Quantrell, Robert John Oliver

2018-05-14T10:15:34Z

2018-05-14T10:15:34Z

2008

Classic techniques designed to understand bacterial gene regulation in vitro are still used today to elucidate genetic mechanisms and pathways. However as much as we can learn from in vitro studies of bacterial behaviour, it is impossible to fully recreate the complex and multifaceted environment the bacterium faces in vivo. To this end the development of techniques to allow us to study gene regulation in vivo is important as these can then be applied to dissect bacterial pathogenesis and should uncover novel therapeutic targets.

Recombinase in vivo expression technology (RIVET) provides a heritable irreversible marker of gene activity in the host and has been applied recently to discover genes important for bacterial virulence in the host. The aim of this project was to develop a novel RIVET system using genes from the enterohaemorrhagic Escherichia coli 0157:H7 (EHEC)ftm operon and the beta-lactamase reporter gene (bid). This system was named FR1VET (Jim recombinase in vivo expression technology).

The basis to FRIVET is a completely synthetic operon placed in single copy in the EHEC chromosome at the Jim locus. The arabinose (ara) inducible promoter was tested in the system initially to validate the system in vitro. The key aim of these in vitro tests was to understand the working tolerances of the FRIVET operon and define appropriate control points. Emphasis was placed on 'setting' the system and controlling the levels of recombination achieved.

Construction of the FRIVET operon on allelic exchange vectors and subsequent exchange into a Shiga-like toxin negative EHEC strain was successful. In vitro tests using the ara promoter proved the system functioned as intended and was experimentally stable. When EHEC promoters, in particular LEE5, were tested in vitro, considerable difficulty was encountered in controlling levels of recombination and setting the system to the off status. This is essential for any use of the system to examine gene expression in vivo. Therefore the emphasis of future work must be on defining appropriate measures for controlling in vitro activity of chosen promoters when preparing constructs for in vivo challenges.

With appropriate modifications the FRIVET system has the potential to produce valuable data about EHEC gene expression in the host and therefore to contribute to our understanding of the complex regulation required to establish colonisation, maintain infection and induce pathology in vivo.

http://hdl.handle.net/1842/29956

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 18

Already catalogued

FRIVET: fim recombinase in vivo expression technology

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy