Team:Colombia/Project

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<center><b><h1  class="curs1">The Project</h1></b></center>
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TO DO: project in a nutshell.
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This year, iGEM Team Colombia has been hard at work developing of a biosensor for the human bacterial pathogen <i>Vibrio cholerae</i>. Our idea focuses on creating a easy-to-use kit capable of detecting this bacterium in different contexts, such as different kinds of food or water, by giving a simple visible color signal as a positive result. This kit would be useful for anyone who wants to detect cholera-infected food and water without having to use the expensive or tedious methods now available, such as immunogenic techniques or direct isolation and culture of bacteria. Possible users are companies in the food industry and quality or research labs.  
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<b> <font size="10"> &nbsp;&nbsp;Context </font> </b>
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<b><font color="#8A0808" size="5" >Cholera</font></b>
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<b> <font size="10"> Context </font> </b>
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Cholera has been a scourge to civilizations the world over since ancient times. According to the World Health Organization’s estimates, there are 3-5 million cholera cases and 100,000-120,000 deaths every year worldwide. Caused by the bacterial pathogen <i>Vibrio cholerae</i>, the disease has been responsible for seven global pandemics in recorded history, plus countless localized outbreaks (World Health Organization, 2014). These outbreaks are triggered by <b><i>V. cholerae</i></b> when present in water sources or food even at very small cell densities, and can have devastating effects, with mortality rates reaching up to an alarming 50% and causing death in a matter of hours if left untreated (Public Health Vigilance Group, 2011). The disease is most prevalent in low-income populations with inadequate health services and water management infrastructure (Public Health Vigilance Group, 2011).
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<b><font color="#8A0808" size="5" >Cholera</font></b>
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Although there is no official reports about confirmed cases in Colombia (due to the weak report system), some of the greatest risk factors are widespread across the country’s rural–and even urban–areas (Public Health Vigilance Group, 2011). Proof of this vulnerability is the impact generated by the arrival of the deadly El Tor <i>V. cholerae</i> strain in 1991, which caused 30,000 cholera cases in two years.
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Cholera has been a scourge to civilizations the world over since ancient times. According to the World Health Organization’s estimates, there are 3-5 million cholera cases and 100,000-120,000 deaths every year worldwide. Caused by the bacterial pathogen <i>Vibrio cholerae</i>, the disease has been responsible for seven global pandemics in recorded history, plus countless localized outbreaks (World Health Organization, 2014). These outbreaks are triggered by <i>V. cholerae</i> when present in water sources or food even at very small cell densities, and can have devastating effects, with mortality rates reaching up to an alarming 50% and causing death in a matter of hours if left untreated (Public Health Vigilance Group, 2011). The disease is most prevalent in low-income populations with inadequate health services and water management infrastructure (Public Health Vigilance Group, 2011). Although no cases have been confirmed in Colombia for a decade, some of the greatest risk factors are widespread across the country’s rural–and even urban–areas (Public Health Vigilance Group, 2011). Proof of this vulnerability is the impact generated by the arrival of the deadly El Tor <i>V. cholerae</i> strain in 1991, which caused 30,000 cholera cases in two years.
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Due to the disease’s alarmingly quick onset upon infection and the fact that rapid access to medical care is often not available in affected areas, prevention is a key factor in combating cholera. Careful monitoring of water sources and food stocks in areas of potential contamination is vital, especially during outbreaks. In spite of this need, there currently is no cost-effective way of detecting <i>V. cholerae</i> easily in environmental samples (Wang et al., 2010). The most effective ways of detecting the pathogen are culturing environmental samples in selective enrichment media in the lab and real-time PCR, which require a lab, trained personnel, and enough time to grow cultures. Rapid detection systems such as immunomagnetic beads or DNA probe hybridization can be unspecific and are based on molecular techniques that can be expensive or difficult to use (Wang et al., 2010). Recent research in environmental cholera detection has focused on improving upon existing techniques, such as adapting immunochromatographic dipstick tests used for stool samples (Chakraborty et al., 2013). Although there have been interesting synthetic biology projects aimed at preventing <i>V. cholerae</i> infection by meddling with the pathogen’s quorum sensing mechanism (Duan & March, 2010), there have been no attempts to build an environmental cholera biosensor using synthetic biology to date.  
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Due to the disease’s alarmingly quick onset upon infection and the fact that rapid access to medical care is often not available in affected areas, prevention is a key factor in combating cholera. Careful monitoring of water sources and food stocks in areas of potential contamination is vital, especially during outbreaks. In spite of this need, there currently is no cost-effective way of detecting <i>V. cholerae</i> easily in environmental samples (Wang et al., 2010). The most effective ways of detecting the pathogen are culturing environmental samples in selective enrichment media in the lab and real-time PCR, which require a lab, trained personnel, and <b>enough time </b> to grow cultures. Rapid detection systems such as immunomagnetic beads or DNA probe hybridization can be unspecific and are based on molecular techniques that can be expensive or difficult to use (Wang et al., 2010). Recent research in environmental cholera detection has focused on improving upon existing techniques, such as adapting immunochromatographic dipstick tests used for stool samples (Chakraborty et al., 2013). Although there have been interesting synthetic biology projects aimed at preventing <i>V. cholerae</i> infection by meddling with the pathogen’s quorum sensing mechanism (Duan & March, 2010), there have been no attempts to build an environmental cholera biosensor using synthetic biology to date.  
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In light of this, our project aims to use synthetic biology to develop a <i>V. cholerae</i> sensor using a new technique. Instead of trying to detect the cholerae toxin, specific sequences of nucleic acid, or antigens, we propose detecting <i>V. cholerae</i> Autoinducer 1 (CAI-1), the bacteria’s species-specific quorum sensing molecule. If we rewire <i>V. cholerae</i>’s own quorum sensing mechanism –used in nature to gauge population levels and regulate pathogenicity– in a harmless <i>E. coli</i> chassis, we can build a cheap and easy-to-use biosensor that gives a color output when it senses the pathogen. This prototype can serve as a proof-of-concept for future quorum sensing-based pathogen biosensors.
In light of this, our project aims to use synthetic biology to develop a <i>V. cholerae</i> sensor using a new technique. Instead of trying to detect the cholerae toxin, specific sequences of nucleic acid, or antigens, we propose detecting <i>V. cholerae</i> Autoinducer 1 (CAI-1), the bacteria’s species-specific quorum sensing molecule. If we rewire <i>V. cholerae</i>’s own quorum sensing mechanism –used in nature to gauge population levels and regulate pathogenicity– in a harmless <i>E. coli</i> chassis, we can build a cheap and easy-to-use biosensor that gives a color output when it senses the pathogen. This prototype can serve as a proof-of-concept for future quorum sensing-based pathogen biosensors.
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<b> <font size="10"> Quorum Sensing <br><br>Mechanism </font> </b>
 
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<b><font color="#8A0808" size="5" >Quorum Sensing Mechanism </font></b>
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To Sherlock Holmes she is always the woman. I have seldom heard him mention her under any other name. In his eyes she eclipses and predominates the whole of her sex. It was not that he felt any emotion akin to love for Irene Adler. All emotions, and that one particularly, were abhorrent to his cold, precise, but admirably balanced mind. He was, I take it, the most perfect reasoning and observing machine that the world has seen; but, as a lover, he would have placed himself in a false position. He never spoke of the softer passions, save with a gibe and a sneer. They were admirable things for the observer—excellent for drawing the veil from men’s motives and actions. But for the trained reasoner to admit such intrusions into his own delicate and finely adjusted temperament was to introduce a distracting factor which might throw a doubt upon all his mental results. Grit in a sensitive instrument, or a crack in one of his own high-power lenses, would not be more disturbing than a strong emotion in a nature such as his. And yet there was but one woman to him, and that woman was the late Irene Adler, of dubious and questionable memory.
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I had seen little of Holmes since the singular chain of events which I have already narrated in a bold fashion under the heading of The Sign of Four. My marriage had, as he foretold, drifted us away from each other. My own complete happiness, and the home-centred interests which rise up around the man who first finds himself master of his own establishment, were sufficient to absorb all my attention; while Holmes, who loathed every form of society with his whole Bohemian soul, remained in our lodgings in Baker Street, buried among his old books, and alternating from week to week between cocaine and ambition, the drowsiness of the drug, and the fierce energy of his own keen nature. He was still, as ever, deeply attracted by the study of crime, and occupied his immense faculties and extraordinary powers of observation in following out those clues, and clearing up those mysteries, which had been abandoned as hopeless by the official police. From time to time I heard some vague account of his doings: of his summons to Odessa in the case of the Trepoff murder, of his clearing up of the singular tragedy of the Atkinson brothers at Trincomalee, and finally of the mission which he had accomplished so delicately and successfully for the reigning family of Holland. Beyond these signs of his activity, however, which I merely shared with all the readers of the daily press, I knew little of my former friend and companion.
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<i>Vibrio cholerae</i>'s quorum sensing mechanism is comprised of two converging circuits: a non-specific branch governed by Auto-Inducer 2 (AI-2), common to a number of Gram-negative and -positive bacteria, and a species-specific branch governed by Cholera Auto-Inducer 1 (CAI-1), which proves to be far more interesting to our project do to its specificity (Svenningsen, Waters & Bassler, 2008). CAI-1 is synthetized by cytoplasmic protein CqsA, released to the extracellular environment, and detected by membrane protein dimer CqsS. At <b>low</b> concentrations of CAI-1, a phosphorylation cascade flows from CqsS through kinase LuxU to transcription factor LuxO. Phosphorylated LuxO acts upon promoter <i>pqrr</i> and, in <i>V. cholerae</i>, induces transcription of genes <i>qrr1</i> to <i>qrr4</i>, which encode a series of small, noncoding RNAs that base-pair to mRNA from gene <i>hapR</i>. With no <i>hapR</i> mRNA, virulence genes are upregulated, a strategy used by the pathogen in order to allow more gut space for its kind. When the pathogen has firmly established itself inside the human gut, virulence genes are therefore no longer needed. Because of this, at high concentrations of CAI-1, CqsS no longer phosphorylates LuxU, and in fact, the whole phosphorylation cascade reverses itself. This dephosphorylating behacior reinforces the signal. Therefore, LuxO stops activating  <i>pqrr</i>, which downregulates transcription of <i>qrr</i> genes and eventually inhibits virulence gene expression down the line (Svenningsen, Waters & Bassler, 2008).
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<b> <font size="10"> Design </font> </b>
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<center>Quorum sensing mechanism of <i>Vibrio cholerae</i>, according to Hammer & Bassler (2007). </center>
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<b><font size="10"> <div name="design">&nbsp;&nbsp;Design</div> </font></b>
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<b><font color="#8A0808" size="5" >Overview</font></b>
<b><font color="#8A0808" size="5" >Overview</font></b>
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One night—it was on the 20th of March, 1888—I was returning from a journey to a patient (for I had now returned to civil practice), when my way led me through Baker Street. As I passed the well-remembered door, which must always be associated in my mind with my wooing, and with the dark incidents of the Study in Scarlet, I was seized with a keen desire to see Holmes again, and to know how he was employing his extraordinary powers. His rooms were brilliantly lit, and, even as I looked up, I saw his tall spare figure pass twice in a dark silhouette against the blind. He was pacing the room swiftly, eagerly, with his head sunk upon his chest, and his hands clasped behind him. To me, who knew his every mood and habit, his attitude and manner told their own story. He was at work again. He had risen out of his drug- created dreams, and was hot upon the scent of some new problem. I rang the bell, and was shown up to the chamber which had formerly been in part my own.
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With <i>V. cholerae</i>'s quorum-sensing mechanism in mind, we designed a genetic circuit capable of detecting CAI-1, processing the signal and producing a visual output. The system is comprised of three parts: the receptor and transduction pathway, an inverter that processes the signal, and an output module with a positive feedback loop.
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<b><font color="#8A0808" size="5" >Receptor and transduction pathway</font></b>
<b><font color="#8A0808" size="5" >Receptor and transduction pathway</font></b>
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We use the same membrane receptor and transduction pathway in <i>V. cholerae</i>, explained above, to detect CAI-1 presence-absence. A phosphorylation cascade is activated in the <b>absence</b> of CAI-1, while a phosphatase reverse cascade is activated in the <b>presence</b> of the signal molecule. Phosphorylated LuxO activates the <i>pqrr4</i> promoter region.
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His manner was not effusive. It seldom was; but he was glad, I think, to see me. With hardly a word spoken, but with a kindly eye, he waved me to an armchair, threw across his case of cigars, and indicated a spirit case and a gasogene in the corner. Then he stood before the fire, and looked me over in his singular introspective fashion.
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<b><font color="#8A0808" size="5" >Signal processing: Inverter</font></b>
<b><font color="#8A0808" size="5" >Signal processing: Inverter</font></b>
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In principle, this step should be as simple as hooking up promoter <i>pqrr</i> to a reporter gene if signal presence were to induce gene expression. However, due to the fact that the pathway's natural design deactivates the promoter in the presence of CAI-1, an inverter device must be used to process the signal. A standard <i>tetR-ptet</i> repressor inverter device was built for this purpose (built from scratch from its constituent parts, because the device in our distribution appeared not to function properly–see <a href="https://2014.igem.org/Team:Colombia/Parts">Parts</a> for details).  
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‘Wedlock suits you,’ he remarked. ‘I think, Watson, that you have put on seven and a half pounds since I saw you.’
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‘Indeed, I should have thought a little more. Just a trifle more, I fancy, Watson. And in practice again, I observe. You did not tell me that you intended to go into harness.
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<b><font color="#8A0808" size="5" >Output: Fluorescent color protein</font></b>
<b><font color="#8A0808" size="5" >Output: Fluorescent color protein</font></b>
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‘I see it, I deduce it. How do I know that you have been getting yourself very wet lately, and that you have a most clumsy and careless servant girl?’
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Again, this system in principle should be a simple reporter gene under control of the inverter device's <i>ptet</i> promoter. However, our <a href="https://2014.igem.org/Team:Colombia/Modeling"> mathematical models</a> predicted a weak signal with this simple scheme. The solution is to add a positive feedback mechanism to reinforce the signal once CAI-1 is detected. Our modeling team recommended the use of <i>tetA</i>, which codes for an activator of promoter <i>ptet</i>. The reporter gene used is <i>amilCP</i>, a blue chromoprotein.
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‘My dear Holmes,’ said I, ‘this is too much. You would certainly have been burned had you lived a few centuries ago. It is true that I had a country walk on Thursday and came home in a dreadful mess; but, as I have changed my clothes, I can’t imagine how you deduce it. As to Mary Jane, she is incorrigible, and my wife has given her notice; but there again I fail to see how you work it out.
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He chuckled to himself and rubbed his long nervous hands together.
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<b> <font size="10"> Results </font> </b>
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Unfortunately, the <i>tetA</i> part did not work properly in the lab. The solution was to put <i>amilCP</i> under control of <i>psid</i>, a different inducible promoter, as well as <i>ptet</i>, the inverter's repressible promoter gene. Gene <i>psp3</i>, concatenated to <i>amilCP</i>, codes for an transcription activator that upregulates <i>psid</i>, thus completing the feedback loop.
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<b> <font size="10"> &nbsp;&nbsp;Results </font> </b>
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We constructed 7 new, RFC10-compatible parts, two of which were not submitted to the registry because we could not transfer them to the appropriate plasmid backbones on time. Among the parts we built are two reporter genes under the control of <i>ptet</i>. One of these was used to build two different versions of the output circuit: one version with <i>psid</i>, thus completing the feedback loop, and another stand-alone version without the inducible promoter. We also built a circuit capable of producing phosphorylated LuxO when induced by arabinose to test the signal processing and output circuits. Additionally, we built our own Quad-Part tetracycline Inverter device, because we found the one in our distribution to be faulted. We also built a <i>pqrr4</i>-containing biobrick; this part, however, did not appear to function as expected. Finally, since we cannot work with actual <i>V. cholerae</i> due to obvious biosecurity concerns, we need a way of testing the circuits we build without having to deal with the pathogen. For this reason, <i>cqsA</i> was cloned in <i>E. coli</i> (BBa_K581011) to use as a positive control for the whole system.
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You can check a table outlining our parts <a href="https://2014.igem.org/Team:Colombia/Parts">here</a>.
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<center>Here is an example of an E: coli DH5α culture carrying psid - pTet - AmilCP - pag activator (PSP3).</center>
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We also participated in the Interlab contest. You can read about our results <a href="https://2014.igem.org/Team:Colombia/Interlab">here</a>.
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<b> <font size="10">References</font> </b>
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<b> <font size="10"> &nbsp;&nbsp;References </font> </b>
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<li>Chakraborty, S., Alam, M., Scobie, H. M., & Sack, D. A. (2013). Adaptation of a simple dipstick test for detection of Vibrio cholerae O1 and O139 in environmental water. Frontiers in microbiology, 4.</li>
<li>Chakraborty, S., Alam, M., Scobie, H. M., & Sack, D. A. (2013). Adaptation of a simple dipstick test for detection of Vibrio cholerae O1 and O139 in environmental water. Frontiers in microbiology, 4.</li>
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<li>Duan, F., & March, J. C. (2010). Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proceedings of the National Academy of Sciences, 107(25), 11260-11264.</li>
<li>Duan, F., & March, J. C. (2010). Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proceedings of the National Academy of Sciences, 107(25), 11260-11264.</li>
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<li>Svenningsen, S. L., Waters, C. M., & Bassler, B. L. (2008). A negative feedback loop involving small RNAs accelerates Vibrio cholerae’s transition out of quorum-sensing mode. Genes & development, 22(2), 226-238.
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<li>Public Health Vigilance Group. (2011) Plan de contingencia del sector salud para la prevención y control de cólera en Colombia [Health sector contingency plan for the prevention and control of cholera in Colombia]. Ministry of Social Protection, Republic of Colombia. Accessed 8 june 2014 from <http://www.minsalud.gov.co/Documentos%20y%20Publicaciones/PLAN%20DE%20CONTINGENCIA%20DEL%20SECTOR%20SALUD%20PARA%20LA%20PREVENCI%C3%93N%20Y%20CONTROL%20DE%20C%C3%93LERA%20EN%20COLOMBIA.pdf></li>
<li>Public Health Vigilance Group. (2011) Plan de contingencia del sector salud para la prevención y control de cólera en Colombia [Health sector contingency plan for the prevention and control of cholera in Colombia]. Ministry of Social Protection, Republic of Colombia. Accessed 8 june 2014 from <http://www.minsalud.gov.co/Documentos%20y%20Publicaciones/PLAN%20DE%20CONTINGENCIA%20DEL%20SECTOR%20SALUD%20PARA%20LA%20PREVENCI%C3%93N%20Y%20CONTROL%20DE%20C%C3%93LERA%20EN%20COLOMBIA.pdf></li>
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<li>Wang, D., Xu, X., Deng, X., Chen, C., Li, B., Tan, H., ... & Kan, B. (2010). Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay. Applied and environmental microbiology, 76(16), 5520-5525.</li>
<li>Wang, D., Xu, X., Deng, X., Chen, C., Li, B., Tan, H., ... & Kan, B. (2010). Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay. Applied and environmental microbiology, 76(16), 5520-5525.</li>
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<li>World Health Organization. (2014) Cholera: Fact sheet No. 107. Media Center Fact Sheets. Retrieved 8 june 2014 from <http://www.who.int/mediacentre/factsheets/fs107/en/>.</li>
<li>World Health Organization. (2014) Cholera: Fact sheet No. 107. Media Center Fact Sheets. Retrieved 8 june 2014 from <http://www.who.int/mediacentre/factsheets/fs107/en/>.</li>
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The Project


This year, iGEM Team Colombia has been hard at work developing of a biosensor for the human bacterial pathogen Vibrio cholerae. Our idea focuses on creating a easy-to-use kit capable of detecting this bacterium in different contexts, such as different kinds of food or water, by giving a simple visible color signal as a positive result. This kit would be useful for anyone who wants to detect cholera-infected food and water without having to use the expensive or tedious methods now available, such as immunogenic techniques or direct isolation and culture of bacteria. Possible users are companies in the food industry and quality or research labs.







  Context


Cholera

Cholera has been a scourge to civilizations the world over since ancient times. According to the World Health Organization’s estimates, there are 3-5 million cholera cases and 100,000-120,000 deaths every year worldwide. Caused by the bacterial pathogen Vibrio cholerae, the disease has been responsible for seven global pandemics in recorded history, plus countless localized outbreaks (World Health Organization, 2014). These outbreaks are triggered by V. cholerae when present in water sources or food even at very small cell densities, and can have devastating effects, with mortality rates reaching up to an alarming 50% and causing death in a matter of hours if left untreated (Public Health Vigilance Group, 2011). The disease is most prevalent in low-income populations with inadequate health services and water management infrastructure (Public Health Vigilance Group, 2011).
Although there is no official reports about confirmed cases in Colombia (due to the weak report system), some of the greatest risk factors are widespread across the country’s rural–and even urban–areas (Public Health Vigilance Group, 2011). Proof of this vulnerability is the impact generated by the arrival of the deadly El Tor V. cholerae strain in 1991, which caused 30,000 cholera cases in two years.

Statistics

Due to the disease’s alarmingly quick onset upon infection and the fact that rapid access to medical care is often not available in affected areas, prevention is a key factor in combating cholera. Careful monitoring of water sources and food stocks in areas of potential contamination is vital, especially during outbreaks. In spite of this need, there currently is no cost-effective way of detecting V. cholerae easily in environmental samples (Wang et al., 2010). The most effective ways of detecting the pathogen are culturing environmental samples in selective enrichment media in the lab and real-time PCR, which require a lab, trained personnel, and enough time to grow cultures. Rapid detection systems such as immunomagnetic beads or DNA probe hybridization can be unspecific and are based on molecular techniques that can be expensive or difficult to use (Wang et al., 2010). Recent research in environmental cholera detection has focused on improving upon existing techniques, such as adapting immunochromatographic dipstick tests used for stool samples (Chakraborty et al., 2013). Although there have been interesting synthetic biology projects aimed at preventing V. cholerae infection by meddling with the pathogen’s quorum sensing mechanism (Duan & March, 2010), there have been no attempts to build an environmental cholera biosensor using synthetic biology to date.

Colombia FoodPoisoning.png


In light of this, our project aims to use synthetic biology to develop a V. cholerae sensor using a new technique. Instead of trying to detect the cholerae toxin, specific sequences of nucleic acid, or antigens, we propose detecting V. cholerae Autoinducer 1 (CAI-1), the bacteria’s species-specific quorum sensing molecule. If we rewire V. cholerae’s own quorum sensing mechanism –used in nature to gauge population levels and regulate pathogenicity– in a harmless E. coli chassis, we can build a cheap and easy-to-use biosensor that gives a color output when it senses the pathogen. This prototype can serve as a proof-of-concept for future quorum sensing-based pathogen biosensors.



Quorum Sensing Mechanism

Vibrio cholerae's quorum sensing mechanism is comprised of two converging circuits: a non-specific branch governed by Auto-Inducer 2 (AI-2), common to a number of Gram-negative and -positive bacteria, and a species-specific branch governed by Cholera Auto-Inducer 1 (CAI-1), which proves to be far more interesting to our project do to its specificity (Svenningsen, Waters & Bassler, 2008). CAI-1 is synthetized by cytoplasmic protein CqsA, released to the extracellular environment, and detected by membrane protein dimer CqsS. At low concentrations of CAI-1, a phosphorylation cascade flows from CqsS through kinase LuxU to transcription factor LuxO. Phosphorylated LuxO acts upon promoter pqrr and, in V. cholerae, induces transcription of genes qrr1 to qrr4, which encode a series of small, noncoding RNAs that base-pair to mRNA from gene hapR. With no hapR mRNA, virulence genes are upregulated, a strategy used by the pathogen in order to allow more gut space for its kind. When the pathogen has firmly established itself inside the human gut, virulence genes are therefore no longer needed. Because of this, at high concentrations of CAI-1, CqsS no longer phosphorylates LuxU, and in fact, the whole phosphorylation cascade reverses itself. This dephosphorylating behacior reinforces the signal. Therefore, LuxO stops activating pqrr, which downregulates transcription of qrr genes and eventually inhibits virulence gene expression down the line (Svenningsen, Waters & Bassler, 2008).

QuorumSensing.gif

Quorum sensing mechanism of Vibrio cholerae, according to Hammer & Bassler (2007).


  Design


Overview

With V. cholerae's quorum-sensing mechanism in mind, we designed a genetic circuit capable of detecting CAI-1, processing the signal and producing a visual output. The system is comprised of three parts: the receptor and transduction pathway, an inverter that processes the signal, and an output module with a positive feedback loop.



Screen Shot 2014-10-17 at 10.48.49 AM.png




Receptor and transduction pathway

We use the same membrane receptor and transduction pathway in V. cholerae, explained above, to detect CAI-1 presence-absence. A phosphorylation cascade is activated in the absence of CAI-1, while a phosphatase reverse cascade is activated in the presence of the signal molecule. Phosphorylated LuxO activates the pqrr4 promoter region.

Colombia Transduction.png






Signal processing: Inverter

In principle, this step should be as simple as hooking up promoter pqrr to a reporter gene if signal presence were to induce gene expression. However, due to the fact that the pathway's natural design deactivates the promoter in the presence of CAI-1, an inverter device must be used to process the signal. A standard tetR-ptet repressor inverter device was built for this purpose (built from scratch from its constituent parts, because the device in our distribution appeared not to function properly–see Parts for details).

Colombia Inverter.png






Output: Fluorescent color protein

Again, this system in principle should be a simple reporter gene under control of the inverter device's ptet promoter. However, our mathematical models predicted a weak signal with this simple scheme. The solution is to add a positive feedback mechanism to reinforce the signal once CAI-1 is detected. Our modeling team recommended the use of tetA, which codes for an activator of promoter ptet. The reporter gene used is amilCP, a blue chromoprotein.

Colombia Output.png

Unfortunately, the tetA part did not work properly in the lab. The solution was to put amilCP under control of psid, a different inducible promoter, as well as ptet, the inverter's repressible promoter gene. Gene psp3, concatenated to amilCP, codes for an transcription activator that upregulates psid, thus completing the feedback loop.






  Results

We constructed 7 new, RFC10-compatible parts, two of which were not submitted to the registry because we could not transfer them to the appropriate plasmid backbones on time. Among the parts we built are two reporter genes under the control of ptet. One of these was used to build two different versions of the output circuit: one version with psid, thus completing the feedback loop, and another stand-alone version without the inducible promoter. We also built a circuit capable of producing phosphorylated LuxO when induced by arabinose to test the signal processing and output circuits. Additionally, we built our own Quad-Part tetracycline Inverter device, because we found the one in our distribution to be faulted. We also built a pqrr4-containing biobrick; this part, however, did not appear to function as expected. Finally, since we cannot work with actual V. cholerae due to obvious biosecurity concerns, we need a way of testing the circuits we build without having to deal with the pathogen. For this reason, cqsA was cloned in E. coli (BBa_K581011) to use as a positive control for the whole system.

You can check a table outlining our parts here.

Partepsid.jpg

Here is an example of an E: coli DH5α culture carrying psid - pTet - AmilCP - pag activator (PSP3).


We also participated in the Interlab contest. You can read about our results here.



  References

  1. Chakraborty, S., Alam, M., Scobie, H. M., & Sack, D. A. (2013). Adaptation of a simple dipstick test for detection of Vibrio cholerae O1 and O139 in environmental water. Frontiers in microbiology, 4.

  2. Duan, F., & March, J. C. (2010). Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proceedings of the National Academy of Sciences, 107(25), 11260-11264.

  3. Svenningsen, S. L., Waters, C. M., & Bassler, B. L. (2008). A negative feedback loop involving small RNAs accelerates Vibrio cholerae’s transition out of quorum-sensing mode. Genes & development, 22(2), 226-238.
  4. Public Health Vigilance Group. (2011) Plan de contingencia del sector salud para la prevención y control de cólera en Colombia [Health sector contingency plan for the prevention and control of cholera in Colombia]. Ministry of Social Protection, Republic of Colombia. Accessed 8 june 2014 from

  5. Wang, D., Xu, X., Deng, X., Chen, C., Li, B., Tan, H., ... & Kan, B. (2010). Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay. Applied and environmental microbiology, 76(16), 5520-5525.

  6. World Health Organization. (2014) Cholera: Fact sheet No. 107. Media Center Fact Sheets. Retrieved 8 june 2014 from .




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