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Electroporation of Mycobacteria
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An efficient electrotransformation method for three Bacillus species.

Wei Quan1, Chen-Lu Liu1, Shao-Xuan Shi1

  • 1College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Key Laboratory of Biopesticide Creation and Resource Utilization for Autonomous Region Higher Education Institutions, Hohhot, 010018, China.

Applied Microbiology and Biotechnology
|December 1, 2025
PubMed
Summary
This summary is machine-generated.

Optimizing electroporation conditions and using glycine significantly enhanced Bacillus transformation efficiency. This provides an efficient method for genetic manipulation of Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus subtilis.

Keywords:
Bacillus amyloliquefaciensBacillus subtilisBacillus velezensisElectroporationResponse surface methodology (RSM)Transformation

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Bacillus strains are vital in food fermentation and agriculture.
  • Efficient genetic manipulation requires optimized transformation methods.
  • Existing methods for Bacillus transformation need improvement for practical applications.

Purpose of the Study:

  • To optimize electroporation conditions for three Bacillus strains: Bacillus amyloliquefaciens YN-J3, Bacillus velezensis JN-Y2, and Bacillus subtilis S-16.
  • To identify effective cell wall agents for enhancing Bacillus transformation efficiency.
  • To establish a robust and efficient system for Bacillus genetic manipulation.

Main Methods:

  • Orthogonal experiments and response surface analysis were employed to optimize electroporation parameters (OD600, competent cell volume, plasmid concentration, field strength).
  • Various cell wall agents were screened for their ability to improve bacterial competence.
  • Transformation efficiency was quantified using CFU/µg DNA.

Main Results:

  • Optimal transformation parameters were determined for each Bacillus strain, leading to high transformation efficiencies (e.g., 22,198.33 CFU/µg DNA for B.a YN-J3).
  • Glycine (50 mg/mL) significantly increased transformation efficiency by 40, 36, and 24 times for B.a YN-J3, B.v JN-Y2, and B.s S-16, respectively.
  • A combined approach of optimized electroporation and glycine treatment yielded a stable and efficient transformation system.

Conclusions:

  • Optimized electroporation protocols combined with glycine treatment offer a highly efficient method for Bacillus strain genetic manipulation.
  • This improved transformation system facilitates advancements in the industrial and agricultural applications of Bacillus species.
  • The study provides a valuable protocol for researchers working with Bacillus transformation.