Difference between revisions of "Team:Pasteur Paris/Improve"

 
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<h1>IMPROVED PARTS</h1>
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                <img class="banner-img" src="https://static.igem.org/mediawiki/2018/2/29/T--Pasteur_Paris--Banner_Improve.jpg">
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<div class="block title"><h1>DETECTION OF <i>S. AUREUS</i> BY OUR BIOFILM - <i>agr</i> system</h1></div>
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<div class="block title"><h1>SECRETION OF RNAIII INHIBITING PEPTIDE</h1></div>
 
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We wanted our interface to produce a protein that would inhibit the development of <i>S. aureus</i> in the environment of the implant, but we didn’t want this protein to be secreted continuously and to accumulate outside the biofilm. We wanted our biofilm to start producing growth inhibiting molecules only in the presence of a pathogen.</p>
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We wanted our interface to produce a protein that would inhibit the development of <i>Staphylococcus aureus</i> in the environment of the implant, and we wanted this peptide (RNA III inhibiting peptide) to be secreted from cytoplasm to the extracellular medium.</p>
 
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We decided to use the Biobrick <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_I746100">BBa_I746100</a> from iGEM Cambridge 2007 Team and to improve it by optimizing it for our chassis:  <i>E. coli</i> BL21 strain.  
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We decided to modify the Biobrick <a href="http://parts.igem.org/Part:BBa_K237002" style="font-weight: bold ; color:#85196a;" target="_blank">BBa_K237002</a> from iGEM SDU-Denmark 2009 Team. This Biobrick enables expression of RNAIII inhibiting peptide, we improved it by <b>adding a secretion signal peptide</b> to adress RIP to  <i>Escherichia coli</i> Type II Secretion System and optimizing it for our chassis <i>E. coli BL21 (DE3) pLysS</i> strain.
 
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We engineered the bacteria composing our biofilm by introducing the genes encoding for AgrC and AgrA proteins, the two proteins responsible for the detection of AIPs. They are encoded under the constitutive promotor BBa_J23107, from <a href="https://2006.igem.org/wiki/index.php/Berkeley">iGEM Berkeley 2006 Team</a>.
 
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When AIPs are detected in the environment by the transmembrane protein AgrC, AgrA is phosphorylated and has an increased affinity for the promoter P2. In our engineered bacteria, P2 encodes for a protein called RIP (RNAIII Inhibiting Peptide), that inhibits the formation of a pathogenic biofilm.
 
  
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                  Following the advice of <b>Dr. Jean-Michel Betton</b>, Research Director in the Structural Biology Department at the Institut Pasteur, we chose to try two different signal sequences to export our peptides, which he knew about and which are known to be efficient:  <b>MalE</b> and <b>DsbA</b>. Once in the periplasm, due to the small size of RIP (7 amino acids), a leaky release through the outer membrane of the bacteria should allow us to obtain RIP in the medium [6].
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                <img src="https://static.igem.org/mediawiki/2018/f/fd/T--Pasteur_Paris--BBa_K2616001.png" style="max-width: 800px;">
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<div class="legend"><b>Figure 1: </b> Schematic diagram of BBa_K2616001</div>
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Latest revision as of 14:28, 10 November 2018

""

SECRETION OF RNAIII INHIBITING PEPTIDE

We wanted our interface to produce a protein that would inhibit the development of Staphylococcus aureus in the environment of the implant, and we wanted this peptide (RNA III inhibiting peptide) to be secreted from cytoplasm to the extracellular medium.

We decided to modify the Biobrick BBa_K237002 from iGEM SDU-Denmark 2009 Team. This Biobrick enables expression of RNAIII inhibiting peptide, we improved it by adding a secretion signal peptide to adress RIP to Escherichia coli Type II Secretion System and optimizing it for our chassis E. coli BL21 (DE3) pLysS strain.

Following the advice of Dr. Jean-Michel Betton, Research Director in the Structural Biology Department at the Institut Pasteur, we chose to try two different signal sequences to export our peptides, which he knew about and which are known to be efficient: MalE and DsbA. Once in the periplasm, due to the small size of RIP (7 amino acids), a leaky release through the outer membrane of the bacteria should allow us to obtain RIP in the medium [6].

Figure 1: Schematic diagram of BBa_K2616001