Difference between revisions of "Team:INSA-UPS France/test"

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{{INSA-UPS_France/Style_new}}
 
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sup{
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}
  
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    <section style="margin-top:200px;">
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</style>
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      <h1 style="text-align: left;margin-top:-110px;font-size:5vw;letter-spacing: 1vw;">Design</h1>
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      <img style="width:25%;min-width: 260px; position:absolute;right:0;top:-200px; " src="https://static.igem.org/mediawiki/2017/e/e3/T--INSA-UPS_France--design_croco.png" alt="">
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      <p style="margin-top: 50px;">
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        Building a synthetic consortium able to deal with the cholera issue led us to investigate on different communication pathways: we had to <b>sense</b> <i>Vibrio cholerae</i> in its natural environment and based on this sensing we had to activate, through <b>transmission</b>, a <b>response</b>: the production of a killing molecule. Moreover, all those actions had to be inserted in the right cellular chassis in order to optimize the system.
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      </p>
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      <p>
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        To wipe out cholera from water, we decided to build a sense-transmit-respond system reacting to <i>V. cholerae</i> and leading to its death.
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      </p>
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    </section>
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    <section style="background: none;">
 
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      <h1 style="text-align:left;">Overview</h1>
 
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      <img src="https://static.igem.org/mediawiki/2017/b/b8/T--INSA-UPS_France--description_loop.png" alt="" style="width:100%;">
 
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    <section>
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  <main class="site-main">
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      <h1 style="text-align: left;">Organisms</h1>
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      <h2><i>E. coli</i></h2>
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      <p>
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        We chose to mimic <i>V. cholerae</i> using <i>E. coli</i> that we modified to produce the CAI-1 of <i>V. cholerae</i>, using the enzyme responsible for its production. <i>E. coli </i> has two main advantages: it is a good molecular biology model and it produces no endogen CAI-1.
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      </p>
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      <img src="https://static.igem.org/mediawiki/2017/f/fc/T--INSA-UPS_France--design_plasmid-coli.png" alt="" style="width: 10%; position:absolute;bottom:0; left:10%;">
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      <p style="margin-left:15%;">
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        The psB1C3 plasmid was chosen for iGEM compatibility.
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      </p>
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    </section>
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    <section>
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      <h2><i>V. harveyi</i></h2>
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      <p>
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        <i>V. harveyi</i> has all the assets to be a good sensor for <i>V. cholerae</i>: it possesses its own pathway of detection of C8-CAI-1, an analogue of CAI-1. A single point mutation allows <i>V. harveyi</i>: to detect <i>V. cholerae</i>’s molecule CAI-1<sup>1</sup>. Moreover, the strain that we are using, JMH626, has been deleted for the enzyme responsible of the production and detection of other quorum sensing molecules, making it a specific sensor of CAI-1<sup>2</sup>. However it cannot be used as  the effector because of its physiological proximity to <i>V. cholerae</i>. Thus we supposed that the production of antimicrobial peptides aimed at<i> V. cholerae</i> would be lethal to it.
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      </p>
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      <img src="https://static.igem.org/mediawiki/2017/f/fa/T--INSA-UPS_France--design_plasmid-harveyi.png" alt="" style="width: 10%; position:absolute;bottom:0; left:10%;">
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      <p style="margin-left:15%;">
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        The pBBR1MCS-4 broad host range plasmid was chosen so we can transfer the system into <i>V.harveyi</i> using a conjugation method.
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      </p>
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    </section>
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    <section>
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      <h2><i>P. pastoris</i></h2>
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      <p>
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        Recognized as a great protein producer and secretor, <i>P. pastoris</i> has already been used to produce a wide range of AMPs<sup>3,4</sup>. Furthermore, the diacetyl/Odr-10 system has been described as a useful tool for prokaryotic/eukaryotic communication<sup>5</sup>.
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      </p>
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      <img src="https://static.igem.org/mediawiki/2017/6/67/T--INSA-UPS_France--design_plasmid-pichia.png" alt="" style="width: 10%; position:absolute;bottom:0; left:10%;">
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      <p style="margin-left:15%;">
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      The pPICZ&alpha; plasmid was chosen because of its &alpha;-factor and its homology sequence allowing it to integrate in a targeted zone in its genome. It is recognized as a good plasmid for protein production in <i>P. pastoris</i>.
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      </p>
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    </section>
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    <h1 style="font-family: 'Quicksand', sans-serif;font-size:34pt;text-align: left;margin:20px 10%;">Modules &amp; Parts</h1>
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      <img src="https://static.igem.org/mediawiki/2017/a/a8/T--INSA-UPS_France--design_blupuriline.png" alt="">
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       <h1>Page under construction</h1>
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     <section class="modules_design" style="border:solid 5px #ae3d3d;margin-top:0px;margin-bottom: 0px;">
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       <h2 style="color:#ae3d3d;">Sense</h2>
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      <p>
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        We chose to take advantage of the intraspecies quorum sensing of <i>V. cholerae</i>: the <b>CAI-1/CqsS system</b>. To mimic this pathway in our laboratory, we had to both produce in vivo the CAI-1 molecule  in a bacteria strain and express the CqsS receptor in an other one.
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      </p>
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      <p>
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        The CAI-1 producing system is inducible in order to avoid toxicity problems. The fact that <b>CqsS*</b> can detect both CAI-1 and C8-CAI-1 led us to choose <i>V. harveyi</i> <b>cqsA gene</b> (Vh-cqsA), instead of <i>V. cholerae</i> gene, that produces C8-CAI-1 for safety reasons.<sup>1</sup>
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      </p>
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     </section>
 
     </section>
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    <section class="modules_design" style="border:solid 5px #468789;margin-top:0px;margin-bottom: 0px;">
 
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      <h2 style="color:#468789;">Transmit</h2>
 
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      <p>
 
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        <i>V. harveyi</i> has the natural pathway leading to the activation or inactivation of <b>pqrr4</b>. At high CAI-1 concentration the promoter is inactivated, thus we needed an inverter, <b>tetR/pTet</b> allowed us to activate the als gene that produces diacetyl at high CAI-1 concentration:
 
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      </p>
 
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      <ul>
 
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        <li>
 
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          If <b>CAI-1</b> is present in high concentration, pqrr is repressed, so TetR no longer inhibits pTet. Thus, the als gene is expressed, producing <b>>diacetyl</b>.</li>
 
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        <li>
 
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          If there is <b>no CAI-1</b>, TetR is produced and repress pTet which inhibits the <b>als gene</b>, ergo diacetyl production.
 
  
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        </li>
 
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      </ul>
 
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      <p>
 
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        We chose to use the <b>diacetyl/Odr-10 binding receptor system</b>, that is known to activate gene expression on yeasts.
 
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      </p>
 
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      <p>
 
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        The constitutive <b>pGAP</b> promoter allows the system to always express the <b>Odr-10 receptor</b> and thus be sensible to diacetyl at any time.
 
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      </p>
 
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    </section>
 
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    <img class="invisible-image" src="https://static.igem.org/mediawiki/2017/8/81/T--INSA-UPS_France--img_vide.png" alt=""  style="width:30%;">
 
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    <section class="modules_design" style="border:solid 5px #f37b6f;margin-top:0px;margin-bottom: 0px;">
 
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      <h2 style="color:#f37b6f;">Respond</h2>
 
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      <p>
 
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        Once the Odr-10 receptor has sensed diacetyl, <b>pFUS</b> is activated and it triggers the Ste12 pathway. Then, the production of antimicrobial peptides (AMP) can start. In order for the cells to excrete the peptides, an <b>&alpha;-factor</b> is needed.
 
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      </p>
 
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    </section>
 
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    <img class="invisible-image" src="https://static.igem.org/mediawiki/2017/8/81/T--INSA-UPS_France--img_vide.png" alt=""  style="width:30%;">
 
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    </div>
 
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    <section style="padding-left:20%;">
 
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      <h1 style="text-align: left;">Experimental plan</h1>
 
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      <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--design_coli.png" alt="" style="width:15%; position:absolute; top:10px; left:10px;">
 
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      <h2><i>E. coli</i></h2>
 
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      <p>
 
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        Quorum sensing molecule production
 
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      </p>
 
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      <ul>
 
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        <li>C8-CAI-1 &amp; CAI-1 NMR</li>
 
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        <li>Bioluminescence</li>
 
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        <li>MS</li>
 
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      </ul>
 
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    </section>
 
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    <section style="padding-left:20%;">
 
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      <img src="https://static.igem.org/mediawiki/2017/5/5c/T--INSA-UPS_France--design_harveyi.png" alt="" style="width:15%; position:absolute; top:10px; left:10px;">
 
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      <h2><i>V. harveyi</i></h2>
 
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      <p>
 
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        Conjugation
 
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      </p>
 
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      <ul>
 
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        <li>Conjugation test with fluorescence</li>
 
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        <li>CqsS* pathway test with fluorescence</li>
 
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      </ul>
 
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      <p>
 
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        diacetyl production
 
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      </p>
 
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      <ul>
 
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        <li>diacetyl NMR (<i>E.coli</i> and <i>V. harveyi</i>)</li>
 
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        <li>pTet characterization in <i>V. harveyi</i> by reporter gene</li>
 
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      </ul>
 
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    </section>
 
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    <section style="padding-left:20%;">
 
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      <img src="https://static.igem.org/mediawiki/2017/2/2d/T--INSA-UPS_France--design_pichia.png" alt="" style="width:15%; position:absolute; top:10px; left:10px;">
 
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      <h2><i>P. pastoris</i></h2>
 
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      <p>
 
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        Antimicrobial peptides (AMP)
 
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      </p>
 
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      <ul>
 
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        <li>AMP activity: growth tests, etc</li>
 
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        <li>AMP purification</li>
 
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      </ul>
 
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      <p>
 
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        diacetyl detection
 
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      </p>
 
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      <ul>
 
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        <li>pFus pathway test with fluorescence</li>
 
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        <li>etc</li>
 
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      </ul>
 
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    </section>
 
 
     <!-- fin section -->     
 
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Revision as of 22:19, 24 September 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Page under construction