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Title: Genetic tools for gene disruption in Rhodococcus
Author: Fernandes, A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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The genetic analysis of the soil actinomycete Rhodococcus has been hampered by a lack of genetic tools. In recent years methods for gene cloning by gain of function into an E. coli or Rhodococcus host have been established. Methods for cloning Rhodococcus genes (particularly into E. coli) are fraught with difficulties, due to restriction/methylation of DNA, integration and ineffectual gene expression in the host. The establishment of a gene disruption system would overcome these difficulties and allow selection of useful phenotypes by loss of function. In this work a recently developed in vitro Tn5-based mutagenesis system was adapted for use of Rhodococcus. Electroporation protocols generating sufficient numbers of transformants were established and a random knockout library was constructed in a Rhodococcus type-strain. Part of this work involved investigations of Rhodococcus cell envelope ultrastructure and the use of growth supplements to aid transformation. Library coverage was investigated by the identification and sequencing of a number of amino acid auxotrophs. The Tn5-based system was applied to a wild-type soil Rhodococcus isolate and a random knockout library was constructed. A number of mutants unable to grow in the presence of toluene and benzene were isolated. A number of transposon delivery vectors based on either Tn5 or IS903 were constructed and problems of transposant selection overcome. For the purposes of construction the sequencing and analysis of two Rhodococcus plasmid replicons was carried out. The IS903-based vector although fully functional in E. coli failed to transpose in Rhodococcus and the possible reasons are discussed. Preliminary characterisation of a putative inducible promoter from Rhodococcus was carried out and the use of reporter genes yfp and luxAB established. The replicative Tn5 delivery vector was adapted to include the promoter/regulator to drive transposase expression however this vector was subjected to deletion in the Rhodococcus host.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available