A synthetic biological sensor utilizes biological elements and assembles them into biological circuits, which generate a measurable signal in the presence of a target environmental input. The presence of vancomycin in nature can lead to the development of resistance, making environmental monitoring crucial for preventing this phenomenon. The two-component VanRS system is a regulatory system of vancomycin-resistant bacteria that recognizes the vancomycin signal in the environment via the receptor histidine kinase VanS. VanS activates the transcriptional regulator VanR, which recruits RNA-polymerase to the target promoter and thus initiates the transcription of genes for vancomycin resistance. In the absence of signal the receptor VanS acts as phosphatase.
In the master's thesis, we wanted to transfer the two-component VanRS system into the Gram-negative E. coli. Since vancomycin does not cross the outer membraneof these bacteria, we use the E. coli strain GKCW104 as the chassis, which is capable of synthesizing pores on the outer membrane in the presence of arabinose, thus enabling vancomycin to enter the periplasm. Using the viability test, we determined the maximum concentration of vancomycin (0,1 μg/mL) that does not affect cell survival over an eight-hour period.
Following the RFC 10 recommendations of the synthetic biology standard RFC 10, we prepared a vector encoding green fluorescent protein (GFP) under the inducible promoter PvanY. The nucleotide sequences vanR and vanS were first obtained from the bacterium E. faecalis using polymerase chain reaction and than inserted under constitutive promotor into a biological circuit with the reporter part already contained. We successfully transformed E. coli GKCW104 with a vector carrying the biosensor components and determined concentration dependence of the signal on final biosensor and the occurrence of non-specific fluorescence on an incomplete biosensor.The appearance of non-specific fluorescence, which was not dependent on the presence of vancomycin, was observed in samples with an incomplete biosensor circuit. The intensity was even higher than in samples with a complete circuit. When studying the response of biosensor cells to different concentrations of vancomycin, the fluorescence intensities did not increase with the concentration of vancomycin, from which we concluded that the prepared biosensor is not capable of reacting to vancomycin in the selected concentration range.
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