FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica

PLoS One. 2012;7(9):e44841. doi: 10.1371/journal.pone.0044841. Epub 2012 Sep 27.

Abstract

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of "scar" DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacteria / drug effects
  • Bacteria / genetics*
  • Base Sequence
  • Chromosomes, Bacterial / genetics*
  • Chromosomes, Bacterial / metabolism
  • Drug Resistance, Bacterial / genetics
  • Enterotoxigenic Escherichia coli / drug effects
  • Enterotoxigenic Escherichia coli / genetics
  • Epitopes / metabolism*
  • Escherichia coli K12 / drug effects
  • Escherichia coli K12 / genetics
  • Gene Deletion
  • Genes, Bacterial / genetics*
  • Genetic Engineering / methods*
  • Kanamycin / pharmacology
  • Mutagenesis*
  • Point Mutation
  • Promoter Regions, Genetic / genetics*
  • Salmonella typhimurium / drug effects
  • Salmonella typhimurium / genetics

Substances

  • Epitopes
  • Kanamycin