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A potential sensor-effector biocircuit to facilitate selective killing of resistant bacteria

By Barbara Rieck


This project focused on an ecological solution to the problem of antimicrobial resistance that depended on promoting the selection of susceptible bacteria over resistant bacteria. If achievable, this could reduce the likelihood of infections being caused by resistant pathogens, thus preserving the life-saving utility of antibiotics. The specific aims were to design and construct genetic switches that were predicted to sense and respond to the presence of resistance-related mRNA, and to validate the selective nature of these switches using gene-reporter systems. Ultimate proof-of-concept would require the building of resistance-sensing lysogenic-lytic switches into the bacteriophage λ genome to generate „anti-resistance‟ phages that selectively kill resistant bacteria. A reporter plasmid encoding cI857 governed by λ PRM promoter and a ß-galactosidase gene expressed from the λ CI857-responsive PR promoter was constructed. Production of the CI857 repressor blocked expression of ß-galactosidase. However, multiple distinct manipulations of the sequence between PRM and the start of cI857, to allow for subsequent introduction antisense orientated DNA fragments, resulted in loss of CI857 production and non-functional CI-Reporters. Only insertion of a restriction site and a Shine-Dalgarno sequence between truncated and intact versions of cI857 and the introduction of a restriction site downstream of cI857 led to functional CI-reporters. Analysis of various E. coli DH5α derivatives, simultaneously harbouring a CI-reporter bearing an antisense-orientated integron-derived DNA fragment and a second plasmid expressing sense integron RNA failed to reveal reduced CI857 production, suggesting negligible antisense effects for the candidate switches and target RNA sequences investigated. Nevertheless, data from an in vitro protein expression experiment utilizing a CI-reporter and an antisense oligonucleotide supported the notion that relevant antisense RNA effects could occur, perhaps only when optimum sense:antisense RNA ratios were achieved. The tools constructed and data derived have laid the groundwork for future pursuit of the proposed anti-resistance phages

Publisher: University of Leicester
Year: 2009
OAI identifier: oai:lra.le.ac.uk:2381/7445

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