Open AccessIntegrative vector for constructing single‐copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat‐stable β‐galactosidase
Author(s) -
Stoß Oliver,
Mogk Axel,
Schumann Wolfgang
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb10348.x
Subject(s) - bacillus subtilis , biology , operon , plasmid , gene , genetics , fusion gene , pbr322 , regulatory sequence , reporter gene , shuttle vector , escherichia coli , regulation of gene expression , microbiology and biotechnology , gene expression , vector (molecular biology) , recombinant dna , bacteria
Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat‐stable β‐galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin‐resistance gene for selection in B. subtilis . Both cassettes are flanked by the 3′‐ and 5′‐end of the amyE gene (encoding α‐amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli , the plasmids contain the pBR322 origin of DNA replication and the β‐lactamase‐encoding gene. Whereas one vector needs a promoter, a Shine‐Dalgarno sequence and the beginning of a gene fused in‐frame to bgaB , the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat‐86 genes. In the first case, heat‐inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol‐inducible regulation of the gene fusion could be demonstrated.