Design of temperature inducible transcription factors and cognate promoters

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dc.contributor.author McWhinnie, Ralph
dc.date.accessioned 2016-05-30T14:29:34Z
dc.date.available 2017-05-14T11:22:05Z
dc.date.copyright 2016 en_US
dc.date.issued 2016-05-30
dc.identifier.uri http://hdl.handle.net/1828/7331
dc.description.abstract The ability to control expression of a gene of interest is an important tool of molecular biologists and genetic engineers. This allows the phenotype associated with the regulated gene or genetic pathway to be partially de-coupled from the genotype and expressed only under condition that lend to induction of the genetic control system employed. Such control is typically implemented through a repressor protein (Eg. TetR, LacI) which will repress transcription when bound to a promoter containing a binding site (operator) recognized specifically by that repressor. Many such repressors and their cognate promoters are well-defined and characterized in model genetic systems, such as Escherichia coli, and may function poorly in other bacterial species. A lack of genetic components that allow the controlled expression of heterologous genes in less well studied bacterial species may limit their bio-industrial potential and the sophistication of engineered phenotypes. The work presented here uses random mutagenesis and selection to isolate mutants of TetR that are inducible by increased culture temperature. Induction of protein expression by temperature change can have benefits over repressors that require small-molecule inducers in bio-industrial applications as reversal of induction and reuse of growth medium are possible. The host range of these, or any, repressor protein is limited by the host range in which its cognate promoter will function. To bypass this limitation and allow use of TetR in Francisella novicida, a method was developed by which TetR-responsive promoters that function in this host could be selected from random DNA sequence flanking the TetR binding site, tetO. Many unique TetR-repressible promoters that function in Francisella were recovered and tightly-regulated expression of both exogenous reporter genes and host virulence genes were demonstrated. This promoter selection technique was also applied to E. coli, which allowed comparison between Francisella-selected promoters and those selected in an E. coli host. Adaption of this process for production of promoters responsive to transcription factors other than TetR would simply require the use of a different operator sequence, suggesting diverse applications for this technique. This success in promoter engineering should enable advances in synthetic biology and genetic engineering in non-model bacterial species. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Transcription en_US
dc.subject Synthetic biology en_US
dc.subject Bacterial genetics en_US
dc.subject Escherichia coli en_US
dc.subject Francisella en_US
dc.subject Promoter en_US
dc.subject Protein engineering en_US
dc.subject TetR en_US
dc.title Design of temperature inducible transcription factors and cognate promoters en_US
dc.type Thesis en_US
dc.contributor.supervisor Nano, Francis E.
dc.degree.department Department of Biochemistry and Microbiology en_US
dc.degree.level Doctor of Philosophy Ph.D. en_US
dc.identifier.bibliographicCitation McWhinnie, R. L., & Nano, F. E. (2014). Synthetic Promoters Functional in Francisella novicida and Escherichia coli. Applied and Environmental Microbiology, 80(1), 226–34. http://doi.org/10.1128/AEM.02793-13 en_US
dc.description.scholarlevel Graduate en_US

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