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

Wei Chen1,2, Ruyi Chen1, Ling He2

  • 1Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China.

Biotechnology Journal
|April 4, 2022
PubMed
Summary

Researchers developed a new lysis gene E counter-selection marker for bacterial chromosome modification. This marker offers high stringency, exceeding current inducible toxin systems for precise genetic engineering.

Keywords:
lysis gene Ecounter-selectioncounter-selection frequencymetabolic engineeringred recombination

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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 PL promoter and cI857 repressor for Escherichia coli.
  • Utilized the Arac/PBAD 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 PL -kil-sd-E cassette achieved counter-selection frequencies as low as 3.2 × 10-8 in E. coli.
  • The PBAD -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.