Production of a plasmid to facilitate gene interruption of the nitric oxide synthase gene from Bacillus subtilis

A genetic interruption of Bacillus subtilis 168's native nitric oxide synthase enzyme (bsNOS) is to be created by inserting a gene for kanamycin resistance (KAN) into the center of the gene. To accomplish this, a plasmid has been constructed (pUC19DeltabsNOS) which includes the bsNOS gene separated into 5' and 3' halves with a KAN gene from p34S between them. The plasmid vector was isolated by digesting Escheria coli plasmid pUC19 with KpnI and PstI, and the KAN gene was cut from Escheria coli plasmid p34S using XbaI. The 5' and 3' halves of the bsNOS gene were amplified using the polymerase chain reaction with custom primers that imparted appropriate digestion sites to the ends of both amplified fragments. The vector and bsNOS fragments were all produced and ligated into a preliminary plasmid form (pNOS20) and amplified. This plasmid was subsequently digested with XbaI, and the KAN fragment was ligated into place between the 5' and 3' XbaI sites. Knockout colonies were generated by amplifying the desired insert from the pUC19DeltabsNOS plasmid using PCR and then using it for transforming competent wild-type Bacillus subtilis via electroporation. Potential Bacillus subtilis DeltabsNOS knockout colonies may have been generated but are pending confirmation.