Source: http://2014.igem.org/Team:Colombia/Project
Timestamp: 2019-04-19 21:21:27+00:00

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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.
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.
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.
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.
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).
Quorum sensing mechanism of Vibrio cholerae, according to Hammer & Bassler (2007).
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.
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.
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).
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.
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.
You can check a table outlining our parts here.
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.
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.
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.
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.
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.
World Health Organization. (2014) Cholera: Fact sheet No. 107. Media Center Fact Sheets. Retrieved 8 june 2014 from .

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