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

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...

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Bibliographic Details
Published in:ACS synthetic biology 2017-01, Vol.6 (1), p.45-54
Main Authors: Monk, Jordan W, Leonard, Sean P, Brown, Colin W, Hammerling, Michael J, Mortensen, Catherine, Gutierrez, Alejandro E, Shin, Nathan Y, Watkins, Ella, Mishler, Dennis M, Barrick, Jeffrey E
Format: Article
Language:English
Subjects:
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
Recombinant Proteins - analysis
Recombinant Proteins - biosynthesis
RNA, Transfer - metabolism
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Summary: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.
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.6b00192