Versatile Methodology for Efficient Large-sized DNA Delivery Between Microorganisms Without In vitro Purification.

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Traditional microbial transformation relies on purified DNA plasmids.
• Purification processes can shear large DNA molecules, limiting their utility.
• A need exists for simplified, efficient DNA delivery methods.

Purpose of the Study:

• To introduce an alternative DNA delivery method using extracellular plasmids.
• To demonstrate the efficacy of the Cell Lysis Technology to provide Transformable Extra-cellular DNA (CELyTED) protocol.
• To adapt CELyTED for transforming both prokaryotic and eukaryotic microorganisms.

Main Methods:

• Utilized extracellular plasmids released from lysed Bacillus subtilis.
• Applied CELyTED for direct transformation of chemically competent Escherichia coli.
• Adapted the protocol for Saccharomyces cerevisiae transformation.
• Optimized conditions for donor host cell lysis.

Main Results:

• Successfully transformed E. coli and S. cerevisiae using lysed donor host cells.
• CELyTED enabled the delivery of DNA plasmids larger than 50 kb.
• The method bypassed the need for biochemical DNA purification.
• Demonstrated versatility for both prokaryotic and eukaryotic microorganisms.

Conclusions:

• CELyTED offers a streamlined approach for genetic transformation.
• The technology facilitates the introduction of large DNA plasmids, preserving their integrity.
• CELyTED presents a foundational platform for synthetic genome applications.
• This method simplifies DNA delivery, enhancing its applicability in diverse microbial systems.