Engineered aminoacyl-tRNA synthetases (aaRS) enable the incorporation of non-canonical amino acids (ncAAs) into proteins, adding functionalities such as bio-orthogonal conjugation, photoactivation, and novel metal coordination. Incorporating novel ncAAs requires identifying orthogonal aminoacyl-tRNA synthetases, engineered to accept the new substrate.

Engineering highly selective orthogonal aminoacyl-tRNA synthetase (aaRS) variants for non-canonical amino acid (ncAAs) incorporation into proteins has proven extremely challenging, but is vitally important, as many of the most impactful applications of ncAAs are intolerant of heterogeneity in the protein sequence and thus require near perfect incorporation efficiency and substrate selectivity (e.g., the development of novel catalytic sites and biologics). We propose using an established, automated workcell for high-throughput fitness landscape measurement developed by collaborators at NIST, to generate an open-source, global genotype-phenotype map spanning all residues in the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase, one of the most widely used tools for genetic code expansion and chemical biology.