BioTechniques (Jun 2002)

One-Step, Highly Efficient Site-Directed Mutagenesis by Toxic Protein Selection

  • W. Xu,
  • Y. Zhang,
  • L.-Y. Yeh,
  • C.R. Ruprecht,
  • F. Wong-Staal,
  • B.A. McFadden,
  • T.R. Reddy,
  • R.M. Ruprecht

DOI
https://doi.org/10.2144/02326st01
Journal volume & issue
Vol. 32, no. 6
pp. 1266 – 1270

Abstract

Read online

A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plasmid. Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers. The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdBgene to inactivate the CcdB protein. The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids. Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%–100% was reached even after one round of transformation.