Supplementary Figure 2 : Coverage of all single-amino-acid substitutions found in the pre-selection single-site saturation mutagenesis library at gentiobiose-responsive positions.

From: Engineering an allosteric transcription factor to respond to new ligands

Supplementary Figure 2

Residues are ordered by protein region as in Supplementary Fig. 8 for comparison. (a) For each position of wild-type LacI, 19 substitutions were synthesized for the single amino-acid substitution library. By next-generation sequencing we measured how many of the 19 possible substitutions were found either before or after negative selection for positions showing response to gentiobiose. Mutants missing from the input library are likely due to synthesis or cloning inefficiencies. (b) Most (>80%) of the positions involved in gentiobiose response were found to have at least 18 of 19 single amino-acid substitutions prior to positive selection. For all 360 positions of LacI (not shown), we found 195 (~54%) positions contained all 19 substitutions, 238 (~66%) contained at least 18, and 306 (85%) contained at least 14 substitutions.