Expanding the Genetic Code of Escherichia coli

  title={Expanding the Genetic Code of Escherichia coli},
  author={Lei Wang and Ansgar Brock and Brad Herberich and Peter G. Schultz},
  pages={498 - 500}
A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-l-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should… 
Adding amino acids to the genetic repertoire.
Expanding the Genetic Code
This methodology provides a powerful tool both for exploring protein structure and function in vitro and in vivo and for generating proteins with new or enhanced properties.
An expanding genetic code.
An expanded genetic code with a functional quadruplet codon.
This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.
Expanding the genetic code for biological studies.
Protein Expression by Expansion of the Genetic Code
The intellectual and practical strategies that have been employed in the challenging, but ultimately successful expansion of the genetic code are discussed.
Incorporation of nonnatural amino acids into proteins.
The genetic code is established by the aminoacylation of transfer RNA, reactions in which each amino acid is linked to its cognate tRNA that, in turn, harbors the nucleotide triplet (anticodon)
Expanding the genetic code.
Using this strategy, the genetic code of Escherichia coli has been expanded to incorporate unnatural amino acids with a fidelity rivaling that of natural amino acids.
An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes.
The feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl-tRNA synthetase and tRNA (TrpRS-tRNATrp) pair is demonstrated and a general strategy to develop additional aaRS- tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes is provided.


A New Orthogonal Suppressor tRNA/Aminoacyl‐tRNA Synthetase Pair for Evolving an Organism with an Expanded Genetic Code
This work has introduced a new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair in E. coli derived from the Saccharomyces cerevisiae tRNAAsp and aspartyl-t RNA Synthetase, and the in vitro and in vivo characteristics of this pair were determined.
Progress toward the evolution of an organism with an expanded genetic code.
  • D. R. Liu, P. Schultz
  • Biology, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 1999
A rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids, and the majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E. coli.
Escherichia coli thymidylate synthase: amino acid substitutions by suppression of amber nonsense mutations.
By using site-directed oligonucleotide mutagenesis, amber nonsense stop codons (5'-TAG-3') have been introduced at 20 sites in the Escherichia coli thymidylate synthase gene. By transforming the thyA
Site-directed mutagenesis with an expanded genetic code.
A biosynthetic method has been developed that makes possible the site-specific incorporation of a large number of amino acids and analogues within proteins to address detailed structure-function questions.
A New Functional Suppressor tRNA/ Aminoacyl-tRNA Synthetase Pair for the in Vivo Incorporation of Unnatural Amino Acids into Proteins
This approach involves the generation of a suppressor tRNA/aminoacyl-tRNA synthetase (tRNACUA/aaRS) pair that is orthogonal to Escherichia coli endogenous tRNAs and insights into the development of additional pairs are provided.
Quadruplet codons: implications for code expansion and the specification of translation step size.
Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet.
A general method for site-specific incorporation of unnatural amino acids into proteins.
The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.
Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of “shifty” four-base codons with a library approach in Escherichia coli1
A general strategy to select tRNAs with the ability to suppress four-base codons from a library of t RNAs with randomized 8 or 9 nt anticodon loops is devised, which resulted in a set of very efficient, non-cross-reactive tRNA/four- base codon pairs for AGGA, UAGA, CCCU and CUAG.
Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria
Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and an aminoacyled tRNAs that function efficiently in suppression of amber codons, and two such aaRS–suppressor tRNA pairs are described.
Rapid evolution of a protein in vitro by DNA shuffling
It is reported here that selected mutants had a minimum inhibitory concentration of 640 μg ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme.