A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile

Area of Science:

• Molecular Biology
• Yeast Genetics
• Synthetic Biology

Background:

• Homologous recombination (HR) in Saccharomyces cerevisiae offers efficient and cost-effective DNA cloning.
• Current HR methods are incompatible with specialized plasmids used in research.
• This limits the widespread adoption of yeast-based cloning techniques.

Purpose of the Study:

• To enhance the versatility of yeast gap-repair cloning.
• To enable compatibility of HR cloning with any DNA plasmid.
• To develop a cost-effective and efficient cloning method for diverse research applications.

Main Methods:

• Introduction of a yeast-cloning cassette (YCC) containing the 2-micron origin of replication (2μm ori) and ura3 gene.
• Assembly of multiple DNA fragments into various DNA vectors using the YCC.
• Demonstration of the method's utility across different plasmid types and model systems.

Main Results:

• The YCC facilitates the assembly of multiple DNA fragments into any DNA vector, overcoming previous limitations.
• A variety of plasmids were successfully constructed for applications including protein production, epitope tagging, mutagenesis, and fluorescent protein expression.
• The method was validated in microbial systems and a vertebrate model, showcasing broad applicability.

Conclusions:

• The developed YCC significantly improves the versatility and compatibility of yeast gap-repair cloning.
• This simple, efficient, and cost-saving method has broad potential for molecular biology and biomedical research.
• The technique facilitates diverse cloning applications, from basic research to applied biotechnology.