Design of Orthogonal Pairs for Protein Translation: Selection Systems for Genetically Encoding Noncanonical Amino Acids in E. coli

The expansion of the genetic code is gradually becoming a core discipline in synthetic biology. Residue-specific incorporation of noncanonical amino acids (ncAAs) into proteins allows facile alteration and enhancement of protein properties. There are two distinct in vivo approaches available for their cotranslational incorporation. For isostructural noncanonical amino acids, residue-specific replacement of canonical amino acids is performed with the supplementation-based incorporation method (SPI) using auxotrophic host strains. On the other hand, orthogonal ncAAs are incorporated into the proteins site specifically in response to stop or quadruplet codons (stop codon suppression (SCS)) using orthogonal aminoacyl-tRNA synthetase/tRNA pairs (o-pair). Frequently used o-pair is based on the tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii (MjTyrRS). To evolve a new orthogonal aminoacyl-tRNA synthetase (aaRS), which recognizes exclusively the noncanonical amino acid, the most straightforward solution is to produce a library of MjTyrRS mutants, containing randomized residues in the amino acid-binding site, on the basis of available crystal structure. The library is transformed into Escherichia coli and three rounds of positive and negative selection are performed in order to select for desired MjTyrRS variant which uniquely charges the tRNA with the ncAA of interest. Here, we provide a protocol with detailed description how to perform positive and negative selection with chloramphenicol acetyltransferase and barnase, respectively.