Engineered semi-orthogonal initiator tRNAs initiate translation at non-canonical start codons

Bioengineering is an emerging discipline within molecular biology. It involves the study,the design and the construction of existing and new biological systems through biomolecular techniques. Being a relatively nascent field, bioengineering faces numerous obstacles in its practice such as reduced synthetic circuit efficiency and control, reduced cellular viability due to the introduction of exogenous circuits and possibilities of biocontainment issues of synthetic designs. Incorporating orthogonality into the design of synthetic circuits has previously been suggested as a promising tool for overcoming these prominent obstacles within the field of bioengineering. Within this thesis, I have developed and built an orthogonal set of initiator tRNAs with the aim of achieving translation initiation from non-conical start codons. I designed and constructed 9 new mutant initiator tRNAs, through altering the anticodon region of the metY gene in Escherichia coli, and tested their efficacy through an anticodon-codon reporter system. I was able to observe successful translation initiation from most of these mutant tRNAs from non-canonical start codons. I believe the results obtained within this dissertation will act as a pilot study for future research, which will explore a more complete set of initiator tRNA variants.