One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases.
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.
Melançon
Charles E
CE
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Schultz
Peter G
PG
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology; D017017:Methanococcus / Q000201:enzymology; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
D018388:Codon, Terminator; D010649:Phenylalanine; D015500:Chloramphenicol O-Acetyltransferase; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; C049181:4-iodophenylalanine