In vivo incorporation of unnatural amino acids

1. A cell comprising:

(a.) an orthogonal tRNA synthetase that aminoacylates endogenous tRNAs of the cell with a reduced efficiency as compared to aminoacylation of the endogenous tRNAs by endogenous tRNA synthetases of the cell, and

(b.) an orthogonal tRNA that is aminoacylated by the endogenous tRNA synthetases with reduced efficiency as compared to aminoacylation of the endogenous tRNAs by the endogenous tRNA synthetases,

wherein said orthogonal tRNA synthetase preferentially aminoacylates said orthogonal tRNA, as compared to the endogenous tRNAs, with an unnatural amino acid in said cell, and

wherein the orthogonal tRNA synthetase preferentially aminoacylates aid orthogonal tRNA with an unnatural amino acid, as compared to a natural amino acid, the orthogonal tRNA synthetase having a lower K m for the unnatural amino acid than for the natural amino acid, and

wheein said orthogonal tRNA recognizes a selector codon of an mRNA in said cell.

2. The cell of claim 1 , wherein said selector codon is selected from a group consisting of: an opal codon, an ochre codon, and a four base codon.

3. The cell of claim 1 , wherein said selector codon is an amber codon.

4. The cell of claim 1 , wherein said unnatural amino acid is incorporated into a protein in said cells.

5. The cell of claim 4 , wherein said protein is human growth hormone.

6. The cell of claim 1 , wherein said unnatural amino acid is selected from a group consisting of: O-methyl-L-tyrosine, L-3-(2-naphthyl)-alanine, 3-methyl-L-phenylalanine, fluorinated phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-L-phenylalanine, p-bromo-L-phenylalanine, p-amino-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, and isopropyl-L-phenylalanine.

7. The cell of claim 1 , wherein the unnatural amino acid is p-azido-L-phenylalanine.

8. The cell of claim 1 , wherein the unnatural amino acid is p-acetyl-L-phenylalanine.

9. The cell of claim 1 , wherein the unnatural amino acid is m-acetyl-L-phenylalanine.

10. The cell of claim 1 , wherein the unnatural amino acid is 4-(2-oxo-propoxy)-L-phenylalanine.

11. The cell of claim 1 , wherein said cell is a prokaryotic cell.

12. The cell of claim 11 , wherein said cell is a bacterium.

13. The cell of claim 12 , wherein said cell is an Escherichia coli cell.

14. The cell of claim 1 , wherein the orthogonal tRNA is encoded by a nucleic acid as set forth in SEQ ID NO.: 1.

15. The cell of claim 1 , wherein the orthogonal tRNA synthetase comprises an amino acid sequence as set forth in SEQ ID NO.: 60.