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.
Highlights
Chromosomal mutagenesis is a critical genetic tool for the study of bacterial systems
We have further developed Flexible Recombineering Using Integration of thyA’’ (FRUIT) to allow for straightforward integration of any DNA sequence by combining recombineering with homologous recombination
We have successfully introduced point mutations, gene deletions, epitope tags and artificial promoters into the genomes of E. coli K-12, Enterotoxigenic E. coli (ETEC), and S. enterica serovar Typhimurium
Summary
Chromosomal mutagenesis is a critical genetic tool for the study of bacterial systems. Recombineering, a method that involves expression of bacteriophage recombination proteins, has transformed our ability to engineer bacterial chromosomes using linear dsDNA (typically generated by PCR) or ssDNA (oligonucleotides) [1]. Existing recombineering methods involve two key components: (i) expression of bacteriophage recombination proteins, and (ii) generation of suitable DNA fragments for recombination. The latter component typically relies on specific DNA templates for PCR-based synthesis of dsDNA. We have further developed FRUIT to allow for straightforward integration of any DNA sequence by combining recombineering with homologous recombination Using these approaches, we have successfully introduced point mutations, gene deletions, epitope tags and artificial promoters into the genomes of E. coli K-12, Enterotoxigenic E. coli (ETEC), and S. enterica serovar Typhimurium
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