Simple and efficient genome recombineering using kil counter-selection in Escherichia coli

Seamless modification of the Escherichia coli genome using positive selection / negative selection is widely used in metabolic engineering and functional genome analysis. Some excellent negative selection systems have been reported, of which tetA-sacB and inducible toxins system are prominent. To expand the existing negative selection toolkit, we constructed a new negative selection marker system based on kil gene of lambda prophage. The selection stringency of kil was measured and compared with the most widely used counter-selection gene, sacB, at the lacI, ack, and dbpa loci using different E. coli strains. At all these loci of tested strains, the selection stringency of kil significantly exceeds that of sacB by 2- to 28-fold. When dsDNA fragments were employed for recombination, the efficiency for isolating the correct recombinant of kil was significantly higher than that of sacB. This new negative selection system does not require special media or extended incubation time. However, our system cannot be used in host strains containing temperature-sensitive kil gene. A Red system providing plasmid without kil gene is recommended for use together with our system. Our counter-selection system is expected to be an addition to the engineering arsenal of E. coli.