Development and optimization of Lysis gene E as a counter-selection marker with high stringency

Seamless modification of bacterial chromosomes is widely performed in both theoretical and practical research. For this purpose, excellent counter-selection marker genes with high stringency are needed.

The lysis gene E was first constructed under the control of the P_L promoter and the cI857 repressor. At 42°C, it could effectively kill Escherichia coli and seamless modification in this bacterium using E as a counter-selection marker was successfully conducted. It also works in another gram-negative strain, Serratia marcescens, under the control of the Arac/P_BAD regulatory system. By combining lysis gene E and kil, the counter-selection frequencies of the P_L -kil-sd-E cassette in E. coli reached 4.9 × 10^-8 and 3.2 × 10^-8 at two test loci, which are very close to frequencies observed with the best counter-selection systems reported, the inducible toxin systems. Under the control of the Arac/P_BAD , the counter-selection frequency of P_BAD -kil-sd-E in S. marcescens reached the level of 10^-7 at four test loci. By expressing the araC gene from plasmid pKDsg-ack, 5- to 17-fold improvements in counter-selection stringency were observed at these loci. A surprisingly low counter-selection frequency of 4.9 × 10^-9 was obtained at the marR-1 locus, which reflects the highest stringency for a counter-selection cassette reported thus far. Similarly, at the araB locus of E. coli, the counter-selection frequency of P_BAD -kil-sd-E was 3 × 10^-9 after introducing plasmid pKDsg-ack.

We have developed and optimized a new universal counter-selection marker based on lysis gene E. The best counter-selection stringency of this new marker exceeds the inducible toxin system several fold. Our work can also provide inspiration for improving counter-selection stringency based on existing markers.