[Preparation and transformation optimization for supercompetent B. subtilis SCK6 cells].

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus subtilis is a safe, Gram-positive aerobic bacterium favored for heterologous protein expression due to its high secretion capacity.
• Low transformation efficiency in B. subtilis hinders its use in directed evolution compared to Escherichia coli.
• Improving transformation efficiency is crucial for advancing B. subtilis applications in biotechnology.

Purpose of the Study:

• To optimize competent cell preparation methods for Bacillus subtilis.
• To enhance transformation efficiency for heterologous protein expression and directed evolution applications.
• To provide a more robust B. subtilis host system for metabolic engineering.

Main Methods:

• Systematic optimization of competent cell preparation parameters for Bacillus subtilis.
• Evaluation of different media formulations, including YN medium.
• Testing various inducer concentrations and durations, specifically xylose.
• Assessing the impact of plasmid source strains, such as E. coli GM272.

Main Results:

• YN medium improved transformation efficiency approximately 4-fold compared to standard LB medium.
• Induction with 1.5% xylose for 2 hours enhanced transformation efficiency by about 2-fold.
• Using plasmids from E. coli GM272 strain increased transformation efficiency by approximately 3-fold.
• Optimized conditions resulted in a transformation efficiency of 10⁶ CFU/μg for pDG1730 plasmid, a 100-fold increase.

Conclusions:

• Optimized competent cell preparation significantly enhances Bacillus subtilis transformation efficiency.
• The improved efficiency facilitates directed evolution of enzymes and metabolic engineering in B. subtilis.
• These findings establish a more effective B. subtilis platform for biotechnological applications.