Patent ID: 8012739

Claim:
A cell comprising an orthogonal tRNA synthetase (ORS) and an orthogonal tRNA (OtRNA), as a pair wherein: (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; (e) k cat /K m for aminoacylation of the OtRNA by the ORS with the unnatural amino acid is higher than k cat /K m 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; and, (f) said cells are cultured in the presence of said unnatural amino acid.