Promoter-Trap Vector pBBR RESO (Broad-Host-Range)
Identify Promoters in a Wide Variety of Gram-Negative Bacteria
Vector map of pBBR RESO
Cloning a promoter sequence into the BglII site causes resolvase mediated kanamycin (KanR) excision (pBBR RESO*) and, thus, irreversible Kan sensitivity. Mob is involved in mobilization, Rep in replication. TnpR: resolvase. CmR: chloramphenicol resistance.
- 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 BglII cloning site immediately upstream of 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 BglII 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 BglII the DNA of interest can also be digested with Sau3A or BamHI, since the overhangs are compatible.
- Not only constitutively but also transiently induced promoters can be detected. Further, the screening for promoter-containing clones does not necessitate a selection pressure onto the reporter gene product.
- Sequence of pBBR RESO (txt)