title: Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments.
abstract: 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.
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tsuge
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Gondo
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Hasebe
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Togashi
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.affiliation: Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Itaya
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*; D020080:Tandem Repeat Sequences / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial