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KMID : 0364820090450020215
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Korean Journal of Microbiology
2009 Volume.45 No. 2 p.215 ~ p.221
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Establishment of an In Vivo Report System for the Evaluation of Amber Suppression Activity in Escherichia coli
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Kim Kyung-Tae
Park Mi-Young
Park Jung-Chan
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Abstract
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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 tRNAGln produced blue colonies in culture plates containing X-Gal as well as dramatically increased β-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor tRNATyr and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of β-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.
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KEYWORD
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amber suppression, aminoacyl-tRNA synthetase, suppressor tRNA, unnatural amino acid
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