A Conjugation Vector Delivery System for Molecular Cloning into Cells of Bacteria of the Genus Bacillus

Abstract

A delivery system for vectors for molecular cloning into bacterial cells of the genus Bacillus has been developed. A specific feature of the developed system is the use of the pBS72 plasmid, which provides the conjugative transfer of the conditionally lethal pKS1mob vector obtained into the cells of the studied bacteria. The ability of vector pKS1mob to be replicated in Escherichia coli and B. subtilis cells at a low temperature (30°C) and the presence of two polylinkers around the kanamycin resistance gene makes it possible to clone target gene fragments into its composition using traditional genetic engineering approaches. Inactivation of the rok gene in the pBS72 plasmid made it possible to transform the strain containing it with the constructed vector pKS1mob with high efficiency. Crossing the B. subtilis 168 donor strain, containing the conjugative pBS72 and pKS1mob mobilizable plasmids, with a recipient strain of the genus Bacillus made it possible to introduce the pKS1mob plasmid into it. The possibility of using the created system for inactivation of the codY gene of Bacillus licheniformis was shown.