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Miscellaneous DNA Vector Systems

RESO01 - Broad-Host-Range Vector pBBR RESO (promoter trap vector)

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Product Description:
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.

Features
Identification of:

  • the promoter sequence of a cloned gene or operon
  • the 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

Advantages:

Not only constitutively but also transiently induced promtors can be detected. Further, the screening for promoter-containing clones does not necessitate a selection pressure onto the reporter gene products.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.

reso

Promoter cloning vector pBBR RESO.Cloning a promoter sequence into the Bgl II 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.

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.