The Second Generation Ogab Method Makes It Feasible for Assembly More Than 50 DNA Fragments at One-Step

Construction of an artificial gene cluster that is comprised of a series of genes responsible for one metabolic pathway has been expected to be one of the potent means to create high performance metabolic pathway in host cell. However, it is still not easy to prepare de novo designed-long DNA with several dozen kb in size, because DNA assembly methods devised so far is not good enough for at once assembly of small DNA blocks that would be prepared by chemical synthesis and following assembly of single stranded DNA. OGAB is multiple gene assembly method devised by us in 2003. This method adopts Bacillus subtilis plasmid transformation system, and is feasible for assembly as much as 15 DNA blocks at one-step. Different from other popular gene assembly methods, for example Golden Gate and Gibson Assembly, are designed to obtain circular form of ligation product in vitro, OGAB method intendedly makes long tandem repeat ligation products of material DNA blocks and assembly vector in vitro, which is achieved only after precise equimolar adjustment of the blocks. However, adjustment of DNA blocks in equimolar is quite difficult as long as there is size variety in those blocks, this is the reason why the maximum number of blocks at one assembly operation is limited to lower that 15. In order to overcome this circumstance, we have recently developed second generation OGAB method by altering original OGAB method. This method is specifically designed for construction of DNA up to 50 kb in size from as much as 50 of small DNA blocks (up to 1 kb) that can be obtained from synthetic DNA service. The key point of this method is preparing the small blocks as equal length by computer-aided manner without taking account for functional boundary of gene. These designed blocks are attached appropriate TypeIIS restriction enzyme site at both ends, and then cloned into conventional E. coli plasmid vector. Since length of the blocks is same and vector is common over the blocks, precise equimolar control of blocks can be easily achieved by just adjusting weight concentration of block-cloned plasmid DNA according to values obtained by a microvolume spectrometer. Resulted equimolar plasmid mixture can be treated as sole kind of plasmid as far as all the plasmids share same TypeIIS site. Thanks to this nature, plasmid vector separation by electrophoresis after relevant restriction enzyme digestion is conveniently finished only in one operation without distortion of equimolarity. Using small blocks thus obtained, assembly of unprecedentedly large number of DNA fragments at once becomes feasible.