Cell

Part:BBa_V1005:Design

Designed by: Tom Knight   Group: Arkin Lab   (2004-07-14)


DB3.1

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Genotype:

F- endA1 recA1 galU galK deoR nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ- What does this mean (?)

Source

Invitrogen

References

  • J Mol Biol. 1992 Aug 5;226(3):735-45.

Cell killing by the F plasmid ccdB protein involves poisoning of DNA-topoisomerase II complexes.

Bernard P, Couturier M.

Departement de Biologie Moleculaire, Universite Libre de Bruxelles, Belgium.

In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.

  • Gene. 1994 Oct 11;148(1):71-4.

Positive-selection vectors using the F plasmid ccdB killer gene.

Bernard P, Gabant P, Bahassi EM, Couturier M.

Departement de Biologie Moleculaire, Universite Libre de Bruxelles, Rhode-Saint-Genese, Belgium.

Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735-745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

  • Gene. 1995 Aug 30;162(1):159-60.

New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers.

Bernard P.

Departement de Biologie Moleculaire, Universite Libre de Bruxelles, Rhode-Saint-Genese, Belgium.

Recently, we described pKIL18/19 positive-selection cloning vectors which rely on the inactivation of the cytotoxic ccdB gene [Bernard et al., Gene 148 (1994) 71-74]. They are new tools for simplifying gene cloning/sequencing procedures, as only recombinant DNA allow the formation of viable colonies. To enhance positive-selection capabilities, putative translational reinitiation codons located within the pKIL18/19 ccdB were modified by site-directed mutagenesis. New pKIL-derived vectors with kanamycin (KmR) or chloramphenicol (CmR) resistance-encoding genes were also constructed. In addition, a new host derived from the DH2 strain was developed.

  • Biotechniques. 1996 Aug;21(2):320-3.

Positive selection of recombinant DNA by CcdB.

Bernard P.

Institut de Biologie, Montpellier, France. pbernard@dbm.ulb.ac.be