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Related Concept Videos

Transformation01:26

Transformation

Microbial communities are dynamic environments where cell lysis releases free DNA into the surroundings. Other cells can take up this extracellular DNA through a process known as transformation.When a cell incorporates this foreign DNA into its genome, resulting in genetic modification, the process is known as transformation. Cells capable of this process are termed competent. Competence can be natural, as observed in certain bacteria and archaea, or artificially induced in the...
Bacterial Phylum Tenericutes01:24

Bacterial Phylum Tenericutes

The phylum Tenericutes, which includes the single class Mollicutes, comprises bacteria that lack cell walls. The term "Mollicutes" derives from the Latin word mollis, meaning "soft." These organisms are among the smallest known and are commonly referred to as mycoplasmas due to the prominence of the genus Mycoplasma, which includes well-known human pathogens. Despite their inability to stain gram-positively (a result of their lack of cell walls), mycoplasmas are phylogenetically related to the...

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Optimized PCR-based Detection of Mycoplasma
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Robust and highly efficient transformation method for a minimal mycoplasma cell.

Masaki Mizutani1, John I Glass2, Takema Fukatsu1,3,4

  • 1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki Prefecture, Japan.

Journal of Bacteriology
|February 4, 2025
PubMed
Summary

This study introduces a highly efficient transformation method for minimal cells like JCVI-syn3B, significantly improving genetic engineering. The new protocol requires minimal DNA and cells, simplifying research with these unique bacteria.

Keywords:
Mycoplasmagrowth curvetransformation

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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.