Double-sieve selection with a mutant library for evolving orthogonal tRNAs or unnatural amino acid-specific synthetases. (A) Evolving orthogonal tRNAs in
E. coli. The negative selection uses toxic barnase gene containing two TAG stop codons at permissive sites. tRNAs non-orthogonal to
E. coli synthetases will be charged with amino acids and suppress the TAG codons to produce a functional barnase, which kills the cell. The positive selection uses the β–lactamase gene containing a TAG codon at a permissive site. tRNAs charged by the cognate synthetase and functional in translation will suppress the TAG codon and express active β–lactamase, which enables cells to survive in ampicillin. Cells containing non-functional tRNAs will be killed by ampicillin. (B) Evolving orthogonal synthetases to be specific for an unnatural amino acid in
E. coli. The positive selection uses the chloramphenicol acetyl transferase (CAT) gene containing a permissive TAG stop codon in the presence of the unnatural amino acid. Mutant synthetases capable of charging the orthogonal
with the unnatural or any natural amino acid will express full length CAT, enabling cells to survive in chloramphenicol (Cm). The negative selection uses the barnase gene containing permissive TAG codons. Synthetase mutants active toward natural amino acids will charge the
to suppress TAG codons in the barnase gene, leading to cell death. In the absence of the unnatural amino acid, synthetase mutants specific for the unnatural amino acid will survive the negative selection. To improve activity and specificity, initial synthetase gene hits can be recombined using DNA shuffling and subject to next round of selection.