A SERIES OF INTEGRATIVE PLASMIDS FOR BACILLUS-SUBTILIS CONTAINING UNIQUE CLONING SITES IN ALL 3 OPEN READING FRAMES FOR TRANSLATIONAL LACZ FUSIONS

Two sets of three plasmids each were constructed based on integrative plasmids for Bacillus subtilis. Each set encodes resistance to either chloramphenicol or kanamycin. The plasmids contain a cassette consisting of the resistance gene, BamHI and SmaI cloning sites, and the promoterless lacZ gene. The cassette is flanked by the 3' and 5' ends of the amyE gene (encoding amylase) allowing integration of the cassette into that locus in the B. subtilis chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of replication and the beta-lactamase-encoding gene. Within a set, each of the three plasmids carries the unique SmaI and BamHI restriction sites in a different reading frame relative to the 'lacZ gene. These sets of plasmids allow the easy construction of translational fusions with lacZ and single-copy integration at the amyE locus of B. subtilis.