Characterization of a novel Escherichia coli recombineering selection/counterselection cassette

Recombineering is a highly efficient DNA cloning and modification technique by using the recombinase-mediated homologous recombination. Selection/counterselection cassette is often used in chromosomal DNA or large episomal DNA manipulation, in which the selection marker is used for the first step cassette selection while deleting the target gene via allelic exchange, and the counterselection marker is used for the second step replacement of the cassette by the foreign DNA fragment. A variety of selection/counterselection cassettes are reported, however, the cassettes suffer from the shortcomings of the requirement of pre-engineered strain or specific culture medium. Herein, we report a novel S-tetR- P_tetA-ccdB-aacC1-S selection/counterselection cassette that sidesteps the disadvantages. As a proof-of-concept, one-step gene cloning (0.7, 1.7, and 4.2 kb) and two-step Escherichia coli chromosomal gene knock-in (0.7 and 4.2 kb) were performed. The gene cloning and gene knock-in efficiencies are high up to 90%. The novel selection/counterselection cassette adds a powerful tool to the recombineering repertoire.