Abstract
We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escherichia coli. This system utilizes an amber stop codon containing chloramphenicol acetyltransferase/uracil phosphoribosyltransferase fusion gene. We demonstrate the utility of the system by identifying a variant of the Methanococcus jannaschii tyrosyl synthetase from a library of 10(9) variants that selectively incorporates para-iodophenylalanine in response to an amber stop codon.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acyl-tRNA Synthetases / genetics*
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Chloramphenicol O-Acetyltransferase / genetics
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Chloramphenicol O-Acetyltransferase / metabolism
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Codon, Terminator / genetics
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Codon, Terminator / metabolism
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Directed Molecular Evolution*
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Escherichia coli / enzymology
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Methanococcus / enzymology
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Pentosyltransferases / genetics
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Pentosyltransferases / metabolism
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Phenylalanine / analogs & derivatives
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Phenylalanine / chemistry
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Phenylalanine / metabolism
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Plasmids*
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Tyrosine-tRNA Ligase / genetics
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Tyrosine-tRNA Ligase / metabolism
Substances
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Codon, Terminator
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Phenylalanine
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Chloramphenicol O-Acetyltransferase
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Pentosyltransferases
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uracil phosphoribosyltransferase
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Amino Acyl-tRNA Synthetases
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Tyrosine-tRNA Ligase
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4-iodophenylalanine