Open AccessA General Method for Fusion of the Escherichia coli lacZ Gene to Chromosomal Genes in Bacillus subtilis
Author(s) -
Jeff Errington
Publication year - 1986
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.352
H-Index - 35
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-132-11-2953
Subject(s) - biology , plasmid , gene , bacillus subtilis , genetics , lac operon , transformation (genetics) , escherichia coli , restriction enzyme , fusion gene , operon , microbiology and biotechnology , bacteria
A series of plasmids has been constructed that can be used to fuse the beta-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or 'cartridge' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac-cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as phi 105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.