Figure 4.

Deep mutational scanning is invaluable for the entire process of directed evolution. It can be used to inspect the quality of the initial aaRS library and quickly identify variants selective for the ncAA. Deep sequencing may be used to ensure even distribution of generated variants after mutagenesis (in purple). After screening or selection is executed in the presence and absence of an ncAA (dark blue rectangle), entire populations of cells are collected and DNA isolated. Individual variants are counted in the populations collected from both selections (light blue rectangle). Variants observed in the populations selected in the presence of ncAA can be then directly tested in an aminoacylation assay in vitro (green). Randomized positions are depicted with “?”.