ArticleMicrobial cell factories2015
A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation.
Article in Microbial cell factories, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed, 8 citations in OpenAlex.
- In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1.PloS one · 2023Article
- Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.Nature communications · 2021Article
- Synthetic biology of microbes synthesizing polyhydroxyalkanoates (PHA).Synthetic and systems biotechnology · 2016Review
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundWith the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. However, for constructions of most circular DNA, there are two contradictions in the ligation/assembly and transformation steps. The ligation/assembly consists of two different reactions: 1) the ligation/assembly between any two pieces of a linear form DNA; 2) the cyclization (or self-ligation) of a single linear form DNA. The first contradiction is that the bimolecular ligation/assembly requires a higher DNA concentration while the cyclization favors a lower one; the second contradiction is that a successful transformation of a ligation/assembly product requires a relatively high DNA concentration again. This study is the first attempt to use linear plasmid and Cyclization After Transformation (CAT) strategy to neutralize those contradictions systematically.
resultsThe linear assembly combined with CAT method was demonstrated to increase the overall construction efficiency by 3-4 times for both the traditional ligation and for the new in vitro recombination-based assembly methods including recombinant DNA, Golden Gate, SLIC (Sequence and Ligation Independent Cloning) and Gibson Isothermal Assembly. Finally, the linear assembly combined with CAT method was successfully applied to assemble a pathway of 7 gene fragments responsible for synthesizing precorrin 3A which is an important intermediate in VB12 production.
conclusionThe linear assembly combined with CAT strategy method can be regarded as a general strategy to enhance the efficiency of most existing circular DNA construction technologies and could be used in construction of a metabolic pathway consisting of multiple genes.
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