Rapid and Inexpensive Evaluation of Nonstandard Amino Acid Incorporation in Escherichia coli

ACS Synth Biol. 2017 Jan 20;6(1):45-54. doi: 10.1021/acssynbio.6b00192. Epub 2016 Sep 26.


By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and systematic comparisons of these critical parameters, we developed a toolkit for characterizing any Escherichia coli OTS that reassigns the amber stop codon (TAG). It assesses OTS performance by comparing how the fluorescence of strains carrying plasmids encoding a fused RFP-GFP reading frame, either with or without an intervening TAG codon, depends on the presence of the nsAA. We used this kit to (1) examine nsAA incorporation by seven different OTSs, (2) optimize nsAA concentration in growth media, (3) define the polyspecificity of an OTS, and (4) characterize evolved variants of amberless E. coli with improved growth rates.

Keywords: amber suppressor tRNA; expanded genetic code; noncanonical amino acid; nsAA measurement kit; unnatural amino acid.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / genetics
  • Amino Acids / metabolism*
  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism
  • Escherichia coli / metabolism*
  • Genetic Code
  • Mass Spectrometry
  • Plasmids / genetics
  • Plasmids / metabolism
  • Protein Biosynthesis
  • RNA, Transfer / metabolism
  • Recombinant Proteins / analysis
  • Recombinant Proteins / biosynthesis


  • Amino Acids
  • Recombinant Proteins
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases