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Title: Rational design, re-engineering and characterisation of tetrahydrofolate riboswitches in bacteria
Author: Leigh, James
ISNI:       0000 0004 5990 1284
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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The ability to independently control the expression of multiple genes in response to small-molecule inducers is highly desirable in the study of metabolic pathways and has numerous applications in the fields of drug discovery and synthetic biology. The existing small-molecule-inducible gene expression tools such as the lac, ara and tet systems have a number of limitations including poor titratability and a tendency to promote cell population heterogeneity. To address these shortcomings, the Micklefield laboratory has developed a novel method for controlling gene expression by re-engineering naturally occurring regulatory elements known as riboswitches. Riboswitches are mRNA-based, small-molecule-responsive genetic switches found in the 5’-UTR of many bacterial genes. We have previously shown that it is possible to re-engineer natural riboswitches, such that they are no longer activated by their cognate metabolite, but instead respond to a non-natural synthetic analogue not ordinarily present in the cell. Our most recent target for re-engineering was the tetrahydrofolate (THF)-responsive riboswitch found to control the folT gene in Firmicutes. The THF riboswitch is unique in that it contains two distinct ligand binding sites within the same aptamer domain, and was considered a candidate for the engineering of a dual-input, repressive gene expression system. To date, the THF riboswitch has not been characterised in vivo and therefore we also sought to further understand the ligand specificity and the role of the two binding sites in eliciting the riboswitch regulatory response. We established an in vivo reporter gene assay in E. coli and demonstrated that the THF riboswitch is able to dose-dependently repress gene expression in response to the THF analogue folinic acid. Ligand binding to the aptamer domain PK site was found to be the primary mechanism by which the regulatory response is mediated in vivo. We designed, synthesised and screened in vivo a series of THF analogues, which identified a potent inhibitor of the wild-type riboswitch, and was confirmed in vitro using ITC. Moreover, the de-repressive activity of the inhibitor compound was exploited to optimise the expression parameters of the wild-type riboswitch. In parallel, we successfully re-engineered one of the two aptamer domain ligand binding sites, such that it selectively bound a non-natural THF analogue whilst rejecting folinic acid as demonstrated in vitro. Additionally, this mutant riboswitch-ligand pair was shown to dose-dependently repress gene expression in vivo. This represents a significant step towards the engineering of a dual-input THF riboswitch gene expression system.
Supervisor: Micklefield, Jason Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available