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MOSAIC: A Highly Efficient, One-Step Recombineering Approach to Plasmid Editing and Diversification
Marijn van den Brink1, Timotheus Y Althuis2, Christophe Danelon1,3
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2629 HZ Delft, The Netherlands.
ACS Synthetic Biology
|June 5, 2025
Summary
MOSAIC is a new, rapid protocol for editing plasmids and creating diverse plasmid libraries in synthetic biology. It uses a novel protein to efficiently incorporate DNA changes, saving time and resources.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Biotechnology
Background:
- Plasmid editing and library construction are crucial for synthetic biology.
- Traditional methods are time-consuming and resource-intensive, involving DNA amplification and cloning.
Purpose of the Study:
- To establish MOSAIC, a highly efficient protocol for plasmid editing and combinatorial plasmid library generation.
- To provide a rapid, resource-effective alternative to existing methods.
Main Methods:
- Utilized the single-stranded DNA annealing protein (SSAP) CspRecT for DNA oligo incorporation.
- Performed plasmid editing and library generation in *Escherichia coli*.
- Integrated a user-friendly Nanopore sequencing validation pipeline.
Main Results:
- Achieved up to 90% single-target plasmid editing efficiency.
- Successfully generated combinatorial plasmid libraries across four target regions in a single transformation.
- Demonstrated a user-friendly validation pipeline with minimal computational requirements.
Conclusions:
- MOSAIC offers a simple, rapid, and resource-effective method for plasmid manipulation.
- This protocol facilitates the generation of large, diverse plasmid libraries for various applications.
- MOSAIC is expected to accelerate research in molecular and synthetic biology.
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