Construction and Manipulation of Giant DNA by a Genome Vector

Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.