- the promoter sequence of a cloned gene or operon
- promoter sequences in random tnpR-gene fusion libraries
- genes induced under harsh environmental conditions, such as various stresses, under which other reporter genes and selection systems (cat, bla) cannot be used
The plasmid pBBR RESO is a broad-host-range promoter cloning vector. In contrast to other known broad-host-range vectors, it is maintained at a medium copy number and has a reasonable size of about 6.8 kb. It stably replicates in any Gram-negative bacterium studied, and is therefore particularly interesting for the isolation and genetic analysis of DNA sequences with promoter activity in the homologous organism.
The reporter system used employs the resolvase-mediated excision of a kanamycin (Kan)-resistance gene flanked by two res sequences. Cloning an active promoter results in Kan-sensitive clones.
pBBR RESO was derived from pBBR1MCS, which itself is a modification of the broad-host-range plasmid pBBR1CM. It contains a chloramphenicol resistance gene (CmR) and a unique Bgl II cloning site immediately upstream the promoterless reporter gene tnpR, encoding the resolvase from transposon Tn3. Two directly repeated res sequences flanking the Kan-gene are located downstream of tnpR. A transcriptional fusion between a DNA fragment cloned into Bgl II and tnpR results in expression of the latter, and resolvase-mediated strand exchange occurs between the res sites. This leads to the irreversible shift from a Kan-resistant to a Kan-sensitive phenotype of the host bacterium.
Clones should be plated on Cm-containing agar and assayed for kanamycin resistance/sensitivity. The only requirement for the use of this system is a resolvase-free background, i. e. the gram-negative strains should not contain any transposon potentially coding for resolvase.
Besides Bgl II the DNA of interest can also be digested with Sau 3A or BamH I, since the overhangs are compatible.