Suite of novel vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the amyE andbglSloci in Bacillus subtilis

Abstract

We report the development of a suite of sixintegrative vectors for construction of single copy transcriptional fusions with the gfpmut3, cfp and iyfp reporter genes in B. subtilis. The promoter fusions are constructed using the highly efficient ligation-independent cloning (LIC) technique making them suitable for high-throughput applications. The plasmids insert into the chromosomeby a double cross-over event at the amyEorbglS loci and integration at each site can be verified by a plate-based screening assay. The vectors allow expression of two different promoters to be determined in the same strain using the cfp and iyfp reporter genes since CFP and iYFP are spectrally distinct and have comparable half-lives of approximately 2 hours in exponentially growing B. subtilis cells. We demonstrate the versatility of these vectors by measuring expression of the tuaAand phoA operons singularly and in combination, during growth in phosphate limiting conditions.