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Establishment of an In Vivo Report System for the Evaluation of Amber Suppression Activity in Escherichia coli  

Kim, Kyung-Tae (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Park, Mi-Young (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Park, Jung-Chan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Publication Information
Korean Journal of Microbiology / v.45, no.2, 2009 , pp. 215-221 More about this Journal
Abstract
Site-specific incorporation of unnatural amino acids into proteins in vivo can be achieved by co-expression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to establish this technique, here we generated an Escherichia coli reporter strain DH10B(Tn:lacZam) by integrating amber mutated lacZ gene into the chromosome of E. coli DH10B strain. In vivo expression of E. coli amber suppressor $tRNA^{Gln}$ produced blue colonies in culture plates containing X-Gal as well as dramatically increased $\beta$-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor $tRNA^{Tyr}$ and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of $\beta$-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.
Keywords
amber suppression; aminoacyl-tRNA synthetase; suppressor tRNA; unnatural amino acid;
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