期刊
ACS SYNTHETIC BIOLOGY
卷 6, 期 1, 页码 45-54出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00192
关键词
expanded genetic code; noncanonical amino acid; unnatural amino acid; amber suppressor tRNA; nsAA measurement kit
资金
- UT Austin Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology
- Welch Foundation [F-1780]
- NSF BEACON Center for the Study of Evolution [DBI-0939454]
- UT Austin Center for Systems and Synthetic Biology, Department of Molecular Biosciences
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 colt 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. colt with improved growth rates.
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