Open AccesspCON4 and pCON5: improved plasmid vectors to study bacterial promoters
Author(s) -
Lorenzo Victor,
Herrero Marta,
Neilands J.B.
Publication year - 1988
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.1574-6968.1988.tb02904.x
Subject(s) - subcloning , operon , plasmid , biology , promoter , lac operon , microbiology and biotechnology , mutagenesis , fusion gene , escherichia coli , dna , site directed mutagenesis , genetics , gene , mutation , gene expression , mutant
Plasmid vectors pCON5 and pCON4 were devised to perform, with a single construction, a number of assay to characterize promoters of Escherichia coli (and related species) which usually require different steps of subcloning, fragment purification and radioactive labeling. Any DNA fragment with promoter activity can be cloned in these plasmids to generate either a lacZ ‐gene fusion (in pCON5) or a lacZ ‐operon fusion (in pCON4). Random and defined mutations to alter the activity or the regulation of the fusion can be obtained by directed saturation mutagenesis of the cloned fragment or site‐directed mutagenesis usign the single‐strand form of the construction. Sequencing of the mutations and in vivo assessment of their effects are carried out without any subloning step. Additional analyses such as determination of the transcription start site(s) and in vitro ‘footprinting’ of DNA‐binding proteins are also possible with the use of 5′‐ 32 P‐labelled primers. As an example, up‐mutations at the promoter region of an iucA'‐'lacZ iron‐regulated fusion were isolated and analyzed with this procedure.