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Title: FRIVET : fim recombinase in vivo expression technology
Author: Quantrell, Robert John Oliver
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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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 O157:H7 (EHEC) fim operon and the beta-lactamase reporter (bla). This system was named FRIVET (fim recombinase in vivo expression technology). The basis to FRIVET is a completely synthetic operon placed in single copy in the EHEC chromosome at the fim 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available