Our goal is to develop a biological tool able to neutralize gram-negative pathogens that are responsible for serious infectious diseases and epidemics. We want to design and engineer a "killer" conjugative plasmid that will be able to propagate within the microbiota and neutralize the target pathogens when transferred into them. To this end, a plasmid featuring high efficiency of transfer and broad host specificity will be equipped with a CRISPR Cas9 system and one or more guide RNAs specifically targeting the pathogens virulence genes. Upon expression in recipient cells, the customized CRISPR Cas9 system will selectively inactivate the targeted organisms by introducing double-strand breaks into its genome .
The combination of inactive Cas9 (dCas9) and conjugative plasmid for the transcriptional control of target genes has already been described (ref. doi: 10.1021/sb500036q). Relying on this publication, we want to push this forward and check for possible field applications. We plan to first demonstrate that the system works between different gram negative species and secondly, to build a fully equipped conjugative plasmid armed with several guide RNAs, an efficient bio containment system for preventing its dissemination in nature, and a tracking device for monitoring its dissemination in a bacterial population.
In the scope of our project we decided to focus on cholera outbreaks. Due to low hygienic standards and limited access to efficient antibiotics, refugee camps are the most exposed places to cholera outbreaks nowadays. This encourages us to design an efficient and low cost bio-based strategy that could be part of the solution to limit such outbreaks.
Many Vibrio cholerae strains exist but only a few of them are responsible for cholera outbreaks due to the expression of the cholera toxin CTX gene. This toxin is an oligomeric protein encoded by the ctxAB operon that is transmitted from virulent to non-virulent V. Cholerae by a filamentous bacteriophage called CTX.
A conjugative functional plasmid, integrating as much existing iGEM biobricks as possible, will be engineered in Escherichia coli. The transformed E.coli strain will be used for conjugation experiments with V. cholerae. An efficient and innovative containment system will be designed using a non natural chemical compound that will be made essential for the replication of the conjugative plasmid. A gene encoding a fluorescent protein will be added as a tracking system for monitoring the plasmid transfer.