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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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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 Danelon^1,3

¹Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2629 HZ Delft, The Netherlands.

ACS Synthetic Biology

|June 5, 2025

Summary

This summary is machine-generated.

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.

Keywords:

MAGE combinatorial libraries multiplex nanopore sequencing plasmid libraries recombineering

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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.