Excision of selectable markers from the Escherichia coli genome without counterselection using an optimized λRed...

The introduction of chromosomal mutations into the E. coli genome using λRed-mediated recombineering includes two consecutive steps-the insertion of an antibiotic resistance gene and the subsequent excision of the marker. The second step usually requires a counterselection method, because the efficiency of recombination is not high enough to find recombinants among non-recombinant cells. Most counterselection methods require the introduction of additional mutations into the genome or the use of expensive chemicals. In this paper, we describe the development of a reliable procedure for the removal of an antibiotic resistance marker from the E. coli genome without the need for counterselection. For this purpose, we used dsDNA cassettes consisting of two regions homologous to the sequences that flank the marker on the chromosome. We optimized the length of the homologous regions, the electroporation conditions, and the duration of recovery for the electroporated cells in order to maximize the recombination efficiency. Using the optimal parameters identified, we obtained a rate of 4-6% recombinants among the transformed cells. This high efficiency allowed us to find marker-less, antibiotic-sensitive recombinants by replica plating without the need for selection.