Translation, folding and function of bacteriophage T4 DNA topoisomerase gene 60

Halablab, Mona-Lissa-Khaled (2022) Translation, folding and function of bacteriophage T4 DNA topoisomerase gene 60. PhD thesis, University of Glasgow.

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Biotechnology enables the design and assembly of customized biological systems to deliver solutions to challenges in medicine, agriculture, sustainable energy production and industrial processes. One current objective within biotechnology, as well as synthetic biology, is to create tools that can be used to manipulate biological systems in a reliable and predictable manner. While many transcription-based and translation-based control devices have been reported, they are based on a limited repertoire of biological component types, and there is a need for new systems that can be used to implement more elaborate devices.

This work explored the potential of reprogramming translation for engineering new gene-regulatory tools. Chapter 1 reviews the standard process of protein synthesis and the numerous translation reprogramming mechanisms that exist in nature, focusing on the role of the RNA message and the nascent translated peptide in these processes. Previous efforts to reprogram translation are also covered, including an examination of potential opportunities for reprogramming translation that have not yet been explored. In Chapter 2, the materials and methods used throughout this work are presented.

In Chapter 3, a reporter system is developed for examining elements of bacteriophage T4 DNA topoisomerase gene 60 mRNA that reprogram protein synthesis by translational bypassing. It is demonstrated that the reporter system is a reliable tool for identifying and verifying previously characterized gene 60 translational bypassing elements and proposes the use of the sequences for reprogramming translation through RNA. In Chapter 4, an experimental framework is developed for engineering de novo translational bypassing devices (based on T4 gene 60 bypassing elements) and their application to regulate protein synthesis in bacteria. Evidence is presented that demonstrates that these engineered devices can regulate gene expression and control the relative stoichiometry of two major distinct protein outputs from one gene. The utility of these devices in expanding our synthetic capabilities to manipulate biological systems is explored, as well as revealing unforeseen details of the bypassing mechanism in T4 gene 60.

A recent focus of synthetic biology has been the control of protein function in cells. The achievement of this goal has focused on split protein complementation approaches, in particular when the N- and C- terminal fragments are fused to inducible protein-protein interaction systems, that enable spatiotemporal control of gene expression. In Chapter 5, a previously predicted protein-protein interaction system between two T4 DNA topoisomerase subunit fragments is demonstrated and its application for generating functional split proteins is explored. Lastly, the concluding remarks on this work are presented in Chapter 6.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Colloms, Dr. Sean D. and Woolhead, Prof. Cheryl A.
Date of Award: 2022
Depositing User: Theses Team
Unique ID: glathesis:2022-82933
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Jun 2022 12:45
Last Modified: 03 Jul 2023 08:42
Thesis DOI: 10.5525/gla.thesis.82933

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