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Title: Detection of bacterial gene expression by a novel isothermic nucleic acid amplification technology
Author: Thomas, Alistair Owen
ISNI:       0000 0001 3517 2580
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2004
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A novel probe-based technique called Signal-Mediated Amplification Reaction Technology (SMART) was optimised for detection of RNA targets in order to quantify gene expression. The SMART assay was used to quantify both 23 S rRNA in P. aeruginosa PAOl and gfpmuti mRNA in the plasmid-borne rpoSwgfpmvXi fusions P. aeruginosa SS429 and SS431. However, the assay was not sufficiently sensitive to detect gfpmvfo mRNA from the chromosomal rpoS::gfpmut3 fusion P. aeruginosa SS336. SDS-PAGE analysis of outer membrane proteins of P. aeruginosa PAOl revealed that cells grown in a reported iron-replete chemically defined medium CDMio were in fact limiting for iron. Modifications to the growth medium such as increased iron concentration, reduction in pH and the addition of citrate and ascorbate all failed to produce an iron-replete phenotype. This was achievable only when MOPSO buffer was replaced with phosphate buffer, indicating that by some unknown mechanism MOPSO can reduce iron availability in minimal media. The effects of nutrient limitation on rpoS expression in P. aeruginosa planktonic and biofilm culture were investigated using direct fluorescence measurement of rpoS::gfpmut3 chromosomal and plasmid fusions. In planktonic culture, nutrient-replete and magnesium-limited conditions resulted in an increase in rpoS expression whilst minimal levels of rpoS expression were seen in both iron-limited and glucose-limited conditions. Furthermore, minimal expression of rpoS was noted in P. aeruginosa biofilms in glucose, magnesium and iron-limited conditions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available