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Related Experiment Video

Updated: Apr 16, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Liquid-based iterative recombineering method tolerant to counter-selection escapes.

Masahiro Tominaga1, Shigeko Kawai-Noma1, Ikuro Kawagishi2

  • 1Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.

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Summary

This study introduces an improved bacterial genome engineering method using a novel counter-selection system. This approach enhances efficiency and robustness, enabling seamless, automatable, multi-locus modifications without manual intervention.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Microbial Genetics

Background:

  • Selection-based recombineering enables precise bacterial genome modification.
  • Current methods are limited by inefficient counter-selectable markers and selection escapees, reducing throughput.
  • Manual removal of escapees is laborious and time-consuming.

Purpose of the Study:

  • To improve the throughput and robustness of selection-based recombineering.
  • To develop a seamless and automatable genome engineering system.
  • To overcome limitations of existing counter-selectable markers.

Main Methods:

  • Developed a rapid, liquid-based counter-selection system utilizing herpes simplex virus thymidine kinase (hsvTK) and a non-natural nucleoside (dP).
  • Implemented gene duplication of hsvTK and controlled population size to eliminate selection escapees.
  • Streamlined the process to require only liquid handling, eliminating colony isolation and genotype confirmation.

Main Results:

  • Achieved highly efficient, rapid counter-selection.
  • Effectively eliminated selection escapees, enabling seamless genome engineering.
  • Completed four rounds of recombineering in 10 days, facilitating multi-locus chromosomal modifications.

Conclusions:

  • The novel hsvTK-based counter-selection system significantly enhances bacterial genome engineering efficiency and robustness.
  • The method allows for seamless, automatable, and multi-locus modifications with minimal manual intervention.
  • This approach broadens accessibility for complex genomic engineering in bacteria.