Difference between revisions of "Team:INSA-UPS France/Design"
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<img style="width:25%;min-width: 260px; position:absolute;right:0;top:-200px; " src="https://static.igem.org/mediawiki/2017/e/e3/T--INSA-UPS_France--design_croco.png" alt=""> | <img style="width:25%;min-width: 260px; position:absolute;right:0;top:-200px; " src="https://static.igem.org/mediawiki/2017/e/e3/T--INSA-UPS_France--design_croco.png" alt=""> | ||
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| − | + | Our project created from ground a synthetic consortium to demonstrate the power of this approach through its application to the cholera thematic. Our proof of concept consortium involves three microorganism: i) an engineered <i>Escherichia coli</i> to mimic<i> Vibrio cholerae</i> ii) a modified </i>Vibrio harveyi</i> to sense the presence of the engineered <i>E. coli</i> and produce diacetyl in response iii) the yeast </i>P. pastoris</i> modified to detect diacetyl and to induce the production of antibacterial peptides (AMP) in order to trigger lysis of<i> V. cholerae</i>. Here is presented a closer view of the molecular details of our project for each micro-organism, and our experimental plan. | |
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<h1 style="text-align: left;">Organisms</h1> | <h1 style="text-align: left;">Organisms</h1> | ||
| − | <h2><i> | + | <h2><i>Escherichia coli</i></h2> |
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| − | + | The bacteria gram negative <i>E. coli</i> was chosen to mimic<i> V. cholerae</i> as we were not allowed to work with pathogens. Moreover <i>E. coli</i> is an easy organism to deal with, especially as it is well documented, easy to transform with exogenous DNA and easy to culture. The strain K-12 MG1655 was transformed with a plasmid allowing expression of the protein CqsA from<i> V. cholerae</i>, the enzyme responsible for the synthesis of CAI-1. However, as a proof of concept in our project, we also transformed our <i>E. coli</i> strain with the gene coding for the CqsA of </i>V. harveyi</i>, a non-pathogen strain, producing the molecule C8-CAI-1 (an analogue of the<i> V. cholerae</i> CAI-1).<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">1</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/15466044" target="_blank">1</a></sup> | |
| − | + | This molecule is a carbohydrate chain based displaying an hydroxyl group on carbon 3 and ketone function on carbon 4. The CqsA synthetase from </i>V. harveyi</i> is able to produce C8-CAI-1 from endogenous <i>E. coli</i> (S)-adenosylmethionine (SAM) and octanoyl-coenzyme. Both quorum sensing producing system are inducible with the pLac promoter. The plasmid backbone is pSB1C3 to maintain compatibility with the iGEM registry. </p> | |
<img src="https://static.igem.org/mediawiki/2017/f/fc/T--INSA-UPS_France--design_plasmid-coli.png" alt="" style="width: 10%; position:absolute;bottom:0; left:10%;"> | <img src="https://static.igem.org/mediawiki/2017/f/fc/T--INSA-UPS_France--design_plasmid-coli.png" alt="" style="width: 10%; position:absolute;bottom:0; left:10%;"> | ||
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Revision as of 14:43, 28 October 2017
iGEM UPS-INSA Toulouse 2017
Our project created from ground a synthetic consortium to demonstrate the power of this approach through its application to the cholera thematic. Our proof of concept consortium involves three microorganism: i) an engineered Escherichia coli to mimic Vibrio cholerae ii) a modified Vibrio harveyi to sense the presence of the engineered E. coli and produce diacetyl in response iii) the yeast P. pastoris modified to detect diacetyl and to induce the production of antibacterial peptides (AMP) in order to trigger lysis of V. cholerae. Here is presented a closer view of the molecular details of our project for each micro-organism, and our experimental plan.
The bacteria gram negative E. coli was chosen to mimic V. cholerae as we were not allowed to work with pathogens. Moreover E. coli is an easy organism to deal with, especially as it is well documented, easy to transform with exogenous DNA and easy to culture. The strain K-12 MG1655 was transformed with a plasmid allowing expression of the protein CqsA from V. cholerae, the enzyme responsible for the synthesis of CAI-1. However, as a proof of concept in our project, we also transformed our E. coli strain with the gene coding for the CqsA of V. harveyi, a non-pathogen strain, producing the molecule C8-CAI-1 (an analogue of the V. cholerae CAI-1).1,1
This molecule is a carbohydrate chain based displaying an hydroxyl group on carbon 3 and ketone function on carbon 4. The CqsA synthetase from V. harveyi is able to produce C8-CAI-1 from endogenous E. coli (S)-adenosylmethionine (SAM) and octanoyl-coenzyme. Both quorum sensing producing system are inducible with the pLac promoter. The plasmid backbone is pSB1C3 to maintain compatibility with the iGEM registry.
V. harveyi has all the assets to be a good sensor for V. cholerae: it possesses its own pathway of detection of C8-CAI-1, an analogue of CAI-1. A single point mutation allows V. harveyi: to detect V. cholerae’s molecule CAI-11. Moreover, the strain that we are using, JMH626, has been deleted for the enzyme responsible of the production and detection of other quorum sensing molecules, making it a specific sensor of CAI-12. However it cannot be used as the effector because of its physiological proximity to V. cholerae. Thus we supposed that the production of antimicrobial peptides aimed at V. cholerae would be lethal to it.
The pBBR1MCS-4 broad host range plasmid was chosen so we can transfer the system into V.harveyi using a conjugation method.
Recognized as a great protein producer and secretor, P. pastoris has already been used to produce a wide range of AMPs3,4. Furthermore, the diacetyl/Odr-10 system has been described as a useful tool for prokaryotic/eukaryotic communication5.
The pPICZα plasmid was chosen because of its α-factor and its homology sequence allowing it to integrate in a targeted zone in its genome. It is recognized as a good plasmid for protein production in P. pastoris.
We chose to take advantage of the intraspecies quorum sensing of V. cholerae: the CAI-1/CqsS system. To mimic this pathway in our laboratory, we had to both produce in vivo the CAI-1 molecule in a bacteria strain and express the CqsS receptor in an other one.
The CAI-1 producing system is inducible in order to avoid toxicity problems. The fact that CqsS* can detect both CAI-1 and C8-CAI-1 led us to choose V. harveyi cqsA gene (Vh-cqsA), instead of V. cholerae gene, that produces C8-CAI-1 for safety reasons.1
V. harveyi has the natural pathway leading to the activation or inactivation of pqrr4. At high CAI-1 concentration the promoter is inactivated, thus we needed an inverter, tetR/pTet allowed us to activate the als gene that produces diacetyl at high CAI-1 concentration:
We chose to use the diacetyl/Odr-10 binding receptor system, that is known to activate gene expression on yeasts.
The constitutive pGAP promoter allows the system to always express the Odr-10 receptor and thus be sensible to diacetyl at any time.
Once the Odr-10 receptor has sensed diacetyl, pFUS is activated and it triggers the Ste12 pathway. Then, the production of antimicrobial peptides (AMP) can start. In order for the cells to excrete the peptides, an α-factor is needed.
Quorum sensing molecule production
Conjugation
diacetyl production
Antimicrobial peptides (AMP)
diacetyl detection
Design
Overview
Organisms
Escherichia coli
V. harveyi
P. pastoris
Modules & Parts
Sense
Transmit
Respond
Experimental plan
E. coli
V. harveyi
P. pastoris
