Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembl...

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Bibliographic Details
Published in:Scientific reports 2015-05, Vol.5 (1), p.10655-10655, Article 10655
Main Authors: Tsuge, Kenji, Sato, Yukari, Kobayashi, Yuka, Gondo, Maiko, Hasebe, Masako, Togashi, Takashi, Tomita, Masaru, Itaya, Mitsuhiro
Format: Article
Language:English
Subjects:
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631/1647/1511
631/326/325/2482
631/553/2690
631/553/552
Bacillus subtilis - genetics
Bacteriophage lambda - genetics
Coefficient of variation
Deoxyribonucleic acid
DNA
DNA Fragmentation
DNA, Bacterial - biosynthesis
DNA, Bacterial - genetics
Genetic transformation
Humanities and Social Sciences
multidisciplinary
Plasmids
Plasmids - genetics
Science
Standardization
Synthetic Biology - methods
Tandem Repeat Sequences - genetics
Transformation, Genetic
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Summary:In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis . Since this method doesn’t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep10655