Construction Of The Integrative Expression Vector For The Glv Operon Promoter Pâ–³glvA Of Bacillus Subtilis And A Preliminary Discussion On Promoter Function

The Bacillus subtilis, next to E.coli, is the best-characterized prokaryotic organism on the genetic, biochemical and physiological level. But the Bacillus subtilis does not contain proper endogenous plasmids. The plasmid vectors for Bacillus subtilis are now taken from other genus bacterium, such as Staphylococcus aureus or lactococcus lactis and so on. Although successful cloning has been reported, it is now clear that these plasmids are far from optimal for Bacillus subtilis, because of low efficiencies of cloning and high levels of replicating instability.Integration of cloned genes into the chromosome is a strategy used to avoid genetic instability. In addition, a efficient expression cassette is necessary for a optimal expression vector in Bacillus subtilis, besides the replicating and genetic stability. The optimal expression cassette includes the promoter element, the terminator element and the appropriate multiple cloning site. It is a strong and controllable promoter that is the key of the high efficient cloning, which is liable to the influence of promoter itself and host strain.The main work in this task is the construction of integration expression vectors Pyzq-3, the vector Pyzq-3 is base on the vector PAX01 bone, assembled from the promoter PA glvA which derived from the glv operon of Bacillus subtilis and exist a site mutation in catabolite repression element , and from the report gene ,the bgaB gene, coding for heat-stable P -Gal which derived from the Bacillus stearothermophilus.In order to discuss the effect of spoOA factor of Bacillus subtilis to the promoter PA glvA, a integrative substitute vector Pyzq-1 by spoOA locus was constructed.The delivery vector Pyzq-1 that can be integrated into spoOA locus within the B. subtilis chromosome by a double crossover has been constructed and is based on the pBluescriptII ks (+) bone. The spoOA-front (474bp) and the spoOA-back (554bp) were generated by PCR using chromosomal DNA of Bacillus subtilis 1094 as a template and the neomycin resistance gene (ned) as a resistant marker in Bacillus subtilis was recovered from the vector of pBEST501.Next, the B.subtilis IA785 was transformed with linear pYZQ-3, and getting the mutant GJYY-03. Then the linear Pyzq-1 transformed the host mutant GJYY-03, resulting in the corresponding mutant GJYY-05. To determine the B-Gal activities of both mutants of GJYY-03 and GJYY-05, the cultures were grown in the medium containing 1%(w/v) glucose, and induced by different concentration maltose 0%, 0.2%, 0.5%, 1% (vv/v ) respectively.To conclusion, the promoter PAglvA has the maximum activity at the 0.5% (w/v)inducer. The B-Gal activities was partially inhibited under the condition of containing 1% glucose in medium or existing a spo0A gene mutation for the host strain. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of the promoter PAglvA.