Journal Article

An efficient electrotransformation method for three Bacillus species

Liu, Zhi-Gang, Zhou, Hong-You, Liu, Chen-Lu, Dong, Bao-Zhu, Hao, Li-Jun, Quan, Wei, Wang, Hai-Xia, Shi, Shao-Xuan

2025

Overview

Bacillus strains are widely used in food fermentation and plant disease control. An efficient transformation method is crucial for genetic manipulation in these organisms. To enhance the transformation efficiency of three Bacillus strains— Bacillus amyloliquefaciens YN-J3 (B.a YN-J3), Bacillus velezensis JN-Y2 (B.v JN-Y2) and  Bacillus subtilis S-16 (B.s S-16), we optimized transformation conditions using orthogonal experiments combined with response surface analysis. Additionally, we tested various cell wall agents to improve competence. Our results showed that the optimal transformation parameters for B.a YN-J3 and B.v JN-Y2 had an OD 600 of 0.70, a competent cell volume of 91 μL, a plasmid concentration of 1040 ng·μL⁻ 1 , and a field strength of 18.1 kV·cm⁻ 1 . For B.s S-16, the optimal conditions were an OD 600 of 0.71, a competent cell volume of 92 μL, a plasmid concentration of 1052 ng·μL⁻ 1 , and a field strength of 18.2 kV·cm⁻ 1 . Under these optimal conditions, the transformation efficiencies for B.a YN-J3, B.v JN-Y2, and B.s S-16 were 22,198.33 CFU·μg⁻ 1 DNA, 24,498.67 CFU·μg⁻ 1 DNA, and 23,305.00 CFU·μg⁻ 1 DNA, respectively. Screening of cell wall agents revealed that 50 mg/mL glycine significantly boosted transformation efficiency by 40, 36, and 24 times for B.a YN-J3, B.v JN-Y2, and B.s S-16, respectively. These findings demonstrate that combining glycine treatment with optimized transformation conditions provides an efficient approach for the genetic manipulation of Bacillus strains. Key points • The electroporation transformation parameters of three Bacillus were optimized by combining orthogonal experiments with response surface methodology • A stable and efficient electroporation transformation system suitable for three types of Bacillus was established. • 3. 50 mg/mL glycine solution can increase the transformation efficiency by 40, 36 and 24 times, respectively.

• Publisher: Springer Berlin Heidelberg,Springer Nature B.V,Springer
• Subject: Bacillus / Bacillus - genetics / Bacillus amyloliquefaciens / Bacillus amyloliquefaciens - genetics / Bacillus subtilis / Bacillus subtilis - genetics / Bacillus velezensis / Biomedical and Life Sciences / Biotechnology / Cell size / Cell Wall - drug effects / Cell walls / Deoxyribonucleic acid / Design / Disease control / DNA / DNA methylation / E coli / Efficiency / Electric fields / Electroporation / Electroporation - methods / Experiments / Fermentation / Fermented food / Field strength / Food plants / genetic engineering / Genetic transformation / Glycine / Life Sciences / Microbial Genetics and Genomics / Microbiology / Optimization / Parameters / plant disease control / Plant diseases / Plasmids / Plasmids - genetics / Reproducibility / Response surface methodology / Response surface methodology (RSM) / Software / species / Strains (organisms) / Surface analysis (chemical) / Transformation / Transformation, Bacterial / Variance analysis