Team:Paris/Addressing overview2 strategy

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== Overview ==
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* [[Team:Paris/Addressing_overview2#Overview |A. ClyA ]]
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* [[Team:Paris/Addressing_overview2_strategy#Overview |B. Our strategy]]
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* [[Team:Paris/Addressing_overview_Construction#Overview |C. Construction]]
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==Addressing the message in the membrane : Our strategy==
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==B. Our strategy==
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Our strategy is to use clyA to export a protein to the outer-membrane of the cell. The protein fused to clyA will be incorporated into the vesicle during the vesiculation process. ClyA contain the signal peptide needed to be exported form the cytoplasm to the periplasm. The overall idea is to fused the protein of interest to clyA.  
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So in a first time in order to see if our ClyA are in OMVs, we fused it we a RFP, for that we add a poly glycine linker to ClyA biobrick design for improving ClyA-RFP fusion. Moreover if we put RFP before ClyA, it seem that there is more fluorescence than ClyA before RFP under [article 1]. You can see the contruction here [[https://2009.igem.org/Team:Paris/Addressing_overview_Construction#Overview]]
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Then if this test we could replace the RFP by the protein of interest for signal transduction,moreover this system couple to fec system can transduct a signal from outer membran to the cytoplasm.
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|
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<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a>
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Our strategy is to use clyA to export a protein to the outer-membrane of the cell. The protein fused to clyA will be incorporated into the vesicle during the vesiculation process and it's also express on the surface. ClyA contain the signal peptide required for the exportation process from the cytoplasm to the periplasm. The overall idea is to fused the protein of interest to clyA.
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-----pas toucher, j'essaye d'assembler ça et les foutre dans les bonnes partie merci^^----
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So in a first time in order to see if our ClyA are localize in OMVs, we fused it we a RFP. For that we add a poly glycine linker to ClyA biobrick to improve ClyA-RFP fusion. Moreover if we put RFP before ClyA, it seem that there is more fluorescence than ClyA before RFP under <sup>[[Team:Paris/Addressing_overview2#References|[1]]]</sup>. You can see the contruction [https://2009.igem.org/Team:Paris/Addressing_overview_Construction#Overview here]
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Que mamalian cell dans article super important
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One step mechanis which secret protein from cytoplasm to outre membrane ou periplasme a verif
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Ça s exprime a la surface mais peu s accumuler dans le periplsme
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voir plus precisement pourquoi y a test ompa et tolc dans l'article
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Then if this test work, we could replace the RFP by the protein of interest for signal transduction, moreover this system couple to fec system can transduct a signal from outer membran to the cytoplasm.
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Clya devient transmembranaire dans la membrane externe de la vésicule. il a le c term a l intérieur et la partie n term a l ext d' ou la meilleur fluorescense. On peut aussi penser qu on puisse adresser 2 sorte de signal, dans le periplasme si on greff notre protéine d' intérêt en c ter et sur les récepteurs de la membrane externe si on lui greffe la protéine en n ter.
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====References====
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<li>[[Team:Paris/Addressing_overview2#1| ^]]J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66.  [http://www.ncbi.nlm.nih.gov/pubmed/18511069  18511069]</li>
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Latest revision as of 02:59, 22 October 2009

iGEM > Paris > ClyA > Our strategy


Addressing the message in the membrane : Our strategy

Our strategy is to use clyA to export a protein to the outer-membrane of the cell. The protein fused to clyA will be incorporated into the vesicle during the vesiculation process and it's also express on the surface. ClyA contain the signal peptide required for the exportation process from the cytoplasm to the periplasm. The overall idea is to fused the protein of interest to clyA.

So in a first time in order to see if our ClyA are localize in OMVs, we fused it we a RFP. For that we add a poly glycine linker to ClyA biobrick to improve ClyA-RFP fusion. Moreover if we put RFP before ClyA, it seem that there is more fluorescence than ClyA before RFP under [1]. You can see the contruction here

Then if this test work, we could replace the RFP by the protein of interest for signal transduction, moreover this system couple to fec system can transduct a signal from outer membran to the cytoplasm.


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References

  1. ^J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. [http://www.ncbi.nlm.nih.gov/pubmed/18511069 18511069]

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