Very nice work on improving CFPS. A few questions for you: 1) Is there a reason you didn't try doing at DoE for varying the components to try to identify which parameters, NTPs, AAs, genetic background etc. were of greatest weight of impact and how they might interact with each other? Could be an interesting way to continue to improve the system.

2) Do all of these trends hold for other proteins besides GFP? Would be nice to test a few other proteins on your idealized lysate to test for universality by testing a couple other target proteins with different folding and biophysical properties.

I also noticed a typo on the y-axis of the graph in panel A in Figure 2. The "," is off by a digit in all the numbers, e.g. 10,0000 instead of 100,000.

Very nice work comparing different HTP screening techniques for binder identification.

A couple comments for clarity: 1) The second paragraph on page 10 (beginning with "To investigate the selection preferences of the two platforms...") is confusing as is. The second sentence has a "." in the middle of it and it is unclear what exactly is trying to be communicated.

2) Figure 2 caption says that dots in panels A and B are color coded by platform of identification, but that is only true for C and D, not A and B. A and B are color coded by validation technique.

3) On page 14, it says "SEC confirmed that the binder was monomeric and non-aggregating (Figure 3C)", however, Figure 3C is a CD plot. I assume this is in reference to a supplemental figure?

Another point on page 14 says "...showed no aggregation on SEC (Figure 3B)...", but Figure 3B is SPR data. Assume this is also in reference to a supplemental figure.

4) Regarding the SPR data, I am concerned about the lack of dissociation observed for TLT1_3042_3 and TLT1_4815_1. Since there is little decrease in signal during the off phase, how can you accurately measure a kd? Are you concerned that the SPR conditions for these variants are causing aggregation or irreversible binding as opposed to simply a slow off rate?

Very beautiful work!

As far as I'm aware there aren't any structures of DksA bound to an RNAP elongation complex. Have you thought about how this interaction would impact your model of ppGpp-binding and RNAP swiveling? Would DksA prevent swiveling given its binding location, or the structural states be in competition? Would be curious to see impact of DksA in vitro as well as what your in vivo results would look like in a ∆dksA background