Precise and scarless genome modification in Escherichia coli using transposase

Various methods have been developed for gene disruption, however, the requirement of extra in vitro manipulation steps or the residual presence of a scar sequence in the host chromosome limits the use of such methods for some purposes. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing the transposase recognition sequence surrounding the antibiotic resistance gene and a counterselection marker by using primers containing extensions homologous to the adjacent regions of the target gene on chromosome. The λ Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with modified gene target. The inserted DNA cassette containing antibiotic resistance gene and counterselection marker can then be excised by using a temperature sensitive plasmid expressing the transposase, which precisely cleaves at the recognition sequence without leaving behind a scar sequence. To demonstrate the wider applicability of this technique, we carried out lacZ gene point mutation repair, two precise disruptions of lacZ gene and constructed a library of lacZ variants having variable β-galactosidase activity by changing its ribosome binding site sequences. This technique can be used in E. coli genome engineering and extended for use in other bacteria.