Latest revision as of 09:49, 12 December 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 chassis^1,2,3. 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. cholerae^8,9. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae^10,11,12, 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. harveyi*¹⁵.**

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

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/

Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. Nature Neuroscience.
https://www.ncbi.nlm.nih.gov/pubmed/26308982

Cameron E, Bashor C & Collins J (2014) A brief history of synthetic biology. Nature Reviews Microbiology https://www.ncbi.nlm.nih.gov/pubmed/24686414

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/

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

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

http://www.emro.who.int/yem/yemeninfocus/situation-reports.html

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

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

https://2014.igem.org/Team:UI-Indonesia

https://2010.igem.org/Team:Sheffield

https://2014.igem.org/Team:UT-Dallas

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

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

https://2013.igem.org/Team:SCUT

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/

Audet M & Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. Cell 151 14–2
https://www.ncbi.nlm.nih.gov/pubmed/2302121

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

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/

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

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

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

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

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

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

Latest revision as of 09:49, 12 December 2017

Description

Synthetic biology: from unique chassis to synthetic consortia

Genesis of our molecular strategy

A microbial consortium chassis against cholera

Mimicking Vibrio cholerae using Escherichia coli

The sensing organism: Vibrio harveyi

The effector organism: Pichia pastoris

Our system

References

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+          Genesis of our molecular strategy
+        </a>
+      </div>
+      <div class="aside-nav__item">
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          A microbial consortium chassis against cholera
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          The sensing organism: <i>Vibrio harveyi</i>
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          The effecting organism: <i>Pichia pastoris</i>
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          Our system
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          References
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      <div class="article_offset" id="a1"></div>
      <section>
-       <h1>Synthetic biology: to the multi-organisms communication and beyond </h1>
+       <h1>Synthetic biology: from unique chassis to synthetic consortia</h1>
        <p>
-        Nature is still developing a wide large diversity of remarkably efficient pathways in order to sense presence of specific chemical, or even physical parameters such as temperature, pressure and light<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/" target="_blank">1</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/26308982" target="_blank">2</a></sup>. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature&rsquo;s tricks, basically by inserting genetic information from microorganisms into a single and unique one, most of the time <i>Escherichia coli</i><sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/24686414" target="_blank">3</a></sup>. However, focusing only on this type of bacteria is not appropriate to reflect the large complexity of living organisms and more, their intimate relationship in Nature. This aspect starts to be a limiting border in the way of the development of the synthetic biology<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/" target="_blank">4</a></sup>.
+       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>
+ <figure>
+      <img src="https://static.igem.org/mediawiki/2017/b/b3/T--INSA-UPS_France--description_sense-effect.png" alt="">
+        <figcaption>
+        <b> Figure 1: General concept of the cell to cell communication. </b> This cascade of events will be developed in our project.
+        </figcaption>
+      </figure>
        <p>
-        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 antimicrobial peptides from crocodile.
+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>
-      <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>
-        During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen<sup><a href="http://www.emro.who.int/yem/yemeninfocus/situation-reports.html" target="_blank">5</a></sup>. Actually, the bacteria <i>Vibrio cholerae</i> that causes cholera disease is usually found in water and infects more than a million of people each year. This terrible situation led us to focus on this problematic and it appeared that current solutions were not efficient enough to deal with this situation.
+       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>
+    </section>
+<div class="article_offset" id="a2"></div>
+    <section>
+      <h1>Genesis of our molecular strategy
+</h1>
        <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="https://www.ncbi.nlm.nih.gov/pubmed/16697733" target="_blank">6</a>,<a href="http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079" target="_blank">7</a></sup>. Additionally, some iGEM teams tried also to deal with the challenging detection of <i>V. cholerae</i><sup><a href="https://2014.igem.org/Team:UI-Indonesia" target="_blank">8</a>,<a href="https://2010.igem.org/Team:Sheffield" target="_blank">9</a>,<a href="https://2014.igem.org/Team:UT-Dallas" target="_blank">10</a></sup>, using <i>E. coli</i>. They based their strategy around the quorum sensing system of <i>V. cholerae</i> in order to detect it, implementing CqsS receptor and the LuxU/O pathway into <i>E. coli</i> in order to activate gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough maybe due to the process complexity of introducing a large amount of DNA information in a single microorganism. That is why we built a synthetic consortium of microorganisms against <i>Vibrio cholerae</i>.
+        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>
+         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>
      </section>
-     <div class="article_offset" id="a2"></div>
+     <div class="article_offset" id="a3"></div>
      <section>
        <h1>A microbial consortium chassis against cholera
 </h1>
        <p>
-        We finally created an artificial consortium chassis to deal with cholera disease. The different partners are described below.
+      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>
+We finally created an artificial consortium chassis to deal with cholera disease. The different partners are deeper described below.
        </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 binds. <i>Vibrio harveyi</i> naturally possesses that pathway. It will lead to the production of a messenger molecule that we chose 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 starts a molecular pathway.This pathway leads to the activation of pFUS1 and will produce our antimicrobial peptides with a secretion cassette. Those peptides will kill <i>Vibrio cholerae</i>.
-        </li>
-      </ul>
@@ Line 168: / Line 181: @@
-     <div class="article_offset" id="a3"></div>
+     <div class="article_offset" id="a4"></div>
      <section>
        <h1>Mimicking <i>Vibrio cholerae</i> using <i>Escherichia coli</i></h1>
        <p>
-         An interesting property of <i>Vibrio cholerae</i> is its quorum sensing autoinducer system based on the production of CAI-1 molecule<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/22001326" target="_blank">11</a></sup>. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is a good reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain <i>Escherichia coli</i> in order to mimic <i>V. cholerae</i>. Thus, we transformed <i>E. coli</i> strain with the CqsA synthase coding gene of <i>Vibrio harveyi</i>, non-pathogen bacteria. CqsA from <i>V. harveyi</i> produces an analog of CAI-1, the molecule C8-CAI-1, from (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A12. Finally, we developed an <i>E. coli</i> strain which produces a marker simulating the presence of the pathogen <i>V. cholerae</i> in the medium. This is the first step of our molecular cascade.
+         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>
+We therefore developed <i>E. coli</i> strains which produce markers simulating the presence of <i>Vibrio </i> species in the medium.
        </p>
      </section>
-     <div class="article_offset" id="a4"></div>
+     <div class="article_offset" id="a5"></div>
      <section>
        <h1>The sensing organism: <i>Vibrio harveyi</i></h1>
        <p>
-        Once we developed <i>E. coli</i> to produce the <i>V. harveyi</i> C8-CAI-1, this molecule as to be detected in the medium. The easiest way to do it is to use directly the quorum sensing of the non-pathogen <i>V. harveyi</i>. This bacteria is an advantageous good engineerable chassis. We identified that <i>V. harveyi</i> already possesses gene expression depending on the binding of C8-CAI-1 on its receptor, CqsS<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">12</a></sup>. For example, pQRR4 is a promoter which activation depends on the presence of C8-CAI-1. To fit with CAI-1 molecule, the CqsS receptor of <i>V. harveyi</i> only needed to be mutated on a single amino acid. We only had to mutate CqsS changing the phenylalanine 175 into a cystein and to integrate the ALS gene under the control of pQRR4 to trigger diacetyl production in presence of CAI-1<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">12</a></sup>.
+       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>
        <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/2017/0/0a/T--INSA-UPS_FRANCE--Design_T1.png" alt="" class="right-img">
          <figcaption>
-         <b>Cascade of events depending on the CAI-1/CqsS binding in V. cholerae<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21219472" target="_blank">12</a></sup></b>. the CAI-1/CqsS binding will start a dephosphorylation cascade leading to the inhibition of pQRR4 and its depending siRNA. The lack of his siRNA will allow the translation of their targeted mRNA.
+         <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>
+       </figcaption>
-      </figure>
+       </figure>
        <p>
-        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, an ubiquitous metabolite. This is the second step of our molecular cascade.
+       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>
-      <figure>
+<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.
+           <b>Figure 3: Production of diacetyl from pyruvate</b>.
          </figcaption>
        </figure>
      </section>
-     <div class="article_offset" id="a5"></div>
+     <div class="article_offset" id="a6"></div>
      <section>
-       <h1>The effecting organism: <i>Pichia pastoris</i></h1>
+       <h1>The effector organism: <i>Pichia pastoris</i></h1>
        <p>
-         The molecular response to the presence of the mimicking vibrio <i>E. coli</i> strain is the production by <i>V. harveyi</i> of diacetyl. We then need a third partner to produce toxic molecule to kill <i>V. cholerae</i>. This last partner needs to be resistant to the toxic molecule so we choose an eukaryotic cell. Team SCUT<sup><a href="https://2013.igem.org/Team:SCUT" target="_blank">13</a></sup> previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, the Odr-10 receptor<sup><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/" target="_blank">14</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/2302121" target="_blank">15</a></sup>. It is a G Protein Coupled Receptor isolated from <i>Caenorhabditis elegans</i> that once activated by diacetyl, lead to the activation of the pFUS1 promoter by Ste12. <i>Pichia pastoris</i> has been chosen as it displays already the Odr-10/pFUS1 pathway.
+         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>
          <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="https://2013.igem.org/Team:SCUT" target="_blank">13</a></sup></b>. Once diacetyl bind to Odr-10 a cascade of activation of Ste proteins will lead to the binding of Ste12 on pFUS1 promoter, and so to the expression of gene of interest.
+           <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>
+      </figcaption>
        </figure>
-      <p>
+       <p>
-        Moreover, <i>P. pastoris</i> is a good protein producing organism<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27905091" target="_blank">16</a>,<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/" target="_blank">17</a></sup>. We engineered the yeast to secret the toxic molecule under the promoter of Ste12. The toxic molecule secreted by <i>P. pastoris</i> is originated from crocodiles<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/21184776" target="_blank">18</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/2059789" target="_blank">19</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460" target="_blank">20</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554" target="_blank">21</a></sup>. Crocodiles display a remarkable and efficient immune system, allowing the reptiles to resist to a large spectrum of diseases. Thus, they produced antimicrobial peptides (AMPs) which are able to lyse bacteria such as <i>V. cholerae</i>. AMPs are cationic pore-forming molecules targeting bacterium membranes, causing bacterial lysis and death<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27837316" target="_blank">22</a></sup>. This is the third step of our cascade.
+       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>
-      <figure>
-        <img src="https://static.igem.org/mediawiki/2017/8/80/T--INSA-UPS_France--Description-kill.png" alt="">
-        <figcaption>
-          <b>Mechanism of action of antimicrobial peptide and their effects on cells<sup><a href="https://www.ncbi.nlm.nih.gov/pubmed/27837316" target="_blank">22</a></sup>. </b> Antimicrobial peptides are making pore formation into the membrane leading to death of the cell. Transmission electron microscopy provide an insight of the effect of the peptide on the cell.
-        </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="https://www.ncbi.nlm.nih.gov/pubmed/21184776" target="_blank">18</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/28159460" target="_blank">20</a>,<a href="https://www.ncbi.nlm.nih.gov/pubmed/24192554" target="_blank">21</a></sup></b>.The three peptides display and minimal inhibitory concentration 50 in the scale of mg/L.
-        </figcaption>
-      </figure>
      </section>
-     <div class="article_offset" id="a6"></div>
+     <div class="article_offset" id="a7"></div>
      <section style="background: none;">
        <h1>Our system</h1>
        <p>
-         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!
+         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>
-       <img style="max-width: 800px;" src="https://static.igem.org/mediawiki/2017/b/b8/T--INSA-UPS_France--description_loop.png" alt="">
+       <img style="max-width: 800px;" src="https://static.igem.org/mediawiki/2017/archive/b/b8/20171101194815%21T--INSA-UPS_France--description_loop.png" alt="">
      </section>
@@ Line 244: / Line 251: @@
      </style>
-     <div class="article_offset" id="a7"></div>
+     <div class="article_offset" id="a8"></div>
      <section>
        <h1>References</h1>
@@ Line 262: / Line 269: @@
             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>
+           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.
+           <a href="https://www.ncbi.nlm.nih.gov/pubmed/27596754
+">https://www.ncbi.nlm.nih.gov/pubmed/27596754</a>
+         </li>
+         <li>
+           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).
+           <a href="https://www.ncbi.nlm.nih.gov/pubmed/26809519
+">https://www.ncbi.nlm.nih.gov/pubmed/26809519</a>
           </li>
           <li>
@@ Line 333: / Line 350: @@
           </li>
         </ol>
+    </section>
+    <style>
+      .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>
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        <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>
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+                  $('*[data-number="8"]').css("font-weight", "normal");
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+                  $('*[data-number="8"]').css("font-weight", "normal");
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+                  $('*[data-number="8"]').css("font-weight", "normal");
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+                  $('*[data-number="8"]').css("font-weight", "normal");
+                  $('*[data-number="8"]').parent().removeClass("selected-item");
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+                  $('*[data-number="8"]').css("font-weight", "normal");
+                  $('*[data-number="8"]').parent().removeClass("selected-item");
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+                  $('*[data-number="8"]').css("font-weight", "normal");
+                  $('*[data-number="8"]').parent().removeClass("selected-item");
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+                    $('*[data-number="1"]').css("font-weight", "normal");
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