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Incorporation of nonstandard amino acids (blue) into proteins in E. coli. An engineered orthogonal aaRS (top) charges a cognate tRNA with a designated amino acid, but does not interact with natural amino acids or E. coli tRNAs (bottom). Similarly, neither the exogenously introduced tRNA (here from M. barkeri) nor the nonstandard amino acid is recognized by any aaRS from E. coli (gray). Translation of the unnatural amino acid occurs opposite an amber stop codon (UAG).
