Fig. 2.

Directed Evolution of PcaV. a. The amino acids H9, H21, W25, Y38, I110, M113, and N114 in the PcaV effector binding site were selected for generation of completely randomized libraries, based on the PcaV-PCA structure (PDB: 4G9Y, 4FHT) [42]. b. Cultures of E. coli with the PcaV DE Libraries were used in a Fluorescence Activated Cell Sorting (FACS) counter-selection method for the enrichment of mutants with altered ligand specificity. Rounds of FACS were made alternating between negative selection of uninduced libraries with sorting of events on the bottom of the GFP fluorescent histogram and positive selection of induced libraries with sorting of events on the top of the GFP fluorescent histogram. Plate-based screening assay was performed on selected colonies from each sorted library and assessed for induction with the effector. The fluorescence was measured and the ratio induced/uninduced was used to select for responsive mutants