Expanding the genetic code of Escherichia coli

The genetic code of every known organism encodes the same twenty common amino acids. Only in rare instances, selenocysteine is added. It is of interest to study if the properties of proteins, or possibly an entire organism, could be enhanced by expanding the genetic code to include additional amino acids with novel chemical or physical properties. This thesis describes the development of a general strategy for the site-specific incorporation of unnatural amino acids directly into proteins in E. coli, and the application of the genetically encoded novel amino acids. This methodology provides powerful new tools for analyzing protein and cellular function both in vitro and in vivo, and should be applicable to other cell types.; Our method involves the generation of additional components for the biosynthetic machinery, including a novel tRNA-codon pair, an aminoacyl-tRNA synthetase, and an amino acid. This new set of components functions orthogonally to the counterparts of the common 20 amino acids; that is, the orthogonal synthetase (and only this synthetase) aminoacylates the orthogonal tRNA (and only this tRNA) with the unnatural amino acid only, and the resulting acylated tRNA inserts the unnatural amino acid in response to only the unique codon.; The amber nonsense codon was chosen to encode the unnatural amino acid. An orthogonal amber suppressor $mutRNATyrCUA$/TyrRS pair was generated in E. coli by first importing a tRNATyr/TyrRS pair from Methanococcus jannaschii (Mj) and then improving the tRNA's orthogonality with a general selection for mutant tRNAs with enhanced orthogonalities. To alter the amino acid specificity of the orthogonal synthetase so that it charges the $mutRNATyrCUA$ with a desired unnatural amino acid, several selection/screen methods were developed. Libraries of Mj-TyrRS mutants with active site residues randomly mutated were generated and subjected to these selections/screens. DNA shuffling was applied between iterating rounds.; A mutant synthetase was evolved that when coexpressed with the $mutRNATyrCUA$, leads to the in vivo incorporation of O -methyl-L-tyrosine into proteins in response to an amber codon with a translational fidelity greater than 99%. The genetic code of E. coli was expanded for the first time. A second evolved synthetase is capable of inserting L-3-(2-naphthyl)-alanine into proteins, suggesting that our methodology should be generalizable to a variety of unnatural amino acids.; In a similar fashion, over 12 new amino acids have been added to the genetic repertoire of E. coli to date. Among them are O-allyl-L-tyrosine containing the alkene functionality, p- and m-acetyl-L-phenylalanine which have been used to selectively and efficiently modify proteins both in vitro and in E. coli, photocrosslinker p-benzoyl-L-phenylalanine for mapping protein-protein interactions, and p-iodo and p-bromo-L-phenylalanine to facilitate the phase determination in protein crystallography.; Finally, an autonomous E. coli with 21 amino acids was generated by creating a biosynthetic pathway for the unnatural amino acid. Such an organism should provide an opportunity to examine whether the addition of a novel amino acid to the genetic repertoire can afford evolutionary advantage to the host.