It is indeed surprising that the amber supressor tRNA also increases translation from the ordinary AUG start codons. In fact, it means that we cannot say whether the amber suppressor tRNA specifically increases translation initiation from amber codons or just increases translation in general, for example through its effect on ribosome maturation, as also mentioned by the authors. It would strengthen this manuscript considerably to do a control experiment where the tRNA expressed from the plasmid is the wildtype metY initiator tRNA, rather than the amber suppressor. The authors cite two papers (ref 19, 20) for showing that the wildtype metY does not initiate translation from amber codons, but that observation has not been tested in the two-plasmid system used here. As it is, we can't say for sure that the modified metY with the amber anticodon actually works as an amber suppressor, since it increases translation from both amber codons and AUG codons. This is a major change that must be addressed.
The results in Figure 1F shows comparisons of the growth of an E. coli strain carrying a plasmid, compared to the same strain without the plasmid. Such a comparison does not necessarily say anything about the effects of the tRNA gene encoded on the plasmid, the differences could just as easily be due to the antibiotics resistance gene expressed from the plasmid, the altered composition of the medium required for selection for the plasmid, or any other change that the introduction of the plasmid has caused. To compare growth rates and yields, one must make the conditions as similar as possible, so one has to compare two strains that both harbor the plasmid backbone, but in one case the plasmid encodes the tRNA, in the control case the tRNA has not been inserted into the plasmid. The authors present no evidence that the altered growth rate and yield are due to the tRNA and not other properties of the plasmid. This must be addressed.
The first reference in the text to Fig 1E mistakenly says "Fig 1F". This must be corrected.
I suggest to remove the first instance of "surprisingly" (Surprisingly, we observed extremely similar trends...")There is nothing in the text that sugggets it is surprising that the five strains show similar trends in bulk flourescence upon induction of the tRNA-fmet-CUA. This is a minor change