doi: 10.1016/j.bmcl.2009.04.007.
Epub 2009 Apr 9.
One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases
Affiliations
- PMID: 19398201
- PMCID: PMC2714362
- DOI: 10.1016/j.bmcl.2009.04.007
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One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases
Bioorg Med Chem Lett.
.
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.
Figures
Dual positive/negative selection vectors.
(A) Chloramphenicol resistance and (B) 5-fluorouracil sensitivity of dual positive/negative selection constructs pRepCM2 (Q98TAG, D181TAG), pRepCM3 (Q98TAG), and pRepCM9 (D181TAG) co-expressed with empty vector, wild-type MjYRS, or pIPheRS.
Sequences of clones selected randomly from (A) the naïve library, and from survivors after (B) the first positive round, (C) first negative round, and (D) second positive round.
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