Scarless DNA Recombineering

The method described here allows editing of the bacterial genome without leaving any secondary changes (scars) behind. This method uses a tripartite selectable and counterselectable cassette comprising an antibiotic-resistance gene (cat or kan) and the tetR repressor gene linked to a P^tet promoter-ccdB toxin gene fusion. In the absence of induction, the tetR gene product represses the P^tet promoter, preventing ccdB expression. The cassette is first inserted at the target site by selecting for chloramphenicol or kanamycin resistance. It is subsequently replaced by the sequence of interest by selecting for growth in the presence of anhydrotetracycline (AHTc), which inactivates the TetR repressor thereby causing CcdB-induced lethality. Unlike other CcdB-based counterselection schemes, which require specifically designed λ-Red delivery plasmids, the system described here uses the popular plasmid pKD46 as the source of λ-Red functions. This protocol allows a wide variety of modifications, including the intragenic insertion of fluorescent or epitope tags, gene replacements, deletions, and single base-pair substitutions, to be made. In addition, the procedure can be used to place the inducible P^tet promoter at a chosen position in the bacterial chromosome.