Translation initiation is a sequential process involving interactions between the 30S small ribosomal subunit, initiation factors and initiator tRNA. The Escherichia coli K-12 strain is unique in the Escherichia because it has two different initiator tRNA sequences, tRNA fMet1 encoded by the metZWV genes and tRNA fMet2 encoded by the metY gene. A mutant of the metY gene was previously made where the anticodon sequence, responsible for specifying the start codon where translation initiation begins, was changed so that it bound to the amber stop codon UAG instead of the usual AUG start codon [1]. This amber initiator tRNA has already been shown to be functional in the K-12 strain [1] [2], but it is unclear whether it would function in other strains normally lacking the tRNA fMet2 variant. In this work, we transformed E. coli K-12, and four other generally regarded as safe (GRAS) laboratory strains, with a plasmid expressing the amber initiator tRNA and evaluated its functionality and growth effects on the bacteria. We performed these tests because, despite these strains all belonging to E. coli phylogenetic group A, it is well known that there is significant variation between even closely related E. coli strains in their metabolism [3] [4] [5], transcriptional response to exogenous DNA expression [6] and rates of amber stop codon suppression [7]. We found that the amber initiator functions similarly across the five strains, effectively initiating translation at the orthogonal UAG start codon and that it had modest growth-slowing effects in the Crooks, W, and K-12 strains. The five tested E. coli strains in this work (K-12, B, C, W, and Crooks) are important workhorses of academic and industrial research and development. The path is now clear to deploy the amber initiator tRNA into these five strains to precisely control gene expression.