Design of growth-coupled measurements of orthogonal aminoacyl-tRNA synthetase function

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.