Peer review of: Chimeric design of pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA (2020)



Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The re-revised manuscript from Ding et. al has addressed many of the concerns that reviewers had 
but requires some additional modifications before publication. The manuscript has a better flow and 
the ideas are more clearly laid out than previous versions. However, the grammar still requires some 
work to make it easier to read and clarify the overall results. 
1. In the abstract it should clearly state that the PylRS system is currently the ideal system for GCE in 
both eukaryotes and prokaryotes. The way it is currently structured does not come across that way 
and could be argued that MjTyrRS is also an ideal system for GCE in prokaryotes. 
2. The authors claim that by simply transplanting the key orthogonal components from the PylRS 
system into an aaRS/tRNA system you get fully orthogonal and high activity. This is a bit of an 
overstatement as the manuscript goes through major detail on how further engineering of the aaRS 
and tRNA is required in most cases (lines 88-92). 
3. The labels on Figure 3F are not consistent, one discusses aaRS and amino acid and the other is 
aaRS and tRNA. It is not clear in the figure or its legend which amino acid is used for chHisRS and 
presuming that tRNAPyl is used with PylRS. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Since I am not one of the original reviewers. All my comments are based on what authors have been 
put together to address previous reviewers' concerns. The authors have provided point-by-point 
responses to the previous concerns. Although most of the previous concerns have been addressed, 
there are still some issues left. 
 
Reviewer 1 
Point 1: 
It is hard to judge from Figure S16B on whether or not 4-azido-Phe was incorporated into the 
proteome. The exposure has been tuned too low so that even makers (assumed for Western blotting) 
cannot be observed. Proper adjustment of exposure is needed for showing both background 
incorporation and a positive control. 
Point 2: 
Concerns have been addressed. 
Point 3: 
Concerns are addressed. 
Point 4: 
Concerns are addressed. 
Point 5: 
Concerns are partially addressed. All integrated LC-MS spectra have quite broad bands. It is no way to 
judge from these spectra that the incorporation is orthogonal. High resolution MS are needed. 
Pont 6: 
Concerns are addressed. 

Point 7: 
Concerns are addressed. 
 
Reviewer 2 
Point 1: 
The title has been changed according to the reviewer's suggestion. 
Point 2: 
Additional data are provided to show 30 degree is optimal. This will be fine for E. coli but highly 
problematic for mammalian cells. Since this is what it is, the authors need to point out in the paper 
that the system will have some disadvantage when used in mammalian systems. 
Point 3: 
Concerns are addressed. 
 
Reviewer 3 
Point 1: 
Concerns are addressed. 
Point 2: 
Concerns are addressed. 
Point 3: 
concerns are addressed. 
Point 4: 
Concerns are addressed. 
Point 5: 
Concerns are addressed. 
Point 6: 
Concerns are addressed. 
Point 7: 
Concerns are addressed. 
Point 8: 
Concerns are addressed. 
Point 9: 
Concerns are partially addressed. Figures 5D, 5G, and S13 need to be redrawn. There are only one 
data points with multiple repeats. It is misleading (more leaning to not right) to draw a count vs ug 
chart. 
Point 10: 
Concerns have been addressed. 
Point 11: 
Concerns have been addressed. 
Point 12: 
No comments.