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Reprogramming the genetic code: defining a new start codon in Escherichia coli

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posted on 28.03.2022, 02:53 authored by Russel Miranda Vincent
Translation maps triplet RNA codons onto single amino acids within a protein. Bacterial translation initiation occurs at a conserved AUG start codon recognised by the initiator tRNA anticodon, with studies demonstrating that codon-anticodon complementarity is sufficient for initiation. Thus far, engineering initiation codons have been neglected aside from one identified mutant amber initiator tRNA (tRNA CUAfmet2) that can start protein synthesis from the UAG stop codon. In this study, I create a new inducible system to express tRNA CUAfmet2 and a new set of fluorescent reporter plasmids to enable population-level studies for the first time. Fluorescence measurements confirmed that tRNA CUAfmet2 initiates translation from UAG start codon with 200-fold increase on inducing tRNA CUAfmet2 compared to the repressed condition with 30-fold increase in reporter expression from an AUG start codon in similar conditions. Time-course measurements indicate different initiation effects as tRNA CUAfmet2 matures. Proteomic analysis of cells expressing tRNA CUAfmet2 reveal a minimal effect on the host proteome with no evidence of peptides initiating from genomic UAG codons. I demonstrate that my tRNA CUAfmet2-expressing plasmid is transportable in all five E. coli strains with no adverse fitness defects. In this thesis, I define tRNA CUAfmet characteristics with future scope for refinement for this orthogonal translation initiation system -- abstract.

History

Table of Contents

Chapter 1: Introduction -- Chapter 2: Methods -- Chapter 3: Results -- Chapter 4: Discussion -- References -- Supplementary material.

Notes

Bibliography: pages i-vii Theoretical thesis.

Awarding Institution

Macquarie University

Degree Type

Thesis MRes

Degree

MRes, Macquarie University, Faculty of Science, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award

2017

Principal Supervisor

Paul R. Jaschke

Rights

Copyright Russel Miranda Vincent 2017. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource [68 pages] colour illustrations

Former Identifiers

mq:71737 http://hdl.handle.net/1959.14/1277568