Lloubès 2004 - [http://www.ncbi.nlm.nih.gov/pubmed/15249060 15249060]</li>
<li> [[Team:Paris/Production_overview_strategy#3 | ^]]Improved methods for producing outer membrane vesicles in gram-negative bacteria. Henry & Lloubès 2004 - [http://www.ncbi.nlm.nih.gov/pubmed/15249060 15249060]</li>
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<li> [[Team:Paris/Production_overview_strategy#2 | ^]]The Tol-Pal proteins of the Escherichia coli cell envelope an energized system required for outer membrane integrity. Lloubès & Journet 2001 - [http://www.ncbi.nlm.nih.gov/pubmed/11501670 11501670]</li>
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Latest revision as of 16:05, 18 October 2009

iGEM > Paris > Vesicle production system > Our Strategy

Overview

B. Our strategy

We want to destabilize the outer membrane to create outer membrane vesicles (OMVs).


The first strategy is the knock out of tol/pal gene witch destabilizes the membrane. It's an active system, whereas the use of conditional mutant is a passive system. Que veux tu dire par actif et passif? Il faut expliquer

A previous study[1] focused on the development of a gene expression system able to induce production of large amounts of OMVs. They used different domains of two protein of Tol-Pal system. This team [1] send us their plasmid and we were able to begin our work quickly.


To achieve our goal, we decided to take advantage of the second domain of TolR (TolRII). Tu ne peux pas dire ca sans expliquer d'abord qu'il ya plusieurs domaines dans TolR et quelles sont leur fonctions... Il faut expliquer pourquoi ca peut être intéressant d'exprimer uniquement des domaines de TolR ou TolA; dire qui a déjà fait ca et quels ont été leur résultats... Il faut aussi expliquer pourquoi dans les constructions on utilise ompAS We don’t use the third domain of TolA because, it doesn’t work so well and TolAIII have lot of PstI domain in his sequence. In order to achieve our project, we will over express specifically designed biobricks containing TolRII fused with OmpA signal which allows it to migrate in the periplasm[2]. So Tol-Pal system will become bad and the membrane integrity will be destabilised. Lot of vesicle could be creating.


If bacteria stay in this conformation, there is lysis. We try to create an ON/OFF system to stop the vesicles creation. In the same framework, we could also over express various Tol ligand (like colicin) to destabilize the membrane. But it doesn’t work. tu ne peux pas faire une affirmation comme ca sans aucune explication ni référence...


References

  1. ^Improved methods for producing outer membrane vesicles in gram-negative bacteria. Henry & Lloubès 2004 - [http://www.ncbi.nlm.nih.gov/pubmed/15249060 15249060]
  2. ^The Tol-Pal proteins of the Escherichia coli cell envelope an energized system required for outer membrane integrity. Lloubès & Journet 2001 - [http://www.ncbi.nlm.nih.gov/pubmed/11501670 11501670]

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