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

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{{INSA-UPS_France/Style_new}}
 
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{{INSA-UPS_France/Header_new}}
 
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<html>
 
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     <section class="home_section home" id="home" style="position:relative;">
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     <section style="margin-top:200px;">
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       <div class="container">
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      <h1 style="text-align: left;margin-top:-110px;font-size:5vw;letter-spacing: 1vw;">Design</h1>
 +
      <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="">
 +
       <p style="margin-top: 50px;">
 +
        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.
 +
      </p>
 +
      <p>
 +
        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.
 +
      </p>
 +
    </section>
  
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      <!-- FIRST IMAGE TO ANIMATE -->
 
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      Croc'n Cholera
 
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    </div>
 
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    <div style="width:100%;position:absolute;font-size:40pt; color:#295a7f;top:70pt;">
 
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      A synthetic microbial consortium
 
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  </div>
 
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  </div>
 
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    <img src="https://static.igem.org/mediawiki/2017/2/2a/T--INSA-UPS_France--Logo.png" alt="" style="width:150px; position:absolute; bottom:20px; left:20px;">
 
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  <div style="position:absolute;bottom:30px; left:170px;font-size:30pt;font-weight:bold;">
 
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    iGEM UPS-INSA Toulouse 2017
 
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  </div>
 
  
 +
    <section style="background: none;">
 +
      <h1 style="text-align:left;">Overview</h1>
 +
      <img src="https://static.igem.org/mediawiki/2017/b/b8/T--INSA-UPS_France--description_loop.png" alt="" style="width:100%;">
 +
    </section>
  
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       </div>
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    <section>
 +
      <h1 style="text-align: left;">Organisms</h1>
 +
      <h2><i>E. coli</i></h2>
 +
      <p>
 +
        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.
 +
      </p>
 +
      <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%;">
 +
      <p style="margin-left:15%;">
 +
        The psB1C3 plasmid was chosen for iGEM compatibility.
 +
      </p>
 +
    </section>
 +
 
 +
 
 +
   
 +
    <section>
 +
      <h2><i>V. harveyi</i></h2>
 +
      <p>
 +
        <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.
 +
      </p>
 +
      <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%;">
 +
      <p style="margin-left:15%;">
 +
        The pBBR1MCS-4 broad host range plasmid was chosen so we can transfer the system into <i>V.harveyi</i> using a conjugation method.
 +
      </p>
 +
    </section>
 +
   
 +
    <section>
 +
      <h2><i>P. pastoris</i></h2>
 +
      <p>
 +
        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>.
 +
      </p>
 +
      <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%;">
 +
      <p style="margin-left:15%;">
 +
      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>.
 +
       </p>
 
     </section>
 
     </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>
 +
 +
    <div class="left_container">
 +
    <div class="left_container__inside">
 +
      <img src="https://static.igem.org/mediawiki/2017/a/a8/T--INSA-UPS_France--design_blupuriline.png" alt="">
 +
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 +
    </div>
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     <div class="right_container">
  
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      <h2 style="color:#ae3d3d;">Sense</h2>
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            <div id="progress-bar_inner">
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      <p>
−
              &nbsp;
+
        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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            </div>
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      </p>
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            <div class="progress-point point-1"></div>
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      <p>
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            <div class="progress-point point-2"></div>
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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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            <div class="progress-point point-4"></div>
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    </section>
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          </div>
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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="">
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          <div class="bubble" id="bubble-1">
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    <section class="modules_design" style="border:solid 5px #468789;margin-top:0px;margin-bottom: 0px;">
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            <p>First steps of synthetic biology</p>
+
      <h2 style="color:#468789;">Transmit</h2>
−
          </div>
+
      <p>
−
          <div class="bubble" id="bubble-2">
+
        <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:
−
            <p>First edition of the iGEM competition</p>
+
      </p>
−
          </div>
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      <ul>
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          <div class="bubble" id="bubble-3">
+
        <li>
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            <p>Boolean algebra in <i>E. coli</i></p>
+
           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>
−
          </div>
+
         <li>
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          <div class="bubble" id="bubble-4">
+
          If there is <b>no CAI-1</b>, TetR is produced and repress pTet which inhibits the <b>als gene</b>, ergo diacetyl production.
−
            <p style="margin-bottom: 0 !important;">Current challenges:</p>
+
−
            <ul style="margin-top: 0 !important;">
+
−
              <li>Cell-free</li>
+
−
              <li>De novo life</li>
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              <li>Multi organisms</li>
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              </ul>
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           </div>
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         </div>
+
  
 +
        </li>
 +
      </ul>
 +
      <p>
 +
        We chose to use the <b>diacetyl/Odr-10 binding receptor system</b>, that is known to activate gene expression on yeasts.
 +
      </p>
 +
      <p>
 +
        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.
 +
      </p>
 
     </section>
 
     </section>
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      <h2 style="color:#f37b6f;">Respond</h2>
 +
      <p>
 +
        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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      <h2><i>E. coli</i></h2>
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         Quorum sensing molecule production
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        <li>C8-CAI-1 &amp; CAI-1 NMR</li>
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        Conjugation
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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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          The living world offers a lot of possibilities, but their use is still limited in synthetic biology.
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         diacetyl production
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          Our idea is about taking advantages of the characteristics of single microorganisms by making them communicate with each other to get the appropriate response.
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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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      <h2><i>P. pastoris</i></h2>
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        Antimicrobial peptides (AMP)
 +
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        <li>AMP activity: growth tests, etc</li>
 +
        <li>AMP purification</li>
 +
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 +
      <p>
 +
        diacetyl detection
 +
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      <ul>
 +
        <li>pFus pathway test with fluorescence</li>
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        <li>etc</li>
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        <h1>A fight against cholera as an application</h1>
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          <i>V. cholerae</i> is responsible for cholera, a disease that contaminates water and affects people in developing countries, war zones and natural disaster zones.
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        Our project is about creating a multi organisms system that is able to sense and wipe cholera out in contaminated water.
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Revision as of 20:09, 21 September 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017


Design

Building a synthetic consortium able to deal with the cholera issue led us to investigate on different communication pathways: we had to sense Vibrio cholerae in its natural environment and based on this sensing we had to activate, through transmission, a response: 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.

To wipe out cholera from water, we decided to build a sense-transmit-respond system reacting to V. cholerae and leading to its death.

Overview

Organisms

E. coli

We chose to mimic V. cholerae using E. coli that we modified to produce the CAI-1 of V. cholerae, using the enzyme responsible for its production. E. coli has two main advantages: it is a good molecular biology model and it produces no endogen CAI-1.

The psB1C3 plasmid was chosen for iGEM compatibility.

V. harveyi

V. harveyi has all the assets to be a good sensor for V. cholerae: it possesses its own pathway of detection of C8-CAI-1, an analogue of CAI-1. A single point mutation allows V. harveyi: to detect V. cholerae’s molecule CAI-11. 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-12. However it cannot be used as the effector because of its physiological proximity to V. cholerae. Thus we supposed that the production of antimicrobial peptides aimed at V. cholerae would be lethal to it.

The pBBR1MCS-4 broad host range plasmid was chosen so we can transfer the system into V.harveyi using a conjugation method.

P. pastoris

Recognized as a great protein producer and secretor, P. pastoris has already been used to produce a wide range of AMPs3,4. Furthermore, the diacetyl/Odr-10 system has been described as a useful tool for prokaryotic/eukaryotic communication5.

The pPICZα plasmid was chosen because of its α-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 P. pastoris.

Modules & Parts

Sense

We chose to take advantage of the intraspecies quorum sensing of V. cholerae: the CAI-1/CqsS system. 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.

The CAI-1 producing system is inducible in order to avoid toxicity problems. The fact that CqsS* can detect both CAI-1 and C8-CAI-1 led us to choose V. harveyi cqsA gene (Vh-cqsA), instead of V. cholerae gene, that produces C8-CAI-1 for safety reasons.1

Transmit

V. harveyi has the natural pathway leading to the activation or inactivation of pqrr4. At high CAI-1 concentration the promoter is inactivated, thus we needed an inverter, tetR/pTet allowed us to activate the als gene that produces diacetyl at high CAI-1 concentration:

  • If CAI-1 is present in high concentration, pqrr is repressed, so TetR no longer inhibits pTet. Thus, the als gene is expressed, producing >diacetyl.
  • If there is no CAI-1, TetR is produced and repress pTet which inhibits the als gene, ergo diacetyl production.

We chose to use the diacetyl/Odr-10 binding receptor system, that is known to activate gene expression on yeasts.

The constitutive pGAP promoter allows the system to always express the Odr-10 receptor and thus be sensible to diacetyl at any time.

Respond

Once the Odr-10 receptor has sensed diacetyl, pFUS 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 α-factor is needed.

Experimental plan

E. coli

Quorum sensing molecule production

  • C8-CAI-1 & CAI-1 NMR
  • Bioluminescence
  • MS

V. harveyi

Conjugation

  • Conjugation test with fluorescence
  • CqsS* pathway test with fluorescence

diacetyl production

  • diacetyl NMR (E.coli and V. harveyi)
  • pTet characterization in V. harveyi by reporter gene

P. pastoris

Antimicrobial peptides (AMP)

  • AMP activity: growth tests, etc
  • AMP purification

diacetyl detection

  • pFus pathway test with fluorescence
  • etc