Stable Positional Cloning of Long Continuous DNA in the Bacillus subtilis Genome Vector

Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time. Two small DNA fragments had to be installed in the vector prior to cloning. The DNA between these two fragments was cloned via homologous recombination. The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp. PCC 6803. Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome. Designated genomic segments as large as 77-kb were cloned by means of a single procedure. Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned. The method is suitable for direct target cloning of large-sized DNA.