A new method for integration and stable DNA amplification in poorly transformable bacilli

We have developed a strategy for the integration and stable amplification of DNA sequences in the chromosome of poorly transformable bacilli, which avoids the presence of a functional plasmid replication system in the integrated DNA. The parental vector for integration contains two plus origins of replication from pUB110 in the same orientation on a single plasmid. Due to the direct repeats, such plasmids produce two individual progeny vectors, one of which is dependent on the other for replication, as it lacks a functional rep gene. We have used such a progeny vector system to integrate and amplify DNA on the chromosome of Bacillus licheniformis, and show that the structure is stable in the absence of selective pressure.