Robust and highly efficient transformation method for a minimal mycoplasma cell

Area of Science:

• Synthetic biology
• Genomics
• Microbiology

Background:

• Mycoplasmas are important models for understanding cellular processes and for bioengineering due to their simple nature.
• Traditional transformation methods for mycoplasmas suffer from low efficiency, requiring substantial amounts of DNA and cells.
• Genetic manipulation is essential for advancing research and applications using these organisms.

Purpose of the Study:

• To develop a robust and highly efficient transformation method for the minimal cell JCVI-syn3B.
• To optimize transformation protocols for minimal cells, reducing the required DNA and cell quantities.
• To establish a convenient transformation method using frozen stocks of cells.

Main Methods:

• Detailed examination of JCVI-syn3B growth states, including pH, color, absorbance, colony-forming units, and transformation efficiency.
• Optimization of transformation conditions focusing on the early exponential growth phase.
• Development of protocols for using minimal culture volumes and reduced plasmid DNA amounts.

Main Results:

• Achieved transformation efficiency of up to 4.4 × 10^-2 transformants per cell per microgram of plasmid DNA.
• Developed a method yielding hundreds to thousands of transformants from small culture volumes (0.2 mL) with approximately 1 × 10⁷-10⁸ cells and 10 ng of plasmid DNA.
• Established a transformation protocol utilizing frozen stocks of JCVI-syn3B cells.

Conclusions:

• The developed transformation method significantly enhances efficiency and simplifies the process for minimal cells.
• These advancements facilitate advanced genetic engineering and biological research using minimal cell platforms.
• The use of frozen stocks offers convenience and reproducibility for transformation experiments.