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

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       <h1 style="vertical-align:bottom;display:table-cell; width:70%;font-size:60pt;letter-spacing: 0.2em;z-index:120;text-align: center;">Description</h1>
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       <h1 style="vertical-align:bottom;display:table-cell; width:70%;font-size:60pt;letter-spacing: 0.2em;z-index:120;text-align: center;">Parts</h1>
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     <h1>New parts submitted to the registry</h1>
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      <div class="aside-nav__item">
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    <table>
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       <a href="#a1" data-number="1">
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       <tr>
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         Synthetic biology
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         <th>Name</th>
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      </a>
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        <th>Function</th>
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      </div>
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        <th>Type</th>
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       <div class="aside-nav__item">
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        <th>Part</th>
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       <a href="#a2" data-number="2">
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        <th>State</th>
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         A microbial consortium chassis against cholera
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       </tr>
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      </a>
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       <tr>
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       </div>
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        <td><a href="">BBa_K2278001</a></td>
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       <div class="aside-nav__item">
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        <td>QS molecule generator </td>
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      <a href="#a3" data-number="3">
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        <td>basic </td>
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         Mimicking <i>Vibrio cholerae</i>
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        <td>
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       </a>
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          <img src="https://static.igem.org/mediawiki/parts/4/40/T--INSA-UPS_France--K2278001.png" alt="">
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         </td>
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      <div class="aside-nav__item">
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        <td>working</td>
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      <a href="#a4" data-number="4">
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       </tr>
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         The sensing organism: <i>Vibrio harveyi</i>
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       <tr>
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       </a>
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        <td><a href="">BBa_K2278002</a></td>
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       </div>
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        <td>QS molecule generator</td>
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      <div class="aside-nav__item">
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        <td>basic</td>
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      <a href="#a5" data-number="5">
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        <td><img src="https://static.igem.org/mediawiki/parts/5/51/T--INSA-UPS_France--K2278002.png" alt=""></td>
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         The effecting organism: <i>Pichia pastoris</i>
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         <td>not released</td>
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       </a>
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       </tr>
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       </div>
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       <tr>
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      <div class="aside-nav__item">
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        <td><a href="">BBa_K2278011</a></td>
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      <a href="#a6" data-number="6">
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        <td>Diacetyl generator </td>
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         Our system
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        <td>basic</td>
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      </a>
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        <td>
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       </div>
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          <img src="https://static.igem.org/mediawiki/parts/0/00/T--INSA-UPS_France--K2278011.png" alt="">
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       <div class="aside-nav__item">
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         </td>
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      <a href="#a7" data-number="7">
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        <td>issues </td>
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         References
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       </tr>
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      </a>
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       <tr>
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       </div>
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        <td><a href="">BBa_K2278021</a></td>
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     </div>
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        <td>D-NY15 AMP generator </td>
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    </div>
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        <td>basic</td>
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        <td>
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          <img src="https://static.igem.org/mediawiki/parts/f/f9/T--INSA-UPS_France--K2278021.png" alt="">
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         </td>
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        <td>Working </td>
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       </tr>
 +
       <tr>
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        <td><a href="">BBa_K2278022</a></td>
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        <td>Leucrocin I AMP generator</td>
 +
        <td>basic</td>
 +
        <td>
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          <img src="https://static.igem.org/mediawiki/parts/2/2b/T--INSA-UPS_France--K2278022.png" alt="">
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         </td>
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        <td>unsuccessful </td>
 +
       </tr>
 +
       <tr>
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        <td><a href="">BBa_K2278023</a></td>
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        <td>coT2 AMP generator</td>
 +
        <td>basic</td>
 +
        <td>
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          <img src="https://static.igem.org/mediawiki/parts/a/aa/T--INSA-UPS_France--K2278023.png" alt="">
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         </td>
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        <td>unsuccessful</td>
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       </tr>
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     </table>
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     <h1>Existing Parts we have contributed to characterized</h1>
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     <h2><a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></h2>
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    <section>
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      <h1>Synthetic biology: to the multi-organisms communication and beyond </h1>
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    <figure>
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      <p>
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       <img src="https://static.igem.org/mediawiki/parts/1/1e/T--INSA-UPS_France--Vh1.png" alt="">
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        Nature is still developing a wide large diversity of remarkably efficient pathways in order to sense presence of specific chemical, or even physical parameters such as temperature, pressure and light<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/" target="_blank">1</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/26308982" target="_blank">2</a></sup>. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature&rsquo;s tricks, basically by inserting genetic information from microorganisms into a single and unique one, most of the time <i>Escherichia coli</i><sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/24686414" target="_blank">3</a></sup>. However, focusing only on this type of bacteria is not appropriate to reflect the large complexity of living organisms and more, their intimate relationship in Nature. This aspect starts to be a limiting border in the way of the development of the synthetic biology<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/" target="_blank">4</a></sup>. 
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       <figcaption>
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      </p>
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        BBa_J04450 biobrick conjugated in <i>Vibrio harveyi</i>
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      <p>
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       </figcaption>
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        Then, our iGEM project focused on a multi organisms communication pathway, especially between prokaryotic and eukaryotic cells. Thus, we developed a strategy using a cascade of events from a sensor cell (<i>Vibrio harveyi</i>) to an effector cell (<i>Pichia pastoris</i>) in order to detect and eradicate a <i>Vibrio cholera</i> mimicking cell (<i>Escherichia coli</i>) using an antimicrobial peptides from crocodile.
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    </figure>
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      </p>
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       <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
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    <p>
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       <h2>Genesis of our molecular strategy</h2>
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      BBa_J04450 was tested in the <i>Vibrio harveyi</i> background. BBa_J04450 biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into <i>Vibrio harveyi</i> to demonstrate the production of RFP in this chassis.  
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       <p>
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    </p>
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        During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen<sup><a href="http://www.emro.who.int/yem/yemeninfocus/situation-reports.html" target="_blank">5</a></sup>. Actually, the bacteria <i>Vibrio cholerae</i> that causes cholera disease is usually found in water and infects more than a million of people each year. This terrible situation led us to focus on this problematic and it appeared that current solutions were not efficient enough to deal with this situation.  
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      </p>
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    <p>
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      <p>
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      <b>To learn more: </b> <a href="http://parts.igem.org/Part:BBa_J04450">http://parts.igem.org/Part:BBa_J04450</a>
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        Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with <i>Vibrio cholerae</i><sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/16697733" target="_blank">6</a>,<a href="http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079" target="_blank">7</a></sup>. Additionally, some iGEM teams tried also to deal with the challenging detection of <i>V. cholerae</i><sup><a href="https://2014.igem.org/Team:UI-Indonesia" target="_blank">8</a>,<a href="https://2010.igem.org/Team:Sheffield" target="_blank">9</a>,<a href="https://2014.igem.org/Team:UT-Dallas" target="_blank">10</a></sup>, using <i>E. coli</i>. They based their strategy around the quorum sensing system of <i>V. cholerae</i> in order to detect it, implementing CqsS receptor and the LuxU/O pathway into <i>E. coli</i> in order to activate gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough maybe due to the process complexity of introducing a large amount of DNA information in a single microorganism. That is why we built a synthetic consortium of microorganism against <i>Vibrio cholerae</i>.
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    <h2><a href="http://parts.igem.org/Part:BBa_K431009">BBa_K431009</a>: glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)</h2>
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  </section>
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  <section>
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    <h1>Parts used in our project but not submitted to the registry </h1>
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      <h1>A microbial consortium chassis against cholera
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</h1>
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      <p>
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        We finally created an artificial consortium chassis to deal with cholera disease. The different partners are described below.
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      <ul>
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        <li>
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          To mimic <i>V. cholerae</i> by producing CAI-1 molecule. This will be done in <i>E. coli</i>
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        </li>
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        <li>
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          A bacteria with a quorum sensing pathway activated on the <i>V. cholerae</i> presence on which CAI-1 bind. <i>Vibrio harveyi</i> naturally possess that pathway. It will lead to the production of a messenger molecule: that we choose to be diacetyl.
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        </li>
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        <li>
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          The diacetyl binds to the Odr-10 receptor that can be expressed on yeast such as <i>Pichia pastoris</i> and start a molecular pathway.This pathway lead to the activation of pFUS1 and will produce our antimicrobial peptide with a secretion cassette. Those peptides will kill <i>Vibrio cholerae</i>.
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      <h1>Mimicking <i>Vibrio cholerae</i> using <i>Escherichia coli</i></h1>
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        An interesting property of <i>Vibrio cholerae</i> is its quorum sensing autoinducer system based on the production of CAI-1 molecule<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/22001326" target="_blank">11</a></sup>. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is a good reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain <i>Escherichia coli</i> in order to mimic <i>V. cholerae</i>. Thus, we transformed <i>E. coli</i> strain with the CqsA synthase coding gene of <i>Vibrio harveyi</i>, non-pathogen bacteria. CqsA from <i>V. harveyi</i> produces an analog of CAI-1, the molecule C8-CAI-1, from (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A12. Finally, we developed an <i>E. coli</i> strain which produces a marker simulating the presence of the pathogen <i>V. cholerae</i> in the medium. This is the first step of our molecular cascade.
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      <h1>The sensing organism: <i>Vibrio harveyi</i></h1>
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      <p>
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        Once we developed <i>E. coli</i> to produce the <i>V. harveyi</i> C8-CAI-1, this molecule as to be detected in the medium. The easiest way to do it is to use directly the quorum sensing of the non-pathogen <i>V. harveyi</i>. This bacteria is an advantageous good engineerable chassis. We identified that <i>V. harveyi</i> already possesses gene expression depending on the binding of C8-CAI-1 on its receptor, CqsS<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">12</a></sup>. For example, pQRR4 is a promoter which activation depends on the presence of C8-CAI-1. To fit with CAI-1 molecule, the CqsS receptor of <i>V. harveyi</i> only needed to be mutated on a single amino acid. We only had to mutate CqsS changing the phenylalanine 175 into a cystein and to integrate the ALS gene under the control of pQRR4 to trigger diacetyl production in presence of CAI-1<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">12</a></sup>.
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      <figure>
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        <figcaption>
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        <b>Cascade of events depending on the CAI-1/CqsS binding in V. cholerae<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">12</a></sup></b>. the CAI-1/CqsS binding will start a dephosphorylation cascade leading to the inhibition of pQRR4 and its depending siRNA. The lack of his siRNA will allow the translation of their targeted mRNA.
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      <p>
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        We checked the metabolism of diacetyl of <i>V. harveyi</i> on KEGG Pathway, and identified that the acetolactate synthase (ALS) alone allowed the production of diacetyl from pyruvate, a ubiquitous metabolite.This is the second step of our molecular cascade.
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      <figure>
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        <img src="https://static.igem.org/mediawiki/parts/0/0a/T--INSA-UPS_France--ALSpathway.png" alt="">
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        <figcaption>
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          <b>Production of diacetyl from pyruvate.</b> The addition of the acetolactate synthase (ALS) can lead to the production of acetolactate which convert itself into diacetyl without enzymatic process.
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      <h1>The effecting organism: <i>Pichia pastoris</i></h1>
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        The molecular response to the presence of the mimicking vibrio <i>E. coli</i> strain is the production by <i>V. harveyi</i> of diacetyl. We then need a third partner to produce toxic molecule to kill <i>V. cholerae</i>. This last partner needs to be resistant to the toxic molecule so we choose an eukaryotic cell. Team SCUT<sup><a href="https://2013.igem.org/Team:SCUT" target="_blank">13</a></sup> previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, the Odr-10 receptor<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/" target="_blank">14</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/2302121" target="_blank">15</a></sup>. It is a G Protein Coupled Receptor isolated from <i>Caenorhabditis elegans</i> that once activated by diacetyl, lead to the activation of the pFUS1 promoter by Ste12. <i>Pichia pastoris</i> has been chosen as it displays already the Odr-10/pFUS1 pathway.  
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        <img src="https://static.igem.org/mediawiki/2017/2/24/T--INSA-UPS_France--Description-communicate.png" alt="">
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        <figcaption>
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          <b>Activation cascade on the dependence of Diacetyl/Odr-10 binding<sup><a href="https://2013.igem.org/Team:SCUT" target="_blank">13</a></sup></b>. Once diacetyl bind to Odr-10 a cascade of activation of Ste proteins will lead to the binding of Ste12 on pFUS1 promoter, and so to the expression of gene of interest.
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        Moreover, <i>P. pastoris</i> is a good protein producing organism<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27905091" target="_blank">16</a>,<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/" target="_blank">17</a></sup>. We engineered the yeast to secret the toxic molecule under the promoter of Ste12. The toxic molecule secreted by <i>P. pastoris</i> is originated from crocodiles<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21184776" target="_blank">18</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/2059789" target="_blank">19</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460" target="_blank">20</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554" target="_blank">21</a></sup>. Crocodiles display a remarkable and efficient immune system, allowing the reptiles to resist to a large spectrum of diseases. Thus, they produced antimicrobial peptides (AMPs) which are able to lyse bacteria such as <i>V. cholerae</i>. AMPs are cationic pore-forming molecules targeting bacterium membranes, causing bacterial lysis and death<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27837316" target="_blank">22</a></sup>. This is the third step of our cascade.
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          <b>Mechanism of action of antimicrobial peptide and their effects on cells<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27837316" target="_blank">22</a></sup>. </b> Antimicrobial peptides are making pore formation into the membrane leading to death of the cell. Transmission electron microscopy provide an insight of the effect of the peptide on the cell.
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          <b>efficiency of the antimicrobial peptide from crocodile on V. cholerae<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21184776" target="_blank">18</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460" target="_blank">20</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554" target="_blank">21</a></sup></b>.The three peptides display and minimal inhibitory concentration 50 in the scale of mg/L.
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        See our <a href="https://2017.igem.org/Team:INSA-UPS_France/Design">Design page</a> for more informations of the genetic engineering we used!
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      <h1>References</h1>     
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554">https://www.ncbi.nlm.nih.gov/pubmed/24192554</a>
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    </section>
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                  $('*[data-number="4"]').parent().removeClass("selected-item");
 
−
                  $('*[data-number="5"]').css("font-weight", "normal");
 
−
                  $('*[data-number="5"]').parent().removeClass("selected-item");
 
−
                  $('*[data-number="6"]').css("font-weight", "normal");
 
−
                  $('*[data-number="6"]').parent().removeClass("selected-item");
 
−
                  $('*[data-number="7"]').css("font-weight", "bold");
 
−
                  $('*[data-number="7"]').parent().addClass("selected-item");
 
−
                }
 
−
              }
 
−
            }
 
−
          }
 
−
        }
 
−
      }
 
−
    }
 
−
    // FIXED ASIDE NAV
 
−
    if(pos>371){
 
−
      $('.left_container').addClass("aside-fixed");
 
−
    }
 
−
    else{
 
−
      $('.left_container').removeClass("aside-fixed");
 
−
    }
 
−
  });
 
  
−
</script>
 
  
−
<script type="text/javascript">
+
<!-- C O N T E N T -->
  
−
    // NAV BAR SUBNAV CLICK
+
</html>
  
−
  $(document).on("click",".cat_name", function(){
 
−
    if($(this).parent().children('.subnav').css("max-height")=="0px"){
 
−
      $('.subnav').css("max-height","0px");
 
−
      $(this).parent().children('.subnav').css("max-height","1000px");
 
−
    }
 
−
    else{
 
−
      $(this).parent().children('.subnav').css("max-height","0px");
 
−
    }   
 
−
  });
 
−
</script>
 
−
 
−
<script type="text/javascript">
 
−
  var contentSections = $('.article_offset'),
 
−
  navigationItems = $('.left_container .aside-nav__item:after');
 
−
 
−
  updateNavigation();
 
−
  $('.main_content').on('scroll', function(){
 
−
    updateNavigation();
 
−
    });
 
−
 
−
 
−
  function updateNavigation() {
 
−
    contentSections.each(function(){
 
−
      $this = $(this);
 
−
 
−
      var activeSection = $('.left_container a[href="#'+$this.attr('id')+'"]').data('number') - 1;
 
−
    });
 
−
  }
 
−
 
−
  function smoothScroll(target) {
 
−
        $('body,html').animate(
 
−
          {'scrollTop':target.offset().top},
 
−
          600
 
−
        );
 
−
  }
 
−
</script>
 
−
 
−
</body>
 
−
</html>
 
 
{{INSA-UPS_France/General_script}}
 
{{INSA-UPS_France/General_script}}
 
{{INSA-UPS_France/Header_script}}
 
{{INSA-UPS_France/Header_script}}

Revision as of 15:40, 22 October 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Parts

New parts submitted to the registry

Name Function Type Part State
BBa_K2278001 QS molecule generator basic working
BBa_K2278002 QS molecule generator basic not released
BBa_K2278011 Diacetyl generator basic issues
BBa_K2278021 D-NY15 AMP generator basic Working
BBa_K2278022 Leucrocin I AMP generator basic unsuccessful
BBa_K2278023 coT2 AMP generator basic unsuccessful

Existing Parts we have contributed to characterized

BBa_J04450

BBa_J04450 biobrick conjugated in Vibrio harveyi

BBa_J04450 was tested in the Vibrio harveyi background. BBa_J04450 biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into Vibrio harveyi to demonstrate the production of RFP in this chassis.

To learn more: http://parts.igem.org/Part:BBa_J04450

BBa_K431009: glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)

Parts used in our project but not submitted to the registry