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Title: Decomposition of the lactose operon
Author: Carmichael, C. S. J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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The immediate aims of the project, as set out in the introduction, were 1) to separate the lacZ and lacY genes of the lactose operon such that they could be controlled/induced independently 2) to maintain the expression construct in the E.coli chromosome. The lacY gene was subcloned into plasmid PBN372 downstream of the S.marsescens trp promoter. The flanking E.coli trp genes were exploited to integrate the construct into the E.coli chromosome at the trpB locus via homologous recombination. Homologous recombinants should be trp{-}. Two approaches were employed to achieve integration: 1) transformation of a recD strain (which was also a lacY deletion) 2) transduction with phage lambda. The first method was unsuccessful, only spontaneous trp- mutations were isolated. The second method yielded several integrants, one of which was used in subsequent growth experiments. Since the constructed strain was rendered trp-, the internal tryptophan concentration could be influenced by the concentration of tryptophan in the medium and the level of induction could be set by the addition of differing amounts of the antirepressor, IAA. The growth rate of the constructed strain in minimal lactose media was comparable with that of wild type E.coli. The permease activity of the constructed strain was seen to vary when assayed in the presence of varying amounts of IAA. The expression of permease was also demonstrated to be independent of galactosidase activity. The constructed strain therefore met all the initial requirements for the experimental system set out in this thesis. Difficulties were encountered during the analysis of permease induction in batch culture. This was due to the competition between antirepressor (IAA) and corepressor (tryptophan). These difficulties would have been minimal had the analysis been carried out in chemostat experiments. It would then have been possible to maintain a constant, low level of tryptophan throughout the experiment. In batch culture, the tryptophan level is constantly changing, initially being relatively high and becoming depleted as the experiment progresses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available