Development and optimization of Lysis gene E as a counter-selection
marker with high selection stringency
Abstract
Seamless modification of bacteria chromosome is widely performed both in
theoretical and in practical research, such as functional genome
analysis and metabolic engineering. For this purpose, excellent
counter-selection marker genes with high selection stringency are urgent
needed. Lysis gene E from bacteriophage PhiX174, which is of universal
and powerful killing effect on Gram-negative strains, was developed and
optimized as a generic counter-selection marker in this paper. Lysis
gene E was firstly constructed under the control of pL promoter. At high
temperature such as 42 °C, inducible expression of Lysis gene E could
effectively promote the death of Escherichia coli MG1655. Seamless
modification using E as a counter-selection marker also successfully
conducted with high ration of positive recombinants. It also works in
another Gram-negative strain Serratia marcescens under the control of
Arac/PBAD regulatory system. Through combining lysis gene E and kil, the
selection stringency frequency of pL-kil-sd-E cassette in E. coli
arrived at 4.9×10−8 and 3.2×10−8 at two test loci, which is very close
to the best counter-selection system, inducible toxins system. Under the
control of Arac/PBAD, selection stringency of PBAD-kil-sd-E in S.
marcescens arrived mostly at the level of 10−7 at four test loci. By
introducing araC gene harboring plasmid pKDsg-ack, 5- to 18- fold
improvement of selection stringency was observed at all these loci, and
a surprising low selection stringency frequency 4.9×10−9 was obtained at
marR-1 locus. This is the lowest selection stringency frequency for
counter-selection reported so far. Similarly, at araB locus of E. coli
selection stringency frequency of PBAD-kil-sd-E was improved 170- fold
to 3×10−9 after introducing plasmid pKDsg-ack. In conclusion, we have
developed and optimized a newly universal counter-selection marker based
on lysis gene E. The best selection stringency of this new marker
exceeds the inducible toxins system several fold. We provide a valuable
counter-selection marker with high selection stringency to the genome
editing toolbox.