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

 
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         Synthetic biology
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         Synthetic Consortia
 
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        <a href="#a2" data-number="2">
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          Genesis of our molecular strategy
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         A microbial consortium chassis against cholera
 
         A microbial consortium chassis against cholera
 
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         Mimicking <i>Vibrio cholerae</i>
 
         Mimicking <i>Vibrio cholerae</i>
 
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         The sensing organism: <i>Vibrio harveyi</i>
 
         The sensing organism: <i>Vibrio harveyi</i>
 
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         The effecting organism: <i>Pichia pastoris</i>
 
         The effecting organism: <i>Pichia pastoris</i>
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        Our system
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        References
 
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     <section>
 
     <section>
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       <h1>Combine to improve: a synthetic biology scale-up</h1>
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       <h1>Synthetic biology: from unique chassis to synthetic consortia</h1>
 
       <p>
 
       <p>
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        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.
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      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>
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<figure>
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      <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
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        <figcaption>
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        <b> Figure 1: General concept of the cell to cell communication. </b> This cascade of events will be developed in our project.
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        </figcaption>
 +
      </figure>
 
       <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?
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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.
 
       </p>
 
       </p>
 
       <p>
 
       <p>
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        For our iGEM project, we decided to focus on the multi organisms aspect, making communication between prokaryotic and eukaryotic possible.
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      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.
 
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<div class="article_offset" id="a2"></div>
 
     <section>
 
     <section>
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       <h1>A core: prokaryotic-eukaryotic communication
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       <h1>Genesis of our molecular strategy
 
</h1>
 
</h1>
 
       <p>
 
       <p>
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         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.
+
         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>
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       <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
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       <p>
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      <p class="left-p">
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        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.  
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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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      <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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        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.
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       <h1>A microbial consortium against cholera</h1>
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       <h1>A microbial consortium chassis against cholera
 +
</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.  
+
      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>
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     </section>
 
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     <div class="article_offset" id="a4"></div>
 
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     <section>
 
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       <h1>Finding a sensor</h1>
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       <h1>Mimicking <i>Vibrio cholerae</i> using <i>Escherichia coli</i></h1>
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       <p class="left-p">
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       <p>
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         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.
+
         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>.  
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      <figure>
 
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        <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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      </figure>
 
 
       <p>
 
       <p>
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        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 therefore developed <i>E. coli</i> strains which produce markers simulating the presence of <i>Vibrio </i> species in the medium.  
 
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       <h1>Finding an effector</h1>
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       <h1>The sensing organism: <i>Vibrio harveyi</i></h1>
 
       <p>
 
       <p>
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        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 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.
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       </p>
 
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      <figure>
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        <img src="https://static.igem.org/mediawiki/2017/0/0a/T--INSA-UPS_FRANCE--Design_T1.png" alt="" class="right-img">
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        <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>
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        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>.  
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      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>
 
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        <img src="https://static.igem.org/mediawiki/parts/0/0a/T--INSA-UPS_France--ALSpathway.png" alt="">
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        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.
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        <figcaption>
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       </p>
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          <b>Figure 3: Production of diacetyl from pyruvate</b>.
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      <h1>The effector organism: <i>Pichia pastoris</i></h1>
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      <p>
 +
        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>
 +
      <figure>
 +
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 +
        <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>
 +
 +
         
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       <h1 style="text-align:left;">Our system</h1>       
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       <h1>Our system</h1>       
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         See design for more informations of the genetic engineering we used!
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      <h1>References</h1>     
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      <ol>
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        <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 />
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        <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/</a>
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}
+
        </li>
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          Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. <i>Nature Neuroscience</i>. <br/>
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/26308982">https://www.ncbi.nlm.nih.gov/pubmed/26308982</a>
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        </li>
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          Cameron E, Bashor C &amp; Collins J (2014) A brief history of synthetic biology. <i>Nature Reviews Microbiology</i>
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/24686414">https://www.ncbi.nlm.nih.gov/pubmed/24686414</a>
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          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 />
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          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/</a>
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          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.
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/27596754
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">https://www.ncbi.nlm.nih.gov/pubmed/27596754</a>
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          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).
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/26809519
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">http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
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          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 />
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        </li>
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          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="http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079">http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079</a>
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        </li>
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        <li>
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          <a href="https://2014.igem.org/Team:UI-Indonesia">https://2014.igem.org/Team:UI-Indonesia</a>
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        </li>
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        <li>
 +
          <a href="https://2010.igem.org/Team:Sheffield">https://2010.igem.org/Team:Sheffield</a>
 +
        </li>
 +
        <li>
 +
          <a href="https://2014.igem.org/Team:UT-Dallas">https://2014.igem.org/Team:UT-Dallas</a>
 +
        </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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/22001326">https://www.ncbi.nlm.nih.gov/pubmed/22001326</a>
 +
        </li>
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        <li>
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          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 />
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472">https://www.ncbi.nlm.nih.gov/pubmed/21219472</a>
 +
        </li>
 +
        <li>
 +
          <a href="https://2013.igem.org/Team:SCUT">https://2013.igem.org/Team:SCUT</a>
 +
        </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 />
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          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/</a>
 +
        </li>
 +
        <li>
 +
          Audet M &amp; Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. <i>Cell</i> <b>151</b> 14–2 <br />
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/2302121">https://www.ncbi.nlm.nih.gov/pubmed/2302121</a>
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        </li>
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        <li>
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          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 />
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          <a href="https://www.ncbi.nlm.nih.gov/pubmed/27905091">https://www.ncbi.nlm.nih.gov/pubmed/27905091</a>
 +
        </li>
 +
        <li>
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          Huang Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris. <i>Experimental and Therapeutic Medicine</i> <br />
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          <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/</a>
 +
        </li>
 +
        <li>
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          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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/21184776">https://www.ncbi.nlm.nih.gov/pubmed/21184776</a>
 +
        </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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/2059789">https://www.ncbi.nlm.nih.gov/pubmed/2059789</a>
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        </li>
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        <li>
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          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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460">https://www.ncbi.nlm.nih.gov/pubmed/28159460</a>
 +
        </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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554">https://www.ncbi.nlm.nih.gov/pubmed/24192554</a>
 +
        </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 />
 +
          <a href="https://www.ncbi.nlm.nih.gov/pubmed/27837316">https://www.ncbi.nlm.nih.gov/pubmed/27837316</a>
 +
        </li>
 +
      </ol>
 +
    </section>
  
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          <td><a href="https://2017.igem.org/Team:INSA-UPS_France/Design">Design</a></td>
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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