A NEW METHOD FOR INTEGRATION AND STABLE DNA AMPLIFICATION IN POORLY TRANSFORMABLE BACILLI

We have developed a strategy for the integration and stable amplificat ion 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 t wo 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 fo r replication, as it lacks a functional rep gene. We have used such a progeny vector system to integrate and amplify DNA on the chromosome o f Bacillus licheniformis, and show that the structure is stable in the absence of selective pressure.