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

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         The effecting organism: <i>Pichia pastoris</i>
 
         The effecting organism: <i>Pichia pastoris</i>
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      </a>
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      </div>
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      <div class="aside-nav__item">
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      <a href="#a6" data-number="6">
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        Our system
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      <div class="aside-nav__item">
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      <a href="#a7" data-number="7">
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        References
 
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     <div class="article_offset" id="a1"></div>
 
     <div class="article_offset" id="a1"></div>
 
     <section>
 
     <section>
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       <h1>Combine to improve: a synthetic biology scale-up</h1>
+
       <h1>Synthetic biology: to the multi-organisms communication and beyond </h1>
 
       <p>
 
       <p>
−
         Nature offers us a wide variety of tools in order to sense precise chemical or physical parameters. For example, enzymes and receptors are specialized for unique or restrained range of molecule, or even for specific light wavelength.<sup>1,2,3</sup> Obviously, as mankind progressed in the comprehension of life science, he wanted to take advantage from this extreme diversity and specialization. Synthetic biology was born thanks to those expectations in the early of the twentieth century<sup>4</sup> and made quite notable breakthrough. However, most of those works were based on the insertion of a lot of DNA information on one single model, typically <i>E. coli</i>. We observed that all those informations are not always compatible with the use of a unique microbial chassis : although standard microbial models are quite modular, they have a particular membrane, specific pathways and different ways to do protein maturation.
+
         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="#a7">1,2</a></sup>. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature’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="#a7">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="#a7">4</a></sup>.
 
       </p>
 
       </p>
 
       <p>
 
       <p>
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         Why should we persist to use a single model when we have access to a wide diversity of organisms?
+
         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.
 
       </p>
 
       </p>
 +
      <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
 +
      <h2>Genesis of our molecular strategy</h2>
 
       <p>
 
       <p>
−
         For our iGEM project, we decided to focus on the multi organisms aspect, making communication between prokaryotic and eukaryotic possible.
+
         During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen<sup><a href="#a7">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.
 +
      </p>
 +
      <p>
 +
        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="#a7">6,7</a></sup>. Additionally, some iGEM teams tried also to deal with the challenging detection of <i>V. cholerae</i><sup><a href="#a7">8,9,10</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>.
 
       </p>
 
       </p>
 
     </section>
 
     </section>
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     <div class="article_offset" id="a2"></div>
 
     <div class="article_offset" id="a2"></div>
 
     <section>
 
     <section>
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       <h1>A core: prokaryotic-eukaryotic communication
+
       <h1>A microbial consortium chassis against cholera
 
</h1>
 
</h1>
 
       <p>
 
       <p>
−
         Our first challenge was to design an eukaryote-prokaryote communication system, so we can have a standardized way of communication, with modulable input and output. This system answers to some of the synthetic biology current challenges<sup>8</sup> that are: device modularization and standardisation with the core, using the diversity of living systems and so, using the right cellular chassis, with the right genetics elements.
+
         We finally created an artificial consortium chassis to deal with cholera disease. The different partners are described below.  
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      </p>
+
−
      <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
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      <p class="left-p">
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        Team SCUT<sup>26</sup> already described a binding-receptor system built on <i>Saccharomyces cerevisiae</i><sup>18</sup>, based on the Odr-10 receptor, a G Protein Coupled Receptor isolated from Caenorhabditis elegans<sup>19</sup>. The pathway is engaged by the activation of Odr-10 and has been shown to start the mating cascade which ends with  the activation of the pFUS1 promoter by Ste12<sup>20</sup>. After a discussion with Diethard Mattanovich , the supervisor of iGEM Team Boku Vienna, it appears from his transcriptomic data that the mating pathway including Ste12 was also present on Pichia pastoris, a yeast gaining more and more interest for being a robust tool for protein and metabolite production.<sup>33</sup> Once we found a molecule factory, we needed to find a way to induce it, whenever the bacteria sensor we were using. Checking the metabolism of diacetyl on KEGG Pathway, we identified that a simple enzyme, the acetolactate synthase (ALS), processes to produce the diacetyl from pyruvate, a ubiquitous metabolite.
+
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      </p>
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      <img class="right-img" src="https://static.igem.org/mediawiki/2017/2/24/T--INSA-UPS_France--Description-communicate.png" alt="">
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      <img class="left-img" src="https://static.igem.org/mediawiki/2017/9/91/T--INSA-UPS_France--description_diacetyl.png" alt="">
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      <p class="right-p">
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        As this mechanism relies on diacetyl detection for pFUS1 activation, we needed  the sensing module to produce this molecule. 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 the sensing module.
+
 
       </p>
 
       </p>
 +
      <ul>
 +
        <li>
 +
          To mimic <i>V. cholerae</i> by producing CAI-1 molecule. This will be done in <i>E. coli</i>
 +
        </li>
 +
        <li>
 +
          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.
 +
        </li>
 +
        <li>
 +
          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>.
 +
        </li>
 +
      </ul>
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 +
     
 
     </section>
 
     </section>
  
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     <section>
 
     <section>
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       <h1>A microbial consortium against cholera</h1>
+
       <h1>Mimicking <i>Vibrio cholerae</i> using <i>Escherichia coli</i></h1>
 
       <p>
 
       <p>
−
         Recently, research teams started to focus on synthetic biology in order to find a way to deal with Vibrio cholerae<sup>5,6</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 succeed7 or showed mixed results<sup>8</sup>. Most of the iGEM teams intended to prevent the cholera infection thanks to gene targeting such as cleaving toxicity genes<sup>9</sup> or inhibiting specific genes on the pathogen<sup>10</sup>. With this lack of successful stories in iGEM on mono-microorganism and the need to have a whole specialised lab in order to build a complex system of synthetic biology, this statement could be perfect to test our core in real conditions and with a useful application.  
+
         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="#a7">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.
 
       </p>
 
       </p>
 
     </section>
 
     </section>
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     <div class="article_offset" id="a4"></div>
 
     <section>
 
     <section>
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       <h1>Finding a sensor</h1>
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       <h1>The sensing organism: <i>Vibrio harveyi</i></h1>
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       <p class="left-p">
+
       <p>
−
         First we had to detect the initial state of the system in order to activate the core. An interesting property of <i>Vibrio cholerae</i> is its quorum sensing system that depends on the CAI-1 molecule. CAI-1 directly reflects the amount of <i>V. cholerae</i> on water, and thus its virulence. Once CAI-1 is at high concentration, it can be detected and then  provides us a good way of detecting <i>V. cholerae</i> once it becomes pathogenic<sup>19</sup>. <i>Vibrio harveyi</i>, a non-pathogenic strain of <i>Vibrio</i>, showed itself as a perfect sensor of the CAI-1 molecule and thus of the quantity of <i>V. cholerae</i>. This strain fulfilled the requirements we wanted as a good synthetic biology chassis: easy to engineer, already has part of our final system such as the CqsS/CAI-1 pathway<sup>19,20</sup>. We only had to mutate the receptor and integrate the als gene to trigger diacetyl production in presence of CAI-1.
+
         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="#a7">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="#a7">12</a></sup>.  
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       </p>
 
       </p>
 
       <figure>
 
       <figure>
 
         <img src="https://static.igem.org/mediawiki/2017/e/eb/T--INSA-UPS_France--Description-sense-quorum_2.png" alt="" class="right-img">
 
         <img src="https://static.igem.org/mediawiki/2017/e/eb/T--INSA-UPS_France--Description-sense-quorum_2.png" alt="" class="right-img">
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         <figcaption><i>Quorum sensing mechanism in</i> V. cholerae</figcaption>
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         <figcaption>
 +
        <b>Cascade of events depending on the CAI-1/CqsS binding in V. cholerae<sup><a href="#a7">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.
 +
        </figcaption>
 
       </figure>
 
       </figure>
 
       <p>
 
       <p>
−
         For safety reasons, we were not able to manipulate <i>V. cholerae</i> in our lab. This is why we had to engineer <i>E. coli</i> to mimick <i>V. cholerae</i> in order to further test our system. The idea was to make <i>E. coli</i> produce the specific quorum sensing molecules of <i>V. cholerae</i>: CAI-1.
+
         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.
 
       </p>
 
       </p>
 +
      <figure>
 +
        <img src="https://static.igem.org/mediawiki/parts/0/0a/T--INSA-UPS_France--ALSpathway.png" alt="">
 +
        <figcaption>
 +
          <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.
 +
        </figcaption>
 +
      </figure>
 
     </section>
 
     </section>
  
 
     <div class="article_offset" id="a5"></div>
 
     <div class="article_offset" id="a5"></div>
 
     <section>
 
     <section>
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       <h1>Finding an effector</h1>
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       <h1>The effecting organism: <i>Pichia pastoris</i></h1>
 
       <p>
 
       <p>
−
         Because of our will to kill gram-negative bacteria, we assessed that a prokaryotic chassis could not be adequate to achieve the desired effect without drawbacks. So the information of <i>V. cholerae</i> detection must be transmitted  to an eukaryotic protein expressor : <i>Pichia pastoris</i>. Once we had both the sensor, and the core, we had to act on the system. In our application, we aim to reduce the amount of <i>V. cholerae</i>, or even better, to totally kill it. We found the solution thanks to an unorthogonal approach: we looked in the environment of <i>V. cholerae</i> in order to find out a competitor, or an organism not affected by it. It appears quite fast that crocodiles have an impressive immune system, that allows them to resist against lots of  diseases vectors infections<sup>22,23,24</sup>. We used from their amazing immune system promising molecules: antimicrobial peptides that have proven to have a good killing efficiency against <i>V. cholerae</i><sup>13,14</sup>. Moreover, this effector, with a good efficiency against bacteria and the need to be secreted, was a perfect effector to implement in our multi organisms system with specialised tasks.
+
         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="#a7">13</a></sup> previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, the Odr-10 receptor<sup><a href="#a7">14,15</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.  
−
 
+
 
       </p>
 
       </p>
 +
      <figure>
 +
        <img src="https://static.igem.org/mediawiki/2017/2/24/T--INSA-UPS_France--Description-communicate.png" alt="">
 +
        <figcaption>
 +
          <b>Activation cascade on the dependence of Diacetyl/Odr-10 binding<sup><a href="#a7">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.
 +
        </figcaption>
 +
      </figure>
 
       <p>
 
       <p>
−
         Those peptides are cationic molecules and can target bacterium membranes, to create pores in it, leading to the lysis of the cells<sup>24</sup>.  
+
         Moreover, <i>P. pastoris</i> is a good protein producing organism<sup><a href="#a7">16,17</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>18,19,20,21</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="#a7">22</a></sup>. This is the third step of our cascade.
 
       </p>
 
       </p>
−
       <img src="https://static.igem.org/mediawiki/2017/8/80/T--INSA-UPS_France--Description-kill.png" alt="">
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       <figure>
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      <p class="left-p">
+
        <img src="https://static.igem.org/mediawiki/2017/8/80/T--INSA-UPS_France--Description-kill.png" alt="">
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        The fact that literature described the robustness of <i>Pichia pastoris</i><sup>16,17</sup> for the production of antimicrobial peptide made us definitively enthousiast about using this yeast as part of the core.  
+
        <figcaption>
−
      </p>
+
          <b>Mechanism of action of antimicrobial peptide and their effects on cells<sup><a href="#a7">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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      <img src="https://static.igem.org/mediawiki/2017/0/0b/T--INSA-UPS_France--Description-kill-MICAMP.png" alt="" class="right-img">
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        </figcaption>
 +
        <img src="https://static.igem.org/mediawiki/2017/0/0b/T--INSA-UPS_France--Description-kill-MICAMP.png" alt="" class="right-img">
 +
        <figcaption>
 +
          <b>efficiency of the antimicrobial peptide from crocodile on V. cholerae<sup><a href="#a7">18,20,21</a></sup></b>.The three peptides display and minimal inhibitory concentration 50 in the scale of mg/L.
 +
        </figcaption>
 +
      </figure>
 +
 
 +
 
 +
     
 
     </section>
 
     </section>
  
 
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       <h1 style="text-align:left;">Our system</h1>       
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       <h1>Our system</h1>       
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       <!--<p>
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       <p>
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         See design for more informations of the genetic engineering we used!
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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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       </p>-->
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       </p>
 
       <img src="https://static.igem.org/mediawiki/2017/b/b8/T--INSA-UPS_France--description_loop.png" alt="">       
 
       <img src="https://static.igem.org/mediawiki/2017/b/b8/T--INSA-UPS_France--description_loop.png" alt="">       
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    </section>
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 +
    <style>
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    <div class="article_offset" id="a7"></div>
 +
    <section>
 +
      <h1>References</h1>     
 +
      <ol>
 +
        <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 />
 +
        <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/</a>
 +
        </li>
 +
        <li>
 +
          Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. <i>Nature Neuroscience</i>. <br/>
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/26308982">https://www.ncbi.nlm.nih.gov/pubmed/26308982</a>
 +
        </li>
 +
        <li>
 +
          Cameron E, Bashor C &amp; Collins J (2014) A brief history of synthetic biology. <i>Nature Reviews Microbiology</i>
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/24686414">https://www.ncbi.nlm.nih.gov/pubmed/24686414</a>
 +
        </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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/</a>
 +
        </li>
 +
        <li>
 +
          <a href="http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
 +
">http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
 +
</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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/16697733">https://www.ncbi.nlm.nih.gov/pubmed/16697733</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>
 +
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          <a href="https://2010.igem.org/Team:Sheffield">https://2010.igem.org/Team:Sheffield</a>
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          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 />
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Revision as of 13:11, 22 October 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017


Description

Synthetic biology
A microbial consortium chassis against cholera
Mimicking Vibrio cholerae
The sensing organism: Vibrio harveyi
The effecting organism: Pichia pastoris
Our system
References

Synthetic biology: to the multi-organisms communication and beyond

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 light1,2. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature’s tricks, basically by inserting genetic information from microorganisms into a single and unique one, most of the time Escherichia coli3. 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 biology4.

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 (Vibrio harveyi) to an effector cell (Pichia pastoris) in order to detect and eradicate a Vibrio cholera mimicking cell (Escherichia coli) using an antimicrobial peptides from crocodile.

Genesis of our molecular strategy

During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen5. Actually, the bacteria Vibrio cholerae 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.

Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with Vibrio cholerae6,7. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae8,9,10, using E. coli. They based their strategy around the quorum sensing system of V. cholerae in order to detect it, implementing CqsS receptor and the LuxU/O pathway into E. coli 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 Vibrio cholerae.

A microbial consortium chassis against cholera

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

  • To mimic V. cholerae by producing CAI-1 molecule. This will be done in E. coli
  • A bacteria with a quorum sensing pathway activated on the V. cholerae presence on which CAI-1 bind. Vibrio harveyi naturally possess that pathway. It will lead to the production of a messenger molecule: that we choose to be diacetyl.
  • The diacetyl binds to the Odr-10 receptor that can be expressed on yeast such as Pichia pastoris 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 Vibrio cholerae.

Mimicking Vibrio cholerae using Escherichia coli

An interesting property of Vibrio cholerae is its quorum sensing autoinducer system based on the production of CAI-1 molecule11. 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 Escherichia coli in order to mimic V. cholerae. Thus, we transformed E. coli strain with the CqsA synthase coding gene of Vibrio harveyi, non-pathogen bacteria. CqsA from V. harveyi produces an analog of CAI-1, the molecule C8-CAI-1, from (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A12. Finally, we developed an E. coli strain which produces a marker simulating the presence of the pathogen V. cholerae in the medium. This is the first step of our molecular cascade.

The sensing organism: Vibrio harveyi

Once we developed E. coli to produce the V. harveyi 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 V. harveyi. This bacteria is an advantageous good engineerable chassis. We identified that V. harveyi already possesses gene expression depending on the binding of C8-CAI-1 on its receptor, CqsS12. 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 V. harveyi 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-112.

Cascade of events depending on the CAI-1/CqsS binding in V. cholerae12. 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.

We checked the metabolism of diacetyl of V. harveyi 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.

Production of diacetyl from pyruvate. The addition of the acetolactate synthase (ALS) can lead to the production of acetolactate which convert itself into diacetyl without enzymatic process.

The effecting organism: Pichia pastoris

The molecular response to the presence of the mimicking vibrio E. coli strain is the production by V. harveyi of diacetyl. We then need a third partner to produce toxic molecule to kill V. cholerae. This last partner needs to be resistant to the toxic molecule so we choose an eukaryotic cell. Team SCUT13 previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, the Odr-10 receptor14,15. It is a G Protein Coupled Receptor isolated from Caenorhabditis elegans that once activated by diacetyl, lead to the activation of the pFUS1 promoter by Ste12. Pichia pastoris has been chosen as it displays already the Odr-10/pFUS1 pathway.

Activation cascade on the dependence of Diacetyl/Odr-10 binding13. 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.

Moreover, P. pastoris is a good protein producing organism16,17. We engineered the yeast to secret the toxic molecule under the promoter of Ste12. The toxic molecule secreted by P. pastoris is originated from crocodiles18,19,20,21. 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 V. cholerae. AMPs are cationic pore-forming molecules targeting bacterium membranes, causing bacterial lysis and death22. This is the third step of our cascade.

Mechanism of action of antimicrobial peptide and their effects on cells22. 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.
efficiency of the antimicrobial peptide from crocodile on V. cholerae18,20,21.The three peptides display and minimal inhibitory concentration 50 in the scale of mg/L.

Our system

See our Design page for more informations of the genetic engineering 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. http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
  6. 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
  7. 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
  8. https://2014.igem.org/Team:UI-Indonesia
  9. https://2010.igem.org/Team:Sheffield
  10. https://2014.igem.org/Team:UT-Dallas
  11. 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
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