New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae

J Ind Microbiol Biotechnol. 2013 May;40(5):523-7. doi: 10.1007/s10295-013-1250-1. Epub 2013 Mar 12.

Abstract

We previously reported the method for introducing gene replacement into Klebsiella pneumoniae through Red-assisted homologous recombination; and it demonstrated that a higher transformation efficiency required long flanking arms at both ends of the linear DNA. The assembly job of the linear DNA is usually time-consuming and laborious. We report here an innovative method for DNA exchange in K. pneumoniae based on PCR-mediated Red recombination. The novel procedure enables rapid gene replacement in K. pneumoniae without prior cloning of the gene of interest; the key modification is to perform PCR reaction to generate linear DNA with extra non-homologous fragments on both ends as mercenary sequences which come from a TA-cloning plasmid. We give a demonstration by deleting the gene dhak1 in K. pneumoniae with high efficiency of about 20 CFU/μg DNA using the new technique.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / genetics
  • Gene Targeting / methods*
  • Homologous Recombination / genetics
  • Klebsiella pneumoniae / genetics*
  • Mutagenesis, Site-Directed / methods
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Transformation, Genetic

Substances

  • DNA