Evolution of antibiotic resistance in mycobacteriaPublic Deposited
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There are existing developed systems for assessing mycobacterial death. Reporter systems have been developed that can distinguish between live and dead M. tuberculosis (Wang et. al 2019). In this example both GFP and RFP were expressed, one from an inducible promoter and one from a constitutive promoter (Wang et al. 2019). This allowed the researchers to distinguish between bacteria that were living and bacteria that had died, as only the live bacteria can express protein from the inducible promoter. Here we sought to develop a live-dead reporter system utilizing GFP and mScarlet. In the reporter system mScarlet would be driven by a constitutive promoter and accumulated protein would be present and remain detectable after cell death. GFP would be expressed by an inducible tet promoter (pmyc1) that can only be expressed by cells that are still alive when the inducer is added (Ehrt et al. 2005). This would allow for distinction between living and dead bacteria, as only the living bacteria would synthesize the inducible protein which can be observed using flow cytometry. This allows for measurement of the proportions of live and dead bacteria. The reporter system can then be used when introducing antibiotics to a bacterial population. This system can then be used as a tool not only to measure bacterial death in a less labor-intensive way but has further application in testing potential drug combinations.
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