The genetic analysis of the soil actinomycete <i>Rhodococcus </i>has been hampered by a lack of genetic tools. In recent years methods for gene cloning by gain of function into an <i>E. coli</i> or <i>Rhodococcus</i> host have been established. Methods for cloning <i>Rhodococcus</i> genes (particularly into <i>E. coli</i>) 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 <i>in vitro</i> Tn5-based mutagenesis system was adapted for use of <i>Rhodococcus</i>. Electroporation protocols generating sufficient numbers of transformants were established and a random knockout library was constructed in a <i>Rhodococcus</i> type-strain. Part of this work involved investigations of <i>Rhodococcus</i> 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 <i>Rhodococcus </i>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 IS<i>903</i> were constructed and problems of transposant selection overcome. For the purposes of construction the sequencing and analysis of two <i>Rhodococcus </i>plasmid replicons was carried out. The IS<i>903</i>-based vector although fully functional in <i>E. coli</i> failed to transpose in <i>Rhodococcus </i>and the possible reasons are discussed. Preliminary characterisation of a putative inducible promoter from <i>Rhodococcus </i>was carried out and the use of reporter genes <i>yfp </i>and <i>luxAB</i> 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 <i>Rhodococcus </i>host.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:598990 |
| Date | January 2001 |
| Creators | Fernandes, A. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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