Recombinational Transfer of 100-Kilobase Genomic DNA to Plasmid in Bacillus subtilis 168

Abstract

Transformation of Bacillus subtilis by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic B. subtilis recombinational repair pathway. The vector, pGETS103, a derivative of the -type replicating plasmid pTB19 of thermophilic Bacillus, had the full length of Escherichia coli plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of B. subtilis. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.

Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome

“…Those findings indicated that appropriately edited DNA cloned in the BGM vector yields DNA suitable for use in different research undertakings. The retrieval of megacloned DNA by the recovery system (14,36) or the genome surgery system (37) renders the BGM vector a tool with which technical breakthroughs may be achieved.…”

Production of Multi-Purpose BAC Clones in the Novel Bacillus subtilis Based Host Systems

“…The third method involves a yet more complicated genetic process referred to as Bacillus Recombinational Transfer (BReT) (Tsuge & Itaya, 2001;Kaneko et al, 2005). Indeed, BReT relies on homologous recombination in B. subtilis and should be as simple as the genetic disconnection procedure described above.…”

Region Dependent Efficiency for Recombinational Transfer of theBacillus subtilis168 Genome

“…1,2) This method, designated the B. subtilis Recombinational Transfer (BReT) method, shown in Fig. 1, presents no theoretical problem of applicability to any genome region.…”