title: FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.
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.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Stringer
authors.forename: Navjot
authors.initials: N
authors.lastname: Singh
authors.forename: Anastasiya
authors.initials: A
authors.lastname: Yermakova
authors.forename: Brianna L
authors.initials: BL
authors.lastname: Petrone
authors.forename: Jayaleka J
authors.initials: JJ
authors.lastname: Amarasinghe
authors.forename: Lucia
authors.initials: L
authors.lastname: Reyes-Diaz
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Mantis
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Wade
mesh_terms: D001419:Bacteria / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D054307:Enterotoxigenic Escherichia coli / Q000187:drug effects / Q000235:genetics; D000939:Epitopes / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000187:drug effects / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D007612:Kanamycin / Q000494:pharmacology; D016296:Mutagenesis; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics
chemical_list: D000939:Epitopes; D007612:Kanamycin