Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector.

Abstract

The Genome of Bacillus subtilis 168 was used for cloning and engineering of large-sized DNAs. A mouse genomic DNA of approximately 120 kb was cloned into a locus of the B. subtilis genome by ordered assembly of 20- to 50-kb mouse DNA segments. Cloned mouse DNA, maintained stably, was engineered through B. subtilis transformation and recombination. Creation of an I-PpoI recognition sequence at both ends of the insert facilitated its isolation by pulsed field gel electrophoresis. The basic concept of genome vector technology is suited to the handling of DNAs larger than 100 kb