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Engineering of RNA sensors and actuators in living cells

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Rostain, William (2017) Engineering of RNA sensors and actuators in living cells. PhD thesis, University of Warwick.

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Official URL: http://webcat.warwick.ac.uk/record=b3109836~S15

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Abstract

The aim of synthetic biology is to create a new discipline of engineering based on biological parts, devices and systems. The availability of predictable, programmable tools to sense and to control gene expression is central to our ability to engineer such systems. Ribonucleic acid (RNA) is an attractive building material to create such programmable tools, as RNA-RNA interactions are predictable and RNA secondary structure prediction software has been developed. Design rules for creating such parts using RNA can be established, based on a standardised approach or on structural design rules into which function is implicitly encoded. In this latter case, RNA folding software can be used to create RNA sequence which satisfy generalisable structural characteristics, but are tailored to a specific application. In this work, new design rules for the creation of RNA-based sensors and actuators are developed. The actuator parts are based on riboregulators, but with a circular topology generated through splicing of a ribozyme. The ability of these circular riboregulators to activate transcription of gene expression in E. coli cells is demonstrated. A method for improving these actuators by directed evolution is then tested. Finally, design rules for creating sensors of RNAs based Clustered Regularly Interspaced Short Palindromic Repeat guide RNAs (CRISPR gRNAs) are developed. These gRNA-based sensors can switch states and repress gene expression through a CRISPR-Cas9 based platform, but only in the presence of an arbitrary "trigger" RNA. The rules developed for creating sensors and actuators are characterised in E. coli, but are based on general principles that could be used in other organisms including eukaryotic cells.

Item Type: Thesis (PhD)
Subjects: Q Science > QH Natural history > QH301 Biology
Library of Congress Subject Headings (LCSH): Synthetic biology, Bioengineering, RNA -- Biotechnology, Gene expression
Official Date: June 2017
Dates:
DateEvent
June 2017Submitted
Institution: University of Warwick
Theses Department: School of Life Sciences
Thesis Type: PhD
Publication Status: Unpublished
Supervisor(s)/Advisor: Jaramillo, Alfonso (Jaramillo Rosales)
Sponsors: France. Délégation générale pour l’armement ; Defence Science and Technology Laboratory (Great Britain) ; European Commission
Format of File: pdf
Extent: xix, 205 pages : illustrations, charts
Language: eng

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