A PLASMID FOR SELECTIVE SELECTION OF HIGHLY SPECIFIC BACILLARY PROMOTERS

Claims

1. A plasmid for the selective selection of clones containing highly specific promoters that function in bacteria of the genus Bacillus, which is a bireplicon vector capable of replicating in E. coli and B. subtilis, with a selective marker of ampicillin resistance for E. coli, and with selective markers of chloramphenicol and trimethoprim resistance for B. subtilis, and containing a dual reporter system luxABCDEcat, represented by the lux operon and the gene for resistance to chloramphenicol cat, which acts as a marker for positive selection of promoters in Bacillus, a Kpn21 site is inserted before the luxABCDEcat reporter system, as well as a sequence encoding a hairpin structure in mRNA, stop codons, a multiple cloning region for insertion of promoters, where a gene encoding a toxic protein for E. coli cells is inserted at the Kpn21 site, but not B. subtilis and acting as a factor of negative selection in E. coli cells, but not in Bacillus.

2. The plasmid of claim 1, wherein the gene that acts as a negative selection factor in E. coli cells but not in Bacillus is the ccdB gene, which encodes the CcdB toxin protein that inhibits DNA gyrase in E. coli cells but not in Bacillus cells.

3. The plasmid of claim 1, wherein the gene that acts as a negative selection factor in E. coli cells but not in Bacillus is the gp46 gene, encoding the endolysin GP46 protein that lyses E. coli but not Bacillus.

4. A method for the selective selection of clones containing highly specific promoters that function in Bacillus but not in E. coli, comprising cloning promoters or cloning random DNA fragments treated with Mbol endonuclease or other endonucleases contained in the multiple cloning region into a plasmid according to claims 1-3 treated with BamHI endonuclease or other endonucleases contained in the multiple cloning region, followed by transformation of E. coli cells, obtaining a library of plasmids containing promoters, isolating a library of plasmids containing promoters, transforming this library into B. subtilis by seeding B. subtilis cells on tryptone medium with trimethoprim and chloramphenicol, selecting clones containing highly specific promoters and resistant to trimethoprim and chloramphenicol.

5. The method according to claim 4, further comprising assessing the strength of the cloned promoters in the selected clones using the plasmid according to claims 1-3 based on the level of luminescence of the clones grown in a liquid nutrient medium, wherein the degree of promoter strength is determined relative to the PymdA promoter.