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

Abstract

Background 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. Main Methods and Major Results The lysis gene E was first constructed under the control of the PL 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/PBAD regulatory system. By combining lysis gene E and kil, the counter‐selection frequencies of the PL‐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/PBAD, the counter‐selection frequency of PBAD‐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 PBAD‐kil‐sd‐E was 3 × 10−9 after introducing plasmid pKDsg‐ack. Conclusions and Implications 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.