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Design of a Fluorescent mRNA Biosensor

Cook, Alexander (2016) Design of a Fluorescent mRNA Biosensor. Master of Science by Research (MScRes) thesis, University of Kent,.

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Abstract

Currently, to measure the amount of mRNA produced during transcription, a post transcription assay is required, such as gel electrophoresis or reverse transcription- quantitative polymerase chain reaction (RT-qPCR) which all require RNA purification steps. Both methods are subject to high error and in addition, RT-qPCR is a long process which can take hours to run. This study describes a method in which post transcriptional mRNA can be qualitatively measured between reaction conditions or quantitatively measured after calibration. The post transcription assay utilises a fluorescent Escherichia coli single stranded binding protein (SSB). In this study, SSB showed similar binding properties to mRNA, to that of its native substrate, ssDNA. A mutant of SSB -SSB G26C- used in previous studies, was fluorescently labelled with 7-Diethylamino-3-((((2-Maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). A fluorescence increase occurs when the SSB is bound to both ssDNA and ssRNA, and this increase is dependent on substrate concentration. This study shows the MDCC-SSB can be used as a comparative measurement of mRNA concentrations following in vitro transcription. After finding this method unsuitable for real time studies, we investigated potential causes for this behaviour and comment on improvements to the biosensor.

Item Type: Thesis (Master of Science by Research (MScRes))
Thesis advisor: Toseland, Christopher
Uncontrolled keywords: SSB Biosensor mRNA Fluorescence ssDNA
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Users 1 not found.
Date Deposited: 28 Sep 2016 13:00 UTC
Last Modified: 29 May 2019 17:54 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/57542 (The current URI for this page, for reference purposes)
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