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Team:Paris/Production overview strategy
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===B. Our strategy=== | ===B. Our strategy=== | ||
| - | + | 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. | ||
| - | 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 | + | 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. | ||
| + | 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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Revision as of 15:16, 10 October 2009
iGEM > Paris > Production > Overview > Our Strategy
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. 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.
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. In the same framework, we could also over express various Tol ligand (like colicin) to destabilize the membrane. But it doesn’t work.
References
- ^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]
- ^Improved methods for producing outer membrane vesicles in gram-negative bacteria. Henry & Lloubès 2004 - [http://www.ncbi.nlm.nih.gov/pubmed/15249060 15249060]
