Simple and highly efficient BAC recombineering using galK selection

TLDR

Recombineering enables modification of E. coli‑cloned DNA via λ Red homologous recombination without restriction enzymes or ligases. The study introduces three new recombineering strains (SW102, SW105, SW106) that allow BAC modification using galK positive/negative selection. The method employs a two‑step galK positive/negative selection in strains lacking galK but carrying a temperature‑sensitive λ prophage, with SW105 and SW106 additionally harboring Cre or Flp for recombination, allowing marker‑free BAC editing.

Abstract

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (λ) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive λ prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.