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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<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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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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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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==B. Our strategy==
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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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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. So, to make sure that there isn't an early saturation phenomenon which will disturb the equilibrium between the vesiculation process and the protein translocation to the periplasm. That's the reason why we thought of overexpressing the important proteins in the Tat pathway (that is to say TatABCE) in order to avoid this early saturation phenomenon. However, in our strategy, the only protein that needs to use the TAT pathway to translocate from the cytoplasm to the periplasm is clyA. Finally, we decided that the overexpression won't be necessary neither for the TAT pathway, nor for the SEC pathway, both of them constitutively expressed in E Coli K12.
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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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