Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

A cell comprising an orthogonal tRNA synthetase (ORS) and an orthogonal tRNA (OtRNA), as a pair having the following provisios:

• (a) said ORS preferentially aminoacylates said OtRNA, wherein preferential aminoacylation is defined in that said ORS aminoacylates endogenous tRNAS of the cell with a reduced efficiency as compared to endogenous tRNA synthetase aminoacylation of said endogenous tRNAS of the cell;
• (b) said ORS preferentially aminoacylates said OtRNA with an unnatural amino acid, as compared to any natural amino acid;
• (c) said OtRNA is aminoacylated by the endogenous tRNA synthetases of the cell with reduced efficiency as compared to aminoacylation of the endogenous tRNAS by said endogenous tRNA synthetases;
• (d) said OtRNA recognizes a selector codon of an mRNA in said cell;
• and wherein kcat/Km for aminoacylation of the OtRNA by the ORS with the unnatural amino acid is higher than kcat/Km for aminoacylation of the OtRNA by the ORS with the natural amino acid, or wherein said unnatural amino acid is incorporated into a growing polypeptide in the cell with a fidelity of greater than 75% in response to the selector codon.