Fig. 2.
General scheme of aaRS selection. In the first round of selection the aaRS library is co-transformed with a plasmid harboring the orthogonal tRNA suppressor (tRNACUA) and reporter gene (cat). Suppression, and therefore growth in the presence of chloramphenicol (Cm) may occur when the tRNA is acylated both with the ncAA, as well as cAA. Surviving clones are isolated and co-transformed with a second reporter plasmid containing a toxic gene with two stop codons (barnase). In the absence of an ncAA, only those clones that do not facilitate read-through using cAAs are selected.