Evolution of bacterial fitness with an expanded genetic code. Escherichia coli

We have carried out directed evolution experiments with an orthogonal translation system that inserts 3-nitro-L-tyrosine across from amber codons, creating a 21 amino acid genetic code in which the amber stop codon ambiguously encodes either 3-nitro-L-tyrosine or stop. The 21 amino acid code is enforced throughthe inclusion of an addicted, essential gene, a beta-lactamase dependent upon 3-nitro-L-tyrosine incorporation. After 2000 generations of directed evolution, the fitness deficit of the original strain was largely repaired through mutations that limited the toxicity of the noncanonical.