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

 
(98 intermediate revisions by 5 users not shown)
Line 1: Line 1:
−
{{INSA-UPS_France/CSS}}
+
{{INSA-UPS_France/Links_new}}
−
{{INSA-UPS_France/Links}}
+
{{INSA-UPS_France/Style_new}}
 +
{{INSA-UPS_France/Header_new}}
  
−
<!-- HEADER -->
 
  
−
{{INSA-UPS_France/Header1}}
 
−
Description
 
−
{{INSA-UPS_France/Header2}}
 
−
 
−
<!-- CONTENT -->
 
 
<html>
 
<html>
  
−
<style>
+
<!-- C O N T E N T -->
−
p{
+
−
font-size: 20px !important;
+
−
font-family: "Scope One", serif !important;
+
−
}
+
−
  .img_center, .image_50, .image_60, .image_80, .image_90{
+
−
    display:block;
+
−
    margin:0px auto;
+
−
  }
+
−
  .image_50{
+
−
    width:50%;
+
−
  }
+
−
  .image_60{
+
−
    width:60%;
+
−
  }
+
−
  .image_80{
+
−
    width:80%;
+
−
  }
+
−
  .image_90{
+
−
    width:90%;
+
−
  }
+
−
  .first_image_container{
+
−
    background:none;
+
−
    text-align:center;
+
−
    margin:0px auto !important;
+
−
  }
+
−
  .first_image_container img{
+
−
    height:600px;
+
−
  }
+
−
  .anchor_offset{
+
−
    position:relative;
+
−
    top:-70px;
+
−
  }
+
 
    
 
    
−
   .table_img{
+
   <main class="site-main">
−
    width:100%;
+
   <div class="main_content">
−
  }
+
   <div class="middle_container">
−
  .table_img td{
+
−
    text-align: center;
+
−
   }
+
−
   sup a{
+
−
    color:#68829e;
+
−
    text-decoration: none;
+
−
    font-weight: bold;
+
−
  }
+
−
  sup a:hover, sup a:active, sup a:visited{
+
−
    color:#505160;
+
−
  }
+
−
  ol li{
+
−
    padding-left: 20px;
+
−
    font-style: italic;
+
−
  }
+
−
  ol li a{
+
−
    color:#68829e;
+
−
    font-weight: bold;
+
−
  }
+
−
  ol li a:hover, ol li a:active, ol li a:visited{
+
−
  color:#505160;
+
−
  }
+
−
  .not_italic{
+
−
    font-style: normal !important;
+
−
  }
+
−
  table td, table tr, table{
+
−
  border:none !important;
+
−
  }
+
−
</style>
+
  
−
   <div class="container_section" >
+
   <div class="section_container">
−
  <section>
+
−
 
+
−
  <div class="article_container no_aside first_image_container">
+
−
    <img src="https://static.igem.org/mediawiki/2017/b/b8/T--INSA-UPS_France--Description_croco_map.png" alt="">
+
−
  </div>
+
  
−
  <div class="article_offset" id="a1">
+
    <section style="display:table;background:none;padding:0px !important;z-index:100; ">
−
  <div class="article_container">
+
      <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>
−
  <h3>Cholera: still a widespread disease</h3>
+
      <img style="vertical-align:bottom;display:table-cell; width:100%;" src="https://static.igem.org/mediawiki/2017/e/e8/T--INSA-UPS_France--description_croco.png" alt="">
−
  <article>
+
    </section>
−
    <p>
+
−
      <b>Cholera is a worldwide diarrheal disease, caused by the ingestion of <i>Vibrio cholerae</i> in contaminated water.</b> Nowadays cholera is still occurring in developing countries, war zones and natural disasters zones. The WHO reported over <b>1 million cases over the year 2015</b> and the mortality was around 1%<sup><a href="#ref1">1</a></sup>. In April 2017, a <b>cholera epidemic burst in Yemen</b>. In August, more than 500,000 cases have already been identified. These epidemics and crisis are sometimes still surpassing the abilities of the non-governmental organizations to help populations<sup><a href="#ref2">2</a></sup> in  the long-term. Drinking water shortages and the lack of hygienic facilities in developing countries are the main reasons explaining current outbreaks.
+
  
−
     </p>
+
    <style>
−
  </article>
+
    /* ASIDE NAV */
−
  </div>
+
      .left_container{
−
  </div>
+
        width:12%;
 +
        position:absolute;
 +
        top:300px;
 +
        padding:0;
 +
        display:inline-block;
 +
        font-family: 'Quicksand', sans-serif;
 +
        font-weight: 400;
 +
        font-size:11pt;
 +
        text-align: right;
 +
      }
 +
 
 +
      .aside-nav__item + .aside-nav__item{
 +
        margin-top:20px;
 +
      }
 +
      .aside-nav__item{
 +
        width:100%;
 +
        position:relative;
 +
      }
 +
      .aside-nav__item:after{
 +
        content:'';
 +
        border:solid #3377a8 2px;
 +
        position:absolute;
 +
        right:-30px;
 +
        top:40%;
 +
        width:10px;
 +
        height:10px;
 +
        border-radius: 10px;   
 +
        margin-left:20px;
 +
      }
 +
      .aside-nav__item.selected-item:after{
 +
        background:#3377a8;
 +
      }
 +
      .aside-fixed{
 +
        position:fixed;
 +
        top: 120px;
 +
        width: 7%;
 +
      }
 +
      .aside-nav__item a, .aside-nav__item a:focus, .aside-nav__item a:visited, .aside-nav__item a:hover{
 +
        text-decoration: none;
 +
        color:black;
 +
      }
 +
      .aside-nav__item a:hover{
 +
        font-weight: 700;
 +
      }
 +
      section img{
 +
        max-width: 400px;
 +
      }
 +
     </style>
 +
 
 +
    <div class="left_container">
 +
    <div class="left_container__inside">
 +
      <div class="aside-nav__item">
 +
      <a href="#a1" data-number="1">
 +
        Synthetic Consortia
 +
      </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
        <a href="#a2" data-number="2">
 +
          Genesis of our molecular strategy
 +
        </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
      <a href="#a3" data-number="3">
 +
        A microbial consortium chassis against cholera
 +
      </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
      <a href="#a4" data-number="4">
 +
        Mimicking <i>Vibrio cholerae</i>
 +
      </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
      <a href="#a5" data-number="5">
 +
        The sensing organism: <i>Vibrio harveyi</i>
 +
      </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
      <a href="#a6" data-number="6">
 +
        The effecting organism: <i>Pichia pastoris</i>
 +
      </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
      <a href="#a7" data-number="7">
 +
        Our system
 +
      </a>
 +
      </div>
 +
      <div class="aside-nav__item">
 +
      <a href="#a8" data-number="8">
 +
        References
 +
      </a>
 +
      </div>
 +
    </div>
 +
    </div>
 +
 
 +
    <style>
 +
    .right_container{
 +
        width:83%;
 +
        margin-left:17%;
 +
    }
 +
    .article_offset{
 +
      margin-bottom:20px;
 +
      border: solid 1px transparent;
 +
    }
 +
    </style>
 +
 
 +
   
 +
    <div class="right_container">
  
−
  <div class="article_offset" id="a2">
+
    <div class="article_offset" id="a1"></div>
−
  <div class="article_container">
+
     <section>
−
    <h3>Current solutions</h3>
+
       <h1>Synthetic biology: from unique chassis to synthetic consortia</h1>
−
     <article>
+
−
       <h4>Therapeutics</h4>
+
 
       <p>
 
       <p>
−
        While direct preventive methods such as vaccination are currently used, they have been shown to have low efficiency<sup><a href="#ref3">3</a></sup>. The most used treatment is the <b>Oral Rehydratation Solution (ORS)</b>, composed of salts and glucose in order to fight the extreme loss of water due to cholera. It can be drunk or injected intravenously depending of the patient and his symptoms<sup><a href="#ref4">4</a></sup>.
+
      Synthetic biology is based on Nature everlasting possibilities, usually by inserting genetic information from microorganisms into a single and unique chassis<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>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/24686414" target="_blank">3</a></sup>. However, a single chassis could present inevitable limits (high genetic burden, incompatibility of some elements with the chassis, too complex design…). These start to be a limit in synthetic biology, its perspectives and applications<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/" target="_blank">4</a></sup>. An emerging solution is the use of synthetic consortium (figure 1). Synthetic consortium have the advantage to require less amount of genetic information into a single chassis to achieve the required process since different microorganisms can share the genetic burden. Moreover, the components of the consortium could be selected to reduce the genetic modifications and increase the chance of success, for example, by taking advantage of already existing signaling or metabolic pathways. Several successes of those synthetic consortia, such as production of bio-electricity<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27596754/" target="_blank">5</a></sup> or shortening bio-manufacturing process like C-vitamin synthesis<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/26809519/" target="_blank">6</a></sup> have provided insight into the strength of this approach.  
 
       </p>
 
       </p>
−
      <table class="table_img">
+
<figure>
−
        <tr>
+
      <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
−
          <td>
+
        <figcaption>
−
            <img src="https://static.igem.org/mediawiki/2017/c/c8/T--INSA-UPS_France--Description-therapeutics_1.png" alt="" style="height:200px;">
+
        <b> Figure 1: General concept of the cell to cell communication. </b> This cascade of events will be developed in our project.
−
          </td>
+
         </figcaption>
−
          <td>
+
       </figure>
−
            <img src="https://static.igem.org/mediawiki/2017/1/1d/T--INSA-UPS_France--Description-therapeutics_2.png" alt="" style="height:200px;">
+
−
          </td>
+
−
         </tr>
+
−
       </table>
+
−
 
+
−
      <h4>Water treatment</h4>
+
 
       <p>
 
       <p>
−
        Moreover, most of the detection or purification methods need professionals to be performed<sup><a href="#ref3">3</a></sup>. Currently the most efficient ways to eradicate  cholera from water include sodium hypochlorite treatment or filters use. These existing prevention methods are expensive and difficult to set up. Finally, the main curative method, rehydrating patient with intravenous salted water, does not wipe out the disease vector. Even if the ORS treatment is really efficient, it would be more convenient for people to use prevention methods. However people living  in remote villages don&rsquo;t have easily access to these systems and it can take them days to reach a camp to be cured. Thus, new methods of prevention and treatment have to be developed.
+
Such approaches are still rare in the iGEM competition, maybe because they require to combine classic strain engineering with information processing strategies. So, our challenge was to demonstrate the power and feasibility of synthetic consortium approach to open new perspectives and applications to iGEMers.
−
        That&rsquo;s why we decided to design  a system that treats water and is suitable for these situations.
+
−
 
+
 
       </p>
 
       </p>
−
      <table class="table_img">
 
−
        <tr>
 
−
          <td>
 
−
            <img src="https://static.igem.org/mediawiki/2017/3/36/T--INSA-UPS_France--Description-treatment_1.png" alt="" style="height:200px;"">
 
−
          </td>
 
−
          <td>
 
−
            <img src="https://static.igem.org/mediawiki/2017/3/39/T--INSA-UPS_France--Description-treatment_2.png" alt="" style="height:200px;"">
 
−
          </td>
 
−
          <td>
 
−
            <img src="https://static.igem.org/mediawiki/2017/d/d6/T--INSA-UPS_France--Description-treatment_3.png" alt="" style="height:200px;"">
 
−
          </td>
 
−
        </tr>
 
−
      </table>
 
−
 
−
      <h4>Research effort in synthetic biology</h4>
 
 
       <p>
 
       <p>
−
        Recently, research teams started to focus on synthetic biology in order to find a way to deal with <i>Vibrio cholerae</i><sup><a href="#ref5">5</a>,<a href="#ref6">6</a></sup>. Additionally, some iGEM teams also took the challenge of detecting <i>V. cholerae</i>, using <i>E. coli</i> as a host for the quorum sensing system, but it seems that this strategy did not succeed<sup><a href="#ref7">7</a></sup> or showed mixed results<sup><a href="#ref8">8</a></sup>. Most of the iGEM teams intended to prevent the cholera infection thanks to <b>gene targeting</b> such as cleaving toxicity genes<sup><a href="#ref9">9</a></sup> or inhibiting specific genes on the pathogen<sup><a href="10">10</a></sup>.  
+
      As a proof of concept, we developed a strategy against cholera. It is based on a cascade of events starting from an engineered <i>Escherichia coli</i> strain mimicking <i>Vibrio cholerae</i>. It triggers a sensor bacterium, <i>Vibrio harveyi</i>, which in turn activates the effector cell (the yeast <i>Pichia pastoris</i>). The later eradicates <i>Vibrio</i> species by producing innovative antimicrobial peptides from crocodile.
 
       </p>
 
       </p>
−
    </article>
+
   
−
  </div>
+
−
  </div>
+
  
−
  <div class="article_offset" id="a3">
+
    </section>
−
  <div class="article_container" id="a3">
+
 
−
    <h3>The crocodile, an unexpected helper</h3>
+
 
−
     <article>
+
<div class="article_offset" id="a2"></div>
−
       <img src="https://static.igem.org/mediawiki/2017/b/bd/T--INSA-UPS_France--Description-crocodile_helper.png" alt="" class="img_center image_80" style="max-width:600px;">
+
     <section>
 +
       <h1>Genesis of our molecular strategy
 +
</h1>
 
       <p>
 
       <p>
−
         <b>Non-salty water is the natural habitat of <i>V. cholerae</i><sup><a href="#ref21">21</a></sup></b>. Fortunatelty, Sobek, the Crocodile God of water and fertility which inspired us for our mascot Sobki, also likes those kind of environment. Indeed, crocodiles have an impressive immune system, that allows them to resist against lots of  diseases vectors infections<sup><a href="#ref22">22</a>,<a href="#ref23">23</a>,<a href="#ref24">24</a></sup>. We took from their amazing immune system  promising molecules: <b>antimicrobial peptides (AMP)</b> that have proven to have a good killing efficiency against <i>V. cholerae</i><sup><a href="#ref13">13</a>,<a href="#ref14">14</a></sup>.
+
         While we were defining our strategy, a cholera epidemia started unfortunately to expand in Yemen<sup><a href="http://www.emro.who.int/yem/yemeninfocus/situation-reports.html" target="_blank">7</a></sup>. This terrible situation led us to focus on this problematic as it appears that current solutions are not efficient enough to deal with this situation. The bacteria <i>V. cholerae</i>, agent of the cholera disease, is usually found in water and infects more than a million people each year.  
 
       </p>
 
       </p>
−
   
 
−
    </article>
 
−
  </div>
 
−
  </div>
 
−
 
−
  <div class="article_offset" id="a4">
 
−
  <div class="article_container">
 
−
    <h3>Our system : Croc'n Cholera</h3>
 
−
    <article>
 
 
       <p>
 
       <p>
−
         In contrast to current prevention systems, the iGEM INSA-UPS team of Toulouse would like to create <b>a low-cost and easy-to-use device that could be able to both detect and destroy cholera to treat water</b>. Our system implies interactions between three microorganisms:
+
         Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with <i>V. cholerae</i><sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/16697733" target="_blank">8</a>,<a href="http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079" target="_blank">9</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">10</a>,<a href="https://2010.igem.org/Team:Sheffield" target="_blank">11</a>,<a href="https://2014.igem.org/Team:UT-Dallas" target="_blank">12</a></sup>, using <i>E. coli</i>. They based their strategy on implementing the quorum sensing detection pathway of <i>V. cholerae</i>  into <i>E. coli</i> to activate reporter gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough, likely due the complexity of introducing a large amount of DNA information in a single microorganism. This is the reason why we built a synthetic consortium of microorganisms. Using multiple microorganisms instead of one will allow us to choose existing species that are already specialized for their tasks, as well as reducing the amount of necessary modification to set up the required functions. The information processing steps have been split in two different microorganisms:  <i>V. harveyi</i> as the sensor and <i>P. pastoris</i> as the effector, with the system triggered by <i>V. cholerae</i> presence.
 
       </p>
 
       </p>
−
      <img src="https://static.igem.org/mediawiki/2017/5/51/T--INSA-UPS_France--Description-project_overview.png" alt="" class="image_80">
+
    </section>
  
−
       <h4>1. Sense</h4>
+
   
 +
    <div class="article_offset" id="a3"></div>
 +
    <section>
 +
       <h1>A microbial consortium chassis against cholera
 +
</h1>
 
       <p>
 
       <p>
−
        Our first goal was to <b>detect <i>V. cholerae</i> in water</b>. To do so, we used its <b>quorum sensing</b> property: at low concentration, <i>V. cholerae</i> CqsS/CAI-1 pathway isn&rsquo;t activated. AlphA is thus activated and HapR is inactivated (responsible for the bacterium virulence) thanks to a phosphorylation cascade. At high concentration, HapR makes <i>V. cholerae</i> virulent<sup><a href="#ref15">15</a></sup>.  
+
      The whole synthetic consortium is composed of three microorganisms. The first one should be <i>V. cholerae</i> but for safety reason, we engineered an <i>E. coli</i> strain to produce a <i>V. cholerae</i> molecular signal. The second bacteria required a quorum sensing pathway to detect <i>V. cholerae</i> signal. <i>V. harveyi</i> naturally possesses such pathway and we engineered it  to make it able to sense <i>V. cholerae</i>. We also engineered <i>V. harveyi</i>  to make it producing a second molecule messenger. The third microorganism, <i>P. pastoris</i> was engineered to detect this messenger and produce in response the secretion of a high amount of antimicrobial peptides that can lyse <i>V. cholerae</i>.
 
       </p>
 
       </p>
 +
      <p>
 +
We finally created an artificial consortium chassis to deal with cholera disease. The different partners are deeper described below.
 +
      </p>
 +
   
 
        
 
        
 +
    </section>
  
−
      <table>
 
−
        <tr>
 
−
          <td>
 
−
            <img src="https://static.igem.org/mediawiki/2017/9/90/T--INSA-UPS_France--Description-sense-quorum_1.png" alt="" style="width:100%;">
 
−
          </td>
 
−
          <td>
 
−
            <img src="https://static.igem.org/mediawiki/2017/e/eb/T--INSA-UPS_France--Description-sense-quorum_2.png" alt="" style="width:100%;">
 
−
          </td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td style="text-align: center;"><i><b>
 
−
            Low CAI-1 concentration
 
−
          </b></i></td>
 
−
          <td style="text-align: center;"><i><b>
 
−
            High CAI-1 concentration
 
−
          </b></i></td>
 
−
        </tr>
 
−
      </table>
 
  
−
       <p style="margin-top:50px;">
+
    <div class="article_offset" id="a4"></div>
−
        For safety reasons, we were not able to manipulate <i>V. cholerae</i> in our lab. This is why we had to <b>engineer <i>E. coli</i> to mimick <i>V. cholerae</i></b> in order to further test our system. The challenge was to make <i>E. coli</i> produce the specific quorum sensing molecules of <i>V. cholerae</i>: CAI-1.
+
    <section>
 +
      <h1>Mimicking <i>Vibrio cholerae</i> using <i>Escherichia coli</i></h1>
 +
       <p>
 +
        An interesting property of <i>V. 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">13</a></sup>. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is an efficient 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>E. coli</i> in order to mimic <i>V. cholerae</i>. <i>E. coli</i> was transformed with the <i>cqsA</i> gene from <i>V. cholerae</i>. Since our sensor was <i>V. harveyi</i>, as a proof of concept, we also transformed an <i>E. coli</i> strain with the <i>cqsA</i> encoding gene of <i>V. harveyi</i>. This Vh_CqsA enzyme synthetizes C8-CAI-1, an analog of <i>V. cholerae</i> CAI-1<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">14</a></sup>.  
 
       </p>
 
       </p>
 
       <p>
 
       <p>
−
        We then used this <b>quorum sensing mechanism in <i>Vibrio harveyi</i></b>, a non-pathogenic strain of Vibrio that is close enough from <i>V. cholerae</i>, to detect cholera in water. We chose this particular strain because it is easy to engineer its receptor to detect and respond to cholera, already having the capacity to detect such quorum sensing molecules.
+
We therefore developed <i>E. coli</i> strains which produce markers simulating the presence of <i>Vibrio </i> species in the medium.  
 
       </p>
 
       </p>
 +
    </section>
  
−
       <h4>2. Communicate</h4>
+
    <div class="article_offset" id="a5"></div>
 +
    <section>
 +
       <h1>The sensing organism: <i>Vibrio harveyi</i></h1>
 
       <p>
 
       <p>
−
        As we wanted to both detect and destroy cholera, one of our challenges was to create an <b>eukaryote-prokaryote communication system</b>, so that <i>V. harveyi</i> could trigger the production of antimicrobial peptides (AMPs) by <i>P. pastoris</i> upon cholera detection.
+
      The easiest way to detect CAI-1 or C8-CAI-1 is to use the quorum sensing pathway of the non-pathogen <i>V. harveyi</i>.  As our project will deal directly with <i>V. cholerae</i> in real situation, the CqsS receptor of <i>V. harveyi</i> will have to also recognize the CAI-1 molecule<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/22001326" target="_blank">13</a></sup>. To do so, a single mutation was introduced in the gene <i>cqsS</i> changing the phenylalanine 175 into a cysteine.
 
       </p>
 
       </p>
 +
      <figure>
 +
        <img src="https://static.igem.org/mediawiki/2017/0/0a/T--INSA-UPS_FRANCE--Design_T1.png" alt="" class="right-img">
 +
        <figcaption>
 +
        <b>Figure 2: Closer view of the C8-CAI-1/CqsS sensing of <i>V. harveyi</i><sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">15</a></sup>.
 +
      </figcaption>
 +
      </figure>
 
       <p>
 
       <p>
−
        Team SCUT already described a binding-receptor system built on <i>Saccharomyces cerevisiae</i><sup><a href="#ref18">18</a></sup>, based on the ODR10 receptor, a G Protein Coupled Receptor isolated from <i>Caenorhabditis elegans</i><sup><a href="#ref19">19</a></sup>. The pathway engaged by the activation of ODR10 has been engineered in order to start the mating cascade which ends with  the activation of the pFUS1 promoter by Ste12<sup><a href="#ref20">20</a></sup>.  
+
      When C8-CAI-1 binds to CqsS this activate a dephosphorylation cascade leading to the inhibition of the pQRR4 promoter and blocking the transcription of siRNA<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">14</a></sup>. In the absence of this siRNA, translation of the targets genes (i.e. virulence genes) is activated (See Figure 2). We used this system to produce the molecule (i.e. diactetyl) used to activate <i> P. pastoris </i>. The <i>als </i> coding sequence, encoding for the acetolactate synthase Als, is involved in the conversion of endogenous pyruvate into diacetyl (Figure 3). <i> als </i> was placed under the control of pQRR4 promoter. In this engineered <i> V. Harveyi </i> strain , diacetyl production will be produced in response to both CAI-1 or C8-CAI-1.  
 
       </p>
 
       </p>
−
      <img src="https://static.igem.org/mediawiki/2017/2/24/T--INSA-UPS_France--Description-communicate.png" alt="" class="image_60" style="margin-bottom:40px;">
+
<figure>
 +
        <img src="https://static.igem.org/mediawiki/parts/0/0a/T--INSA-UPS_France--ALSpathway.png" alt="">
 +
        <figcaption>
 +
          <b>Figure 3: Production of diacetyl from pyruvate</b>.
 +
        </figcaption>
 +
      </figure>
 +
   
 +
     
 +
    </section>
 +
 
 +
    <div class="article_offset" id="a6"></div>
 +
    <section>
 +
      <h1>The effector organism: <i>Pichia pastoris</i></h1>
 
       <p>
 
       <p>
−
         As this mechanism relies on <b>diacetyl detection</b> for pFUS1 activation, we needed our sensing module to produce this molecule. Diacetyl is not produced by wild type <i>Vibrio harveyi</i> (Kegg pathway), but online tools predict that the missing enzyme to catalyse the production of this molecule is the acetolactate synthase (ALS). Indeed, this enzyme catalyses acetolactate production, which is then oxidized into diacetyl. Thus we decided to add the gene responsible for ALS production in the genetic construction we put in <i>V. harveyi</i>.
+
         The function of the third partner is to efficiently produce a killing molecule (i.e. anti-microbial peptides, AMPs) to lyse both <i>V. cholerae</i> and <i>V. harveyi</i>. This microorganism has to be resistant to the AMPs that are specific to prokaryotic cells. Therefore we chose an eukaryotic cell. Last this eukaryotic microorganism has to communicate with prokaryotic cell. Team SCUT <sup><a href="https://2013.igem.org/Team:SCUT" target="_blank">15</a></sup> previously described a binding-receptor system involving diacetyl and a eukaryotic receptor, Odr-10 <sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/" target="_blank">16</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/2302121" target="_blank">17</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 through the endogeneous Ste12 pathway (Figure 4). For all this reasons, we chose <i>P. pastoris</i> as the effector organism since it already possess the ste12 pathway and is good protein producer <sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27905091" target="_blank">18</a>,<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/" target="_blank">19</a></sup>.
 
       </p>
 
       </p>
−
       <img src="https://static.igem.org/mediawiki/2017/2/22/T--INSA-UPS_France--Description-communicate-ALS.png" alt="" class="image_60" style="min-width:400px;max-width:700px;">
+
       <figure>
 +
        <img src="https://static.igem.org/mediawiki/2017/2/24/T--INSA-UPS_France--Description-communicate.png" alt="">
 +
        <figcaption>
 +
          <b>Figure 4: The diacetyl/Odr-10 activation cascade.<sup><a href="https://2013.igem.org/Team:SCUT" target="_blank">15</a></sup></b>.
 +
      </figcaption>
 +
      </figure>
 +
      <p>
 +
      We engineered the yeast to secret the AMPs under control of the pFUS1 promotor. We choose the AMPs from crocrodiles  <sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21184776" target="_blank">20</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/2059789" target="_blank">21</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460" target="_blank">22</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554" target="_blank">23</a></sup>. Indeed crocodiles display a remarkable and efficient defense system, allowing the reptiles to resist to a large spectrum of bacterial infection. 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">24</a></sup>.
 +
      </p>
 +
 
 +
         
 +
    </section>
  
−
       <h4>3. Treat</h4>
+
    <div class="article_offset" id="a7"></div>
 +
    <section style="background: none;">
 +
       <h1>Our system</h1>    
 
       <p>
 
       <p>
−
         Instead of using gene targeting, we decided to eradicate the pathogen bacteria thanks to <b>crocodile antimicrobial peptides (AMPs)</b><sup><a href="#ref13">13</a>,<a href="#ref14">14</a></sup>. Those peptides are cationic molecules and can target bacterium membranes, to create pores in it, leading to the lysis of the cells<sup><a href="#ref24">24</a></sup>.
+
         See our <a href="https://2017.igem.org/Team:INSA-UPS_France/Design">Design page</a> for more informations about the genetic elements we used!
 
       </p>
 
       </p>
−
       <img src="https://static.igem.org/mediawiki/2017/8/80/T--INSA-UPS_France--Description-kill.png" alt="" class="img_center" style="min-width:400px; max-width:800px; width:50%;">
+
       <img style="max-width: 800px;" src="https://static.igem.org/mediawiki/2017/archive/b/b8/20171101194815%21T--INSA-UPS_France--description_loop.png" alt="">    
−
      <p>
+
     </section>
−
        Since these peptides have a high killing efficiency on <i>V. cholerae</i> it was obvious that our sensor, which is another species of <i>Vibrio</i>, could not stand any close contact with AMPs  and would be killed by them. That’s why we needed to find another organism to produce it. We thus used a <b>robust yeast</b> as our effector, <i>Pichia pastoris</i><sup><a href="#ref16">16</a>,<a href="#ref17">17</a></sup> which is also famous for being a high protein producer.
+
−
      </p>
+
−
      <img src="https://static.igem.org/mediawiki/2017/0/0b/T--INSA-UPS_France--Description-kill-MICAMP.png" alt="" class="image_50">
+
−
      <p style="text-align: center;">
+
−
        <i><b>
+
−
        Minimal inhibitory concentration 50 of selected antimicrobial peptides against <i>Vibrio cholerae</i><sup><a href="#ref13">13</a>,<a href="#ref14">14</a>,<a href="#ref22">22</a></sup>.
+
−
        </b></i>
+
−
      </p>
+
−
     </article>
+
−
  </div>
+
−
  </div>
+
  
−
  <div class="article_offset" id="a5">
+
    <style>
−
  <div class="article_container">
+
      ol{
−
     <h3>References</h3>
+
        text-align:justify;
−
    <article>
+
      }
 +
    </style>
 +
 
 +
    <div class="article_offset" id="a8"></div>
 +
     <section>
 +
      <h1>References</h1>    
 
       <ol>
 
       <ol>
−
        <li>
+
        <li>Brogi S, Tafi A, D&eacute;saubry L &amp; Nebigil CG (2014) Discovery of GPCR ligands for probing signal transduction pathways. <i>Frontiers in Pharmacology</i> <br />
−
        <div class="anchor_offset" id="ref1"></div>
+
        <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/</a>
−
          Ali M, Nelson AR, Lopez AL &amp; Sack DA (2015) Updated Global Burden of Cholera in Endemic Countries. PLOS Neglected Tropical Diseases 9 e0003832
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
          Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. <i>Nature Neuroscience</i>. <br/>
−
           <div class="anchor_offset" id="ref2"></div>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/26308982">https://www.ncbi.nlm.nih.gov/pubmed/26308982</a>
−
           <a href="http://www.who.int/hac/crises/yem/en/">WHO 23 SEPTEMBER 2016, 91thYEAR / No 38, 2016, 91, 433&ndash;440</a>
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
           Cameron E, Bashor C &amp; Collins J (2014) A brief history of synthetic biology. <i>Nature Reviews Microbiology</i>
−
          <div class="anchor_offset" id="ref3"></div>
+
           <a href="https://www.ncbi.nlm.nih.gov/pubmed/24686414">https://www.ncbi.nlm.nih.gov/pubmed/24686414</a>
−
          Clemens JD, Nair GB, Ahmed T, Qadri F &amp; Holmgren J (2017) Cholera. The Lancet
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
          Hennig S, R&ouml;del G &amp; Ostermann K (2015) Artificial cell-cell communication as an emerging tool in synthetic biology applications. <i>Journal of Biological Engineering</i> <br />
−
          <div class="anchor_offset" id="ref4"></div>
+
          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/</a>
−
          Davies HG, Bowman C &amp; Luby SP (2017) Cholera - management and prevention. Journal of Infection
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
          Liu T, Yu Y, Chen T and Chen W. (2016). A synthetic microbial consortium of Shewanella and Bacillusfor enhanced generation of bioelectricity. <i>Biotechnology and Bioengineering</i>, 114(3), pp.526-532.
−
          <div class="anchor_offset" id="ref5"></div>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/27596754
−
          Focareta A, Paton JC, Morona R, Cook J &amp; Paton AW (2006) A Recombinant Probiotic for Treatment and Prevention of Cholera. Gastroenterology 130 1688&ndash;1695
+
">https://www.ncbi.nlm.nih.gov/pubmed/27596754</a>
−
        </li>
+
        </li>
−
        <li>
+
        <li>  
−
          <div class="anchor_offset" id="ref6"></div>
+
          Wang, E., Ding, M., Ma, Q., Dong, X. and Yuan, Y. (2016). Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation. <i>Microbial Cell Factories</i>, 15(1).
−
          Holowko MB, Wang H, Jayaraman P &amp; Poh CL (2016) Biosensing <span class="not_italic">Vibrio cholerae</span> with Genetically Engineered <span class="not_italic">Escherichia coli</span>. ACS Synthetic Biology
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/26809519
−
        </li>
+
">https://www.ncbi.nlm.nih.gov/pubmed/26809519</a>
−
        <li>
+
        </li>
−
          <div class="anchor_offset" id="ref7"></div>
+
        <li>
−
          <a href="https://2014.igem.org/Team:UI-Indonesia">iGEM UI-Indonesia 2014</a>
+
          <a href="http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
−
        </li>
+
">http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
−
        <li>
+
</a>
−
          <div class="anchor_offset" id="ref8"></div>
+
        </li>
−
          <a href="https://2010.igem.org/Team:Sheffield">iGEM Sheffield 2010</a>
+
        <li>
−
        </li>
+
          Focareta A, Paton JC, Morona R, Cook J &amp; Paton AW (2006) A Recombinant Probiotic for Treatment and Prevention of Cholera. <i>Gastroenterology</i> <b>130</b> 1688&ndash;1695 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/16697733">https://www.ncbi.nlm.nih.gov/pubmed/16697733</a>
−
          <div class="anchor_offset" id="ref9"></div>
+
        </li>
−
          <a href="https://2014.igem.org/Team:UT-Dallas">iGEM UT-Dallas 2014</a>
+
        <li>
−
        </li>
+
          Holowko MB, Wang H, Jayaraman P &amp; Poh CL (2016) Biosensing Vibrio cholerae with Genetically Engineered <i>Escherichia coli</i>. <i>ACS Synthetic Biology</i>
−
        <li>
+
          <a href="http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079">http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079</a>
−
          <div class="anchor_offset" id="ref10"></div>
+
        </li>
−
          <a href="https://2016.igem.org/Team:Dundee">iGEM Dundee 2016</a>
+
        <li>
−
        </li>
+
          <a href="https://2014.igem.org/Team:UI-Indonesia">https://2014.igem.org/Team:UI-Indonesia</a>
−
        <li>
+
        </li>
−
          <div class="anchor_offset" id="ref11"></div>
+
        <li>
−
          Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF &amp; Bassler BL (2011) Signal production and detection specificity in <span class="not_italic">Vibrio</span> CqsA/CqsS quorum-sensing systems: <span class="not_italic">Vibrio</span> quorum-sensing systems. Molecular Microbiology 79 1407&ndash;1417
+
          <a href="https://2010.igem.org/Team:Sheffield">https://2010.igem.org/Team:Sheffield</a>
−
        </li>
+
        </li>
−
        <li>
+
        <li>
−
          <div class="anchor_offset" id="ref12"></div>
+
          <a href="https://2014.igem.org/Team:UT-Dallas">https://2014.igem.org/Team:UT-Dallas</a>
−
          Bolitho ME, Perez LJ, Koch MJ, Ng W-L, Bassler BL &amp; Semmelhack MF (2011) Small molecule probes of the receptor binding site in the <span class="not_italic">Vibrio cholerae</span> CAI-1 quorum sensing circuit. Bioorganic &amp; Medicinal Chemistry 19 6906&ndash;6918
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
          Bolitho ME, Perez LJ, Koch MJ, Ng W-L, Bassler BL &amp; Semmelhack MF (2011) Small molecule probes of the receptor binding site in the <i>Vibrio cholerae</i> CAI-1 quorum sensing circuit. <i>Bioorganic &amp; Medicinal Chemistry</i> <b>19</b> 6906&ndash;691 <br />
−
          <div class="anchor_offset" id="ref13"></div>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/22001326">https://www.ncbi.nlm.nih.gov/pubmed/22001326</a>
−
          Prajanban B, Jangpromma N, Araki T &amp; Klaynongsruang S (2017) Antimicrobial effects of novel peptides cOT2 and sOT2 derived from <span class="not_italic">Crocodylus siamensis</span> and <span class="not_italic">Pelodiscus sinensis</span> ovotransferrins. Biochimica et Biophysica Acta (BBA) - Biomembranes 1859 860&ndash;869
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
          Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF &amp; Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. <i>Molecular Microbiology</i> <b>79</b> 1407&ndash;1417 <br />
−
          <div class="anchor_offset" id="ref14"></div>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472">https://www.ncbi.nlm.nih.gov/pubmed/21219472</a>
−
          Yaraksa N, Anunthawan T, Theansungnoen T, Daduang S, Araki T, Dhiravisit A &amp; Thammasirirak S (2014) Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts. Journal of Antibiotics 67 205
+
        </li>
−
        </li>
+
        <li>
−
        <li>
+
          <a href="https://2013.igem.org/Team:SCUT">https://2013.igem.org/Team:SCUT</a>
−
          <div class="anchor_offset" id="ref15"></div>
+
        </li>
−
          Ng W-L, Perez L, Cong J, Semmelhack MF &amp; Bassler BL (2012) Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in <span class="not_italic">Vibrios</span>. PLoS Pathogens 8 e1002767
+
        <li>
−
        </li>
+
          Zhang Y, Chou JH, Bradley J, Bargmann CI &amp; Zinn K (1997) The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells. <i>Proceedings of the National Academy of Sciences</i> <b>94</b> 12162–12167 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/</a>
−
          <div class="anchor_offset" id="ref16"></div>
+
        </li>
−
          Huang Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in <span class="not_italic">Pichia pastoris</span>. Experimental and Therapeutic Medicine
+
        <li>
−
        </li>
+
          Audet M &amp; Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. <i>Cell</i> <b>151</b> 14–2 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/2302121">https://www.ncbi.nlm.nih.gov/pubmed/2302121</a>
−
          <div class="anchor_offset" id="ref17"></div>
+
        </li>
−
          Zhang Y, Teng D, Mao R, Wang X, Xi D, Hu X &amp; Wang J (2014) High expression of a plectasin-derived peptide NZ2114 in <span class="not_italic">Pichia pastoris</span> and its pharmacodynamics, postantibiotic and synergy against <span class="not_italic">Staphylococcus aureus</span>. Applied Microbiology and Biotechnology 98 681&ndash;694
+
        <li>
−
        </li>
+
          Kang Z, Huang H, Zhang Y, Du G &amp; Chen J (2017) Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications. <i>World Journal of Microbiology and Biotechnology</i> <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/27905091">https://www.ncbi.nlm.nih.gov/pubmed/27905091</a>
−
          <div class="anchor_offset" id="ref18"></div>
+
        </li>
−
          <a href="https://2013.igem.org/Team:SCUT">iGEM SCUT 2013</a>
+
        <li>
−
        </li>
+
          Huang Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris. <i>Experimental and Therapeutic Medicine</i> <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/</a>
−
          <div class="anchor_offset" id="ref19"></div>
+
        </li>
−
          Zhang Y, Chou JH, Bradley J, Bargmann CI &amp; Zinn K (1997) The <span class="not_italic">Caenorhabditis elegans</span> seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells. Proceedings of the National Academy of Sciences 94 12162&ndash;12167
+
        <li>
−
        </li>
+
          Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T &amp; Thammasirirak S (2011) Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. <i>Developmental &amp; Comparative Immunology</i> <b>35</b> 545–553 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/21184776">https://www.ncbi.nlm.nih.gov/pubmed/21184776</a>
−
          <div class="anchor_offset" id="ref20"></div>
+
        </li>
−
          Audet M &amp; Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. Cell 151 14&ndash;23
+
        <li>
−
        </li>
+
          Preecharram S, Jearranaiprepame P, Daduang S, Temsiripong Y, Somdee T, Fukamizo T, Svasti J, Araki T &amp; Thammasirirak S (2010) Isolation and characterisation of crocosin, an antibacterial compound from crocodile (Crocodylus siamensis) plasma: CROCODILE PLASMA ANTIBACTERIAL COMPOUND. <i>Animal Science Journal</i> <b>81</b> 393–401 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/2059789">https://www.ncbi.nlm.nih.gov/pubmed/2059789</a>
−
          <div class="anchor_offset" id="ref21"></div>
+
        </li>
−
          Robert Koch and the cholera vibrio: a centenary. N Howard-Jones, British Medical Journal (Clinical Research Edition)
+
        <li>
−
        </li>
+
          Prajanban B, Jangpromma N, Araki T &amp; Klaynongsruang S (2017) Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins. <i>Biochimica et Biophysica Acta (BBA) - Biomembranes</i> <b>1859</b> 860–869 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460">https://www.ncbi.nlm.nih.gov/pubmed/28159460</a>
−
          <div class="anchor_offset" id="ref22"></div>
+
        </li>
−
          Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T &amp; Thammasirirak S (2011) Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Developmental &amp; Comparative Immunology 35 545&ndash;553
+
        <li>
−
        </li>
+
          Yaraksa N, Anunthawan T, Theansungnoen T, Daduang S, Araki T, Dhiravisit A &amp; Thammasirirak S (2014) Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts. <i>Journal of Antibiotics</i> <b>67</b> 205 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554">https://www.ncbi.nlm.nih.gov/pubmed/24192554</a>
−
          <div class="anchor_offset" id="ref23"></div>
+
        </li>
−
          Preecharram S, Jearranaiprepame P, Daduang S, Temsiripong Y, Somdee T, Fukamizo T, Svasti J, Araki T &amp; Thammasirirak S (2010) Isolation and characterisation of crocosin, an antibacterial compound from crocodile (<span class="not_italic">Crocodylus siamensis</span>) plasma: CROCODILE PLASMA ANTIBACTERIAL COMPOUND. Animal Science Journal 81 393&ndash;401
+
        <li>
−
        </li>
+
          Mar&iacute;n-Medina N, Ram&iacute;rez DA, Trier S &amp; Leidy C (2016) Mechanical properties that influence antimicrobial peptide activity in lipid membranes. <i>Applied Microbiology and Biotechnology</i> <b>100</b> 10251&ndash;10263 <br />
−
        <li>
+
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/27837316">https://www.ncbi.nlm.nih.gov/pubmed/27837316</a>
−
          <div class="anchor_offset" id="ref24"></div>
+
        </li>
−
          Mar&iacute;n-Medina N, Ram&iacute;rez DA, Trier S &amp; Leidy C (2016) Mechanical properties that influence antimicrobial peptide activity in lipid membranes. Applied Microbiology and Biotechnology 100 10251&ndash;10263
+
      </ol>  
−
        </li>
+
    </section>
−
      </ol>
+
 
−
        
+
    <style>
−
     </article>
+
      .links_end{
 +
        background: none;
 +
      }
 +
      .links_end table{
 +
        width:100%;
 +
      }
 +
      .links_end th, .links_end td{
 +
        font-size:18pt;
 +
        padding:10px;
 +
      }
 +
      .links_end td{
 +
       
 +
        border:solid #eee 1px;
 +
       }
 +
     </style>
 +
 
 +
    <section class="links_end">
 +
      <table>
 +
        <tr>
 +
          <th colspan="3">
 +
            Strategy pages
 +
          </th>
 +
        </tr>
 +
        <tr>
 +
          <td><i>Description</i></td>
 +
          <td><a href="https://2017.igem.org/Team:INSA-UPS_France/Design">Design</a></td>
 +
          <td><a href="https://2017.igem.org/Team:INSA-UPS_France/Parts">Parts</a></td>
 +
        </tr>
 +
      </table>
 +
    </section>
 +
 
 +
    </div>
 +
    <!-- fin section -->   
 +
   
 
   </div>
 
   </div>
 
   </div>
 
   </div>
 +
   
 +
  <footer>
 +
   
 +
    <div class="footer_sponsors">
 +
          <a href="http://www.lisbp.fr/en/index.html"><img src="https://static.igem.org/mediawiki/2017/3/3e/T--INSA-UPS_France--Logo_lisbp.png" alt=""></a>
 +
          <a href="http://www.toulouse-white-biotechnology.com/en/"><img src="https://static.igem.org/mediawiki/2017/5/56/T--INSA-UPS_France--Logo_twb.png" alt=""></a>
 +
          <a href="http://fondation.insa-lyon.fr/"><img src="https://static.igem.org/mediawiki/2017/6/6d/T--INSA-UPS_France--Logo_fondationinsal.png" alt=""></a>
 +
          <a href="https://fr-fr.facebook.com/insaadnconceptclub/"><img src="https://static.igem.org/mediawiki/2017/b/b7/T--INSA-UPS_France--Logo_ia2c.png" alt=""></a>
 +
          <a href="http://www.biomerieux.com/en"><img src="https://static.igem.org/mediawiki/2017/8/83/T--INSA-UPS_France--Logo_biomerieux.png" alt=""></a>
 +
          <a href="http://www.sunwaterlife.com/en/"><img src="https://static.igem.org/mediawiki/2017/0/09/T--INSA-UPS_France--Logo_sunwaterlife.png" style="width:150px;" alt=""></a>
 +
          <a href="http://www.univ-tlse3.fr/home/home-page-en-379161.kjsp?RH=ACCUEIL"><img src="https://static.igem.org/mediawiki/2017/2/28/T--INSA-UPS_France--Logo_ups.png" alt=""></a>
 +
          <a href="http://www.fsi.univ-tlse3.fr/"><img src="https://static.igem.org/mediawiki/2017/a/ab/T--INSA-UPS_France--Logo_fsi.png" alt=""></a>
 +
          <a href="https://www.kbs-frb.be/"><img src="https://static.igem.org/mediawiki/2017/c/cf/T--INSA-UPS_France--Logo_ernestsolvay.png" alt=""></a>
 +
          <a href="http://www.insa-toulouse.fr/fr/institution/fondation-insa-toulouse.html#"><img src="https://static.igem.org/mediawiki/2017/6/62/T--INSA-UPS_France--Logo_fondationinsat.png" alt=""></a>
 +
      <a href="http://www.adisseo.com/en/"><img src="https://static.igem.org/mediawiki/2017/9/96/T--INSA-UPS_France--Logo_adisseo.png" alt=""></a>
 +
      <a href="http://www.insa-toulouse.fr/fr/index.html"><img src="https://static.igem.org/mediawiki/2017/2/2d/T--INSA-UPS_France--Logo_insatoulouse.png" style="width:150px;" alt=""></a>
 +
      <a href="https://www.veolia.com/en"><img src="https://static.igem.org/mediawiki/2017/9/91/T--INSA-UPS_France--Logo_veolia.png" alt=""></a>
 +
      <a href="https://www.france-science.org/-Homepage-English-.html"><img src="https://static.igem.org/mediawiki/2017/1/1a/T--INSA-UPS_France--Logo_ambassade.jpg" alt=""></a>
 +
      <a href="https://www-lbme.biotoul.fr/"><img src="https://static.igem.org/mediawiki/2017/5/51/T--INSA-UPS_France--Logo_LBME.png" alt=""></a>
 +
      <a href="https://www6.toulouse.inra.fr/metatoul_eng/"><img src="https://static.igem.org/mediawiki/2017/1/16/T--INSA-UPS_France--Logo_metatoul.png" alt=""></a>
 +
      <a href="http://www.univ-tlse3.fr/associations-+/do-you-have-a-project--378066.kjsp?RH=1238417866394"><img src="https://static.igem.org/mediawiki/2017/5/5b/T--INSA-UPS_France--Logo_fsdie.png" alt=""></a>
 +
      <a href="http://en.univ-toulouse.fr/our-strengths"><img src="https://static.igem.org/mediawiki/2017/9/93/T--INSA-UPS_France--Logo_fsie.jpg" alt=""></a>
  
  
−
   </section>
+
      <a href="https://www.crous-toulouse.fr/english/"><img src="https://static.igem.org/mediawiki/2017/3/37/T--INSA-UPS_France--Logo_crous.png" alt=""></a> 
 +
 
 +
    </div>
 +
    <div class="icons">
 +
      <a id="fbIcon" href="https://www.facebook.com/IGEM-Toulouse-1604834019761538/?fref=ts"><i class="fa fa-facebook fa-3x"></i></a>
 +
      <a id="twitterIcon" href="https://twitter.com/iGEM_Toulouse"><i class="fa fa-twitter fa-3x"></i></a>
 +
      <a id="instaIcon" href="https://www.instagram.com/igem_toulouse/"><i class="fa fa-instagram fa-3x"></i></a>
 +
      <a id="contactIcon" href="mailto:igem.toulouse@gmail.com"><i class="fa fa-envelope fa-3x"></i></a> 
 +
    </div>
 +
 
 +
 
 +
   </footer>
 
   </div>
 
   </div>
 +
  </main>
  
−
  <!-- VERTICAL NAV -->
 
  
−
<aside class="vertical_nav">
+
<!-- C O N T E N T -->
  
−
    <div class="aside_circle"><a href="#a1" data-number="1">About cholera</a></div>
+
<script type="text/javascript">
−
    <div class="aside_circle"><a href="#a2" data-number="2">Current solutions</a></div>
+
  $('.main_content').scroll(function(){
−
    <div class="aside_circle"><a href="#a3" data-number="3">The crocodile, an unexpected helper</a></div>
+
    var pos = $('.main_content').scrollTop();
−
    <div class="aside_circle"><a href="#a4" data-number="4">Our system: Croc&rsquo; n Cholera</a></div>
+
    // BOLD ASIDE NAV
−
    <div class="aside_circle"><a href="#a5" data-number="6">References</a></div>
+
    if(pos>$("#a1").position().top-10){
 +
      $('*[data-number="1"]').css("font-weight", "bold");
 +
      $('*[data-number="1"]').parent().addClass("selected-item");
 +
      $('*[data-number="2"]').css("font-weight", "normal");
 +
      $('*[data-number="2"]').parent().removeClass("selected-item");
 +
      $('*[data-number="3"]').css("font-weight", "normal");
 +
      $('*[data-number="3"]').parent().removeClass("selected-item");
 +
      $('*[data-number="4"]').css("font-weight", "normal");
 +
      $('*[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", "normal");
 +
                  $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
      if(pos>$("#a2").position().top-10){
 +
        $('*[data-number="1"]').css("font-weight", "normal");
 +
        $('*[data-number="1"]').parent().removeClass("selected-item");
 +
        $('*[data-number="2"]').css("font-weight", "bold");
 +
        $('*[data-number="2"]').parent().addClass("selected-item");
 +
        $('*[data-number="3"]').css("font-weight", "normal");
 +
        $('*[data-number="3"]').parent().removeClass("selected-item");
 +
        $('*[data-number="4"]').css("font-weight", "normal");
 +
        $('*[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", "normal");
 +
                  $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
        if(pos>$("#a3").position().top-10){
 +
          $('*[data-number="1"]').css("font-weight", "normal");
 +
          $('*[data-number="1"]').parent().removeClass("selected-item");
 +
          $('*[data-number="2"]').css("font-weight", "normal");
 +
          $('*[data-number="2"]').parent().removeClass("selected-item");
 +
          $('*[data-number="3"]').css("font-weight", "bold");
 +
          $('*[data-number="3"]').parent().addClass("selected-item");
 +
          $('*[data-number="4"]').css("font-weight", "normal");
 +
          $('*[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", "normal");
 +
                  $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
          if(pos>$("#a4").position().top-10){
 +
            $('*[data-number="1"]').css("font-weight", "normal");
 +
            $('*[data-number="1"]').parent().removeClass("selected-item");
 +
            $('*[data-number="2"]').css("font-weight", "normal");
 +
            $('*[data-number="2"]').parent().removeClass("selected-item");
 +
            $('*[data-number="3"]').css("font-weight", "normal");
 +
            $('*[data-number="3"]').parent().removeClass("selected-item");
 +
            $('*[data-number="4"]').css("font-weight", "bold");
 +
            $('*[data-number="4"]').parent().addClass("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", "normal");
 +
                  $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
            if(pos>$("#a5").position().top-10){
 +
              $('*[data-number="1"]').css("font-weight", "normal");
 +
              $('*[data-number="1"]').parent().removeClass("selected-item");
 +
              $('*[data-number="2"]').css("font-weight", "normal");
 +
              $('*[data-number="2"]').parent().removeClass("selected-item");
 +
              $('*[data-number="3"]').css("font-weight", "normal");
 +
              $('*[data-number="3"]').parent().removeClass("selected-item");
 +
              $('*[data-number="4"]').css("font-weight", "normal");
 +
              $('*[data-number="4"]').parent().removeClass("selected-item");
 +
              $('*[data-number="5"]').css("font-weight", "bold");
 +
              $('*[data-number="5"]').parent().addClass("selected-item");
 +
              $('*[data-number="6"]').css("font-weight", "normal");
 +
              $('*[data-number="6"]').parent().removeClass("selected-item");
 +
              $('*[data-number="7"]').css("font-weight", "normal");
 +
                  $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
              if(pos>$("#a6").position().top-10){
 +
                $('*[data-number="1"]').css("font-weight", "normal");
 +
                $('*[data-number="1"]').parent().removeClass("selected-item");
 +
                $('*[data-number="2"]').css("font-weight", "normal");
 +
                $('*[data-number="2"]').parent().removeClass("selected-item");
 +
                $('*[data-number="3"]').css("font-weight", "normal");
 +
                $('*[data-number="3"]').parent().removeClass("selected-item");
 +
                $('*[data-number="4"]').css("font-weight", "normal");
 +
                $('*[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", "bold");
 +
                $('*[data-number="6"]').parent().addClass("selected-item");
 +
                $('*[data-number="7"]').css("font-weight", "normal");
 +
                  $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
                if(pos>$("#a7").position().top-10){
 +
                  $('*[data-number="1"]').css("font-weight", "normal");
 +
                  $('*[data-number="1"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="2"]').css("font-weight", "normal");
 +
                  $('*[data-number="2"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="3"]').css("font-weight", "normal");
 +
                  $('*[data-number="3"]').parent().removeClass("selected-item");
 +
                  $('*[data-number="4"]').css("font-weight", "normal");
 +
                  $('*[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");
 +
                  $('*[data-number="8"]').css("font-weight", "normal");
 +
                  $('*[data-number="8"]').parent().removeClass("selected-item");
 +
                  if(pos>$("#a8").position().top-10){
 +
                    $('*[data-number="1"]').css("font-weight", "normal");
 +
                    $('*[data-number="1"]').parent().removeClass("selected-item");
 +
                    $('*[data-number="2"]').css("font-weight", "normal");
 +
                    $('*[data-number="2"]').parent().removeClass("selected-item");
 +
                    $('*[data-number="3"]').css("font-weight", "normal");
 +
                    $('*[data-number="3"]').parent().removeClass("selected-item");
 +
                    $('*[data-number="4"]').css("font-weight", "normal");
 +
                    $('*[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", "normal");
 +
                    $('*[data-number="7"]').parent().removeClass("selected-item");
 +
                    $('*[data-number="8"]').css("font-weight", "bold");
 +
                    $('*[data-number="8"]').parent().addClass("selected-item");
 +
                  }
 +
                }
 +
              }
 +
            }
 +
          }
 +
        }
 +
      }
 +
    }
 +
    // FIXED ASIDE NAV
 +
    if(pos>371){
 +
      $('.left_container').addClass("aside-fixed");
 +
    }
 +
    else{
 +
      $('.left_container').removeClass("aside-fixed");
 +
    }
 +
  });
  
−
</aside>
+
</script>
  
 +
<script type="text/javascript">
  
 +
    // NAV BAR SUBNAV CLICK
  
 +
  $(document).on("click",".cat_name", function(){
 +
    if($(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>
 
</html>
−
 
+
{{INSA-UPS_France/General_script}}
−
<!-- FOOTER -->
+
{{INSA-UPS_France/Header_script}}
−
{{INSA-UPS_France/Footer}}
+
−
{{INSA-UPS_France/scroll_Descr}}
+

Latest revision as of 09:49, 12 December 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017


Description

Synthetic Consortia
Genesis of our molecular strategy
A microbial consortium chassis against cholera
Mimicking Vibrio cholerae
The sensing organism: Vibrio harveyi
The effecting organism: Pichia pastoris
Our system
References

Synthetic biology: from unique chassis to synthetic consortia

Synthetic biology is based on Nature everlasting possibilities, usually by inserting genetic information from microorganisms into a single and unique chassis1,2,3. However, a single chassis could present inevitable limits (high genetic burden, incompatibility of some elements with the chassis, too complex design…). These start to be a limit in synthetic biology, its perspectives and applications4. An emerging solution is the use of synthetic consortium (figure 1). Synthetic consortium have the advantage to require less amount of genetic information into a single chassis to achieve the required process since different microorganisms can share the genetic burden. Moreover, the components of the consortium could be selected to reduce the genetic modifications and increase the chance of success, for example, by taking advantage of already existing signaling or metabolic pathways. Several successes of those synthetic consortia, such as production of bio-electricity5 or shortening bio-manufacturing process like C-vitamin synthesis6 have provided insight into the strength of this approach.

Figure 1: General concept of the cell to cell communication. This cascade of events will be developed in our project.

Such approaches are still rare in the iGEM competition, maybe because they require to combine classic strain engineering with information processing strategies. So, our challenge was to demonstrate the power and feasibility of synthetic consortium approach to open new perspectives and applications to iGEMers.

As a proof of concept, we developed a strategy against cholera. It is based on a cascade of events starting from an engineered Escherichia coli strain mimicking Vibrio cholerae. It triggers a sensor bacterium, Vibrio harveyi, which in turn activates the effector cell (the yeast Pichia pastoris). The later eradicates Vibrio species by producing innovative antimicrobial peptides from crocodile.

Genesis of our molecular strategy

While we were defining our strategy, a cholera epidemia started unfortunately to expand in Yemen7. This terrible situation led us to focus on this problematic as it appears that current solutions are not efficient enough to deal with this situation. The bacteria V. cholerae, agent of the cholera disease, is usually found in water and infects more than a million people each year.

Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with V. cholerae8,9. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae10,11,12, using E. coli. They based their strategy on implementing the quorum sensing detection pathway of V. cholerae into E. coli to activate reporter gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough, likely due the complexity of introducing a large amount of DNA information in a single microorganism. This is the reason why we built a synthetic consortium of microorganisms. Using multiple microorganisms instead of one will allow us to choose existing species that are already specialized for their tasks, as well as reducing the amount of necessary modification to set up the required functions. The information processing steps have been split in two different microorganisms: V. harveyi as the sensor and P. pastoris as the effector, with the system triggered by V. cholerae presence.

A microbial consortium chassis against cholera

The whole synthetic consortium is composed of three microorganisms. The first one should be V. cholerae but for safety reason, we engineered an E. coli strain to produce a V. cholerae molecular signal. The second bacteria required a quorum sensing pathway to detect V. cholerae signal. V. harveyi naturally possesses such pathway and we engineered it to make it able to sense V. cholerae. We also engineered V. harveyi to make it producing a second molecule messenger. The third microorganism, P. pastoris was engineered to detect this messenger and produce in response the secretion of a high amount of antimicrobial peptides that can lyse V. cholerae.

We finally created an artificial consortium chassis to deal with cholera disease. The different partners are deeper described below.

Mimicking Vibrio cholerae using Escherichia coli

An interesting property of V. cholerae is its quorum sensing autoinducer system based on the production of CAI-1 molecule13. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is an efficient reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain E. coli in order to mimic V. cholerae. E. coli was transformed with the cqsA gene from V. cholerae. Since our sensor was V. harveyi, as a proof of concept, we also transformed an E. coli strain with the cqsA encoding gene of V. harveyi. This Vh_CqsA enzyme synthetizes C8-CAI-1, an analog of V. cholerae CAI-114.

We therefore developed E. coli strains which produce markers simulating the presence of Vibrio species in the medium.

The sensing organism: Vibrio harveyi

The easiest way to detect CAI-1 or C8-CAI-1 is to use the quorum sensing pathway of the non-pathogen V. harveyi. As our project will deal directly with V. cholerae in real situation, the CqsS receptor of V. harveyi will have to also recognize the CAI-1 molecule13. To do so, a single mutation was introduced in the gene cqsS changing the phenylalanine 175 into a cysteine.

Figure 2: Closer view of the C8-CAI-1/CqsS sensing of V. harveyi15.

When C8-CAI-1 binds to CqsS this activate a dephosphorylation cascade leading to the inhibition of the pQRR4 promoter and blocking the transcription of siRNA14. In the absence of this siRNA, translation of the targets genes (i.e. virulence genes) is activated (See Figure 2). We used this system to produce the molecule (i.e. diactetyl) used to activate P. pastoris . The als coding sequence, encoding for the acetolactate synthase Als, is involved in the conversion of endogenous pyruvate into diacetyl (Figure 3). als was placed under the control of pQRR4 promoter. In this engineered V. Harveyi strain , diacetyl production will be produced in response to both CAI-1 or C8-CAI-1.

Figure 3: Production of diacetyl from pyruvate.

The effector organism: Pichia pastoris

The function of the third partner is to efficiently produce a killing molecule (i.e. anti-microbial peptides, AMPs) to lyse both V. cholerae and V. harveyi. This microorganism has to be resistant to the AMPs that are specific to prokaryotic cells. Therefore we chose an eukaryotic cell. Last this eukaryotic microorganism has to communicate with prokaryotic cell. Team SCUT 15 previously described a binding-receptor system involving diacetyl and a eukaryotic receptor, Odr-10 16,17. It is a G Protein Coupled Receptor isolated from Caenorhabditis elegans that once activated by diacetyl, lead to the activation of the pFUS1 promoter through the endogeneous Ste12 pathway (Figure 4). For all this reasons, we chose P. pastoris as the effector organism since it already possess the ste12 pathway and is good protein producer 18,19.

Figure 4: The diacetyl/Odr-10 activation cascade.15.

We engineered the yeast to secret the AMPs under control of the pFUS1 promotor. We choose the AMPs from crocrodiles 20,21,22,23. Indeed crocodiles display a remarkable and efficient defense system, allowing the reptiles to resist to a large spectrum of bacterial infection. Thus, they produced antimicrobial peptides (AMPs) which are able to lyse bacteria such as V. cholerae. AMPs are cationic pore-forming molecules targeting bacterium membranes, causing bacterial lysis and death 24.

Our system

See our Design page for more informations about the genetic elements we used!

References

  1. Brogi S, Tafi A, Désaubry L & Nebigil CG (2014) Discovery of GPCR ligands for probing signal transduction pathways. Frontiers in Pharmacology
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/
  2. Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. Nature Neuroscience.
    https://www.ncbi.nlm.nih.gov/pubmed/26308982
  3. Cameron E, Bashor C & Collins J (2014) A brief history of synthetic biology. Nature Reviews Microbiology https://www.ncbi.nlm.nih.gov/pubmed/24686414
  4. Hennig S, Rödel G & Ostermann K (2015) Artificial cell-cell communication as an emerging tool in synthetic biology applications. Journal of Biological Engineering
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/
  5. Liu T, Yu Y, Chen T and Chen W. (2016). A synthetic microbial consortium of Shewanella and Bacillusfor enhanced generation of bioelectricity. Biotechnology and Bioengineering, 114(3), pp.526-532. https://www.ncbi.nlm.nih.gov/pubmed/27596754
  6. Wang, E., Ding, M., Ma, Q., Dong, X. and Yuan, Y. (2016). Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation. Microbial Cell Factories, 15(1). https://www.ncbi.nlm.nih.gov/pubmed/26809519
  7. http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
  8. Focareta A, Paton JC, Morona R, Cook J & Paton AW (2006) A Recombinant Probiotic for Treatment and Prevention of Cholera. Gastroenterology 130 1688–1695
    https://www.ncbi.nlm.nih.gov/pubmed/16697733
  9. Holowko MB, Wang H, Jayaraman P & Poh CL (2016) Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli. ACS Synthetic Biology http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079
  10. https://2014.igem.org/Team:UI-Indonesia
  11. https://2010.igem.org/Team:Sheffield
  12. https://2014.igem.org/Team:UT-Dallas
  13. Bolitho ME, Perez LJ, Koch MJ, Ng W-L, Bassler BL & Semmelhack MF (2011) Small molecule probes of the receptor binding site in the Vibrio cholerae CAI-1 quorum sensing circuit. Bioorganic & Medicinal Chemistry 19 6906–691
    https://www.ncbi.nlm.nih.gov/pubmed/22001326
  14. Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417
    https://www.ncbi.nlm.nih.gov/pubmed/21219472
  15. https://2013.igem.org/Team:SCUT
  16. Zhang Y, Chou JH, Bradley J, Bargmann CI & Zinn K (1997) The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells. Proceedings of the National Academy of Sciences 94 12162–12167
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/
  17. Audet M & Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. Cell 151 14–2
    https://www.ncbi.nlm.nih.gov/pubmed/2302121
  18. Kang Z, Huang H, Zhang Y, Du G & Chen J (2017) Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications. World Journal of Microbiology and Biotechnology
    https://www.ncbi.nlm.nih.gov/pubmed/27905091
  19. Huang Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris. Experimental and Therapeutic Medicine
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/
  20. Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T & Thammasirirak S (2011) Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Developmental & Comparative Immunology 35 545–553
    https://www.ncbi.nlm.nih.gov/pubmed/21184776
  21. Preecharram S, Jearranaiprepame P, Daduang S, Temsiripong Y, Somdee T, Fukamizo T, Svasti J, Araki T & Thammasirirak S (2010) Isolation and characterisation of crocosin, an antibacterial compound from crocodile (Crocodylus siamensis) plasma: CROCODILE PLASMA ANTIBACTERIAL COMPOUND. Animal Science Journal 81 393–401
    https://www.ncbi.nlm.nih.gov/pubmed/2059789
  22. Prajanban B, Jangpromma N, Araki T & Klaynongsruang S (2017) Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins. Biochimica et Biophysica Acta (BBA) - Biomembranes 1859 860–869
    https://www.ncbi.nlm.nih.gov/pubmed/28159460
  23. Yaraksa N, Anunthawan T, Theansungnoen T, Daduang S, Araki T, Dhiravisit A & Thammasirirak S (2014) Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts. Journal of Antibiotics 67 205
    https://www.ncbi.nlm.nih.gov/pubmed/24192554
  24. Marín-Medina N, Ramírez DA, Trier S & Leidy C (2016) Mechanical properties that influence antimicrobial peptide activity in lipid membranes. Applied Microbiology and Biotechnology 100 10251–10263
    https://www.ncbi.nlm.nih.gov/pubmed/27837316
Strategy pages
Description Design Parts