Ho, Yan-Kay;
(2025)
Investigating Genetic Code Plasticity
in Transient Expression Systems
Towards alternative genetic codes:
development of bacterial circuits for the robust and
efficient detection of transient sense-to-sense codon reassignment.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The natural genetic code, bar some exceptions, is universal. It dictates a specific and unambiguous set of rules regarding the assignment between RNA triplet (genetic information) and amino acid (protein product), which is enforced by transfer RNAs (tRNAs) and aminoacyl-tRNA synthetases (aaRSs). The latter ensures that amino acids are specifically charged to their cognate tRNAs, whilst the former establishes the physical bridge between amino acid and the associated messenger RNA (mRNA) triplet. However, these links can be rewritten by engineering the identity elements of tRNAs and/or by altering the substrate specificity of aaRSs to produce a modified genetic code. Alternative viable genetic codes, with reassigned codons, could be used to create enhanced biocontainment systems that are unable to contaminate the environment with engineered traits. Leucine-to-histidine reassignment (using the mRNA sequence as the frame of reference) can be accomplished by altering the anticodon region of E. coli histidyltRNA (EcotRNAHis) such that it base-pairs with leucine codons, whilst maintaining compatibility with its cognate histidyl-tRNA synthetase (EcoHisRS). Equivalent modifications to E. coli leucyl-tRNA (EcotRNALeu) should generate the complementary histidine-to-leucine reassignment. Such sense-to-sense codon reassignments are expected to produce a significant degree of toxicity in vivo, making it difficult to detect reassignments in heterologously-expressed histidine/leucine-recoded reporters. By incorporating serine recombinase phage integrase PhiC31 (ΦC31) with these codon-modified reporters, a biological toggle circuit platform was developed that produces a secondary non-toxic and permanent response to augment any initial transient signals of codon reassignment. I demonstrate that the developed platform is functional, and that leucine-to-histidine reassignment can be robustly detected. The genetic toggle switch assay, the first-of-its-kind, can be adapted to other codon reassignments and therefore represents a feasible route towards testing and characterising alternative sense-to-sense codon reassignments. Despite significant optimisation, the counter histidine-to-leucine reassignment, which would create the first viable orthogonal genetic code using the native translational machinery, was not successful in the time-frame of the project.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Investigating Genetic Code Plasticity in Transient Expression Systems Towards alternative genetic codes: development of bacterial circuits for the robust and efficient detection of transient sense-to-sense codon reassignment |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10213311 |
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