A universal instinct for designing thermoregulated promotors in gram-positive bacteria
Khazak, V.E.; Veĭko, V.P.; Sorokin, A.V.
Molekuliarnaia Biologiia 25(5): 1197-1206
1991
ISSN/ISBN: 0026-8984 PMID: 1836528 Document Number: 382569
The construction of plasmid pVKH300, which is useful for modifying any promoter into the thermoregulated form in B. subtilis cells, is presented. The main features of the plasmid are the presence of effectively expressed in B. subtilis lambda C1857 gene and recognition site of BglII restriction enzyme between OR2 and OR3 lambda phage operator sites. Promoterless alpha-amylase gene of B. amyloliquefaciens is used as a reporter gene for promoter cloning into BglII site of pVKH300. Examples of promoter-containing DNA fragments cloning with pVKH300 as vector are presented. It was found that the best regulated promoter, in a plasmid named pVKH332, was cloned in such a way that the distance between central nucleotides of OR2 and OR3 is equal to integer number of DNA helix turns (84 b.p. in the case).