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

Area of Science:

• Molecular Biology
• Microbial Genetics
• Synthetic Biology

Background:

• Seamless modification of bacterial chromosomes is crucial for research and applications.
• Effective counter-selection marker genes with high stringency are essential for this process.

Purpose of the Study:

• To develop and optimize a novel, highly stringent counter-selection marker for bacterial chromosome modification.
• To evaluate the efficacy of the lysis gene E-based marker in different bacterial strains and genetic contexts.

Main Methods:

• Constructed lysis gene E under the control of P_L promoter and cI857 repressor for Escherichia coli.
• Utilized the Arac/P_BAD regulatory system for lysis gene E expression in Serratia marcescens.
• Combined lysis gene E with kil and employed the araC gene expressed from a plasmid to enhance stringency.

Main Results:

• Lysis gene E effectively killed E. coli at 42°C, enabling seamless modification.
• The P_L -kil-sd-E cassette achieved counter-selection frequencies as low as 3.2 × 10^-8 in E. coli.
• The P_BAD -kil-sd-E system in S. marcescens reached frequencies of 10^-7, with 5- to 17-fold improvements upon expressing araC.
• A remarkably low counter-selection frequency of 4.9 × 10^-9 was achieved in E. coli using the optimized system, representing the highest stringency reported.

Conclusions:

• A new universal counter-selection marker based on lysis gene E has been successfully developed and optimized.
• The optimized lysis gene E marker demonstrates superior counter-selection stringency compared to existing inducible toxin systems.
• This work provides a valuable tool for bacterial genetics and offers insights for improving other counter-selection strategies.