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Updated: Apr 14, 2026
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Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells
Published on: April 4, 2016
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A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation.
Ying-Ying Guo1, Zhen-Yu Shi2, Xiao-Zhi Fu3
1MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China. guoyingying@973pha.org.
Microbial Cell Factories
|April 22, 2015
Summary
This study introduces a linear DNA assembly method combined with the Cyclization After Transformation (CAT) strategy, significantly improving the efficiency of creating large circular DNA structures for synthetic biology applications.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Genetic Engineering
Background:
- Synthetic biology demands efficient assembly of multiple DNA fragments into large circular DNA.
- Existing circular DNA construction methods face contradictions between ligation/assembly and transformation steps regarding DNA concentration.
- This study addresses these contradictions using a novel linear plasmid and Cyclization After Transformation (CAT) strategy.
Purpose of the Study:
- To systematically neutralize contradictions in circular DNA construction.
- To enhance the efficiency of assembling multiple DNA fragments in a single step.
- To present a general strategy for improving existing DNA assembly technologies.
Main Methods:
- Utilized a linear plasmid assembly approach.
- Implemented the Cyclization After Transformation (CAT) strategy.
- Applied the combined method to various in vitro recombination-based assembly techniques.
Main Results:
- The linear assembly with CAT method increased construction efficiency by 3-4 times across multiple assembly techniques (recombinant DNA, Golden Gate, SLIC, Gibson Isothermal Assembly).
- Successfully assembled a 7-gene pathway for synthesizing precorrin 3A, an intermediate in VB12 production.
Conclusions:
- The linear assembly combined with CAT strategy is a general method to enhance circular DNA construction efficiency.
- This strategy is applicable to metabolic pathway construction involving multiple genes.
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