Fig. 3.

Evolving substrate specificity of aminoacyl-tRNA synthetases (aa-RSs) for ncAAs. a In prokaryotic expression systems, a two-step evolution procedure for aa-RSs is well established. After choosing an aa-RS/tRNA pair orthogonal to the host, a library containing randomized resi- dues in the amino acid binding site of the enzyme is constructed. This population of aa-RS variants (grey and red spheres) is subjected together with its cognate tRNA (black cross) to a round of positive selection and screened for activity with either canonical (grey circle) or non-canonical (red star) amino acids, by virtue of their ability to suppress an introduced stop codon and so allow to complete translation of a gene that is essential for survival. To eliminate aa-RSs that recognize natural amino acids (grey spheres) a negative selection in the absence of the ncAA is performed. Here, nonsense suppression of a cell-toxic gene takes place, thus allowing for survival of clones that carry aa-RSs specific for the ncAA only (red spheres). Scheme was adapted from Davis and Chin 2012. b In eukaryotic expression systems, evolution of aa-RSs is more complicated but follows the same principles. The main difference is that the TAG stop codons are introduced not directly into genes that are responsible for survival or death of clones during the selection but into the gene of a transcription factor (GAL4) that drives the expression of reporter genes (HIS3, URA3) causing growth or death of the cells. For details of the procedure please refer to the main text.