Characterising N-terminal amino acid incorporation and protein stability using a mutant i-tRNA(AAC)

Synthetic biology is a rapidly evolving field that harnesses principles derived from computational and biomolecular science to create novel biological systems. However, current hurdles such as reduced circuit control and efficiency prevent its full potential being realised for applications in industry, medicine, and research. One prospective solution to circumvent these issues is the idea of developing an orthogonal central dogma of biology. Orthogonal translation initiation from a non-canonical start codon is possible by simultaneously introducing a non-canonical start codon and a mutant initiator tRNA with a complimentary anticodon into cells, but little is known about which amino acids are incorporated into translated proteins. In this thesis, I explore the initiation fidelity of a mutant initiator tRNA with an AAC anticodon which specifically initiates from a noncanonical GUU start codon. Proteomic analysis shows that the mutant initiator tRNA substitutes valine for methionine as the first amino acid in reporter proteins, resulting in improved protein stability. This study serves as a roadmap for the measurement of other non-canonical initiator tRNA activities and the development of a system allowing for improved control of protein levels in vivo, bringing synthetic biology closer to achieving an orthogonal central dogma -- abstract.