Gene cluster or operon design by ordered gene assembly in Bacillus Subtilis (OGAB) method

In the post genome sequence era, metabolic engineering conducted by a number of genes is attracting great attention for not only basic research but also for application in production of beneficial materials for our lives. To carry this out, methods to handle a number of genes at a time are a prerequisite. However, the poor ability of Escherichia coli-based genetic methods to manipulate the reconstruction makes the steps timeconsuming and laborious. Bacillus subtilis, a gram-positive soil bacterium, develops natural competence in certain physiological conditions. In the competence, this bacterium can take up exogenous DNAs, and can recombine these DNAs with the genome if there is a homologous sequence. By using this trait, a novel method to carry out gene assembly quickly and with greatest fidelity using B. subtilis was developed. The method, named Ordered Gene Assembly in B. subtilis (OGAB), facilitates the connection of more than 10 DNA fragments in plasmid in one-step using an intrinsic B. subtilis plasmid transformation system. In this chapter, the principle of and actual protocol for the OGAB method are written. Moreover, two types of gene assembly in terms of metabolic engineering are also described.