Part:BBa_K1318000

CcdB toxin

All ccdb parts have been withdrawn from the Registry. Samples of parts containing the ccdB gene cannot be requested. - iGEM HQ

Usage and Biology

CcdB is a toxin that poisons and kills Escherichia coli by inhibiting the DNA gyrase and inducing double strand breaks. CcdB comes from the CcdBA operon where it its transcribed alongside CcdA, an antoxin that inhibits CcdB toxicity, allowing cells to survive.

Sequence and Features

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 216
  

Characterization

In order to characterize the CcdB biobrick, we made an activity screening. The CcdB biobrick was inserted into a pBAD33 vector under the control of the ara promoter (arabinose-induced and glucose-repressed). The CcdA antitoxin was also inserted into a pKK233 vector under the control of the lac promoter (lactose/IPTG-induced). We transformed E.coli cells with one or both of these vectors and plated them on LB medium with arabinose/IPTG or glucose/IPTG.

Figure 1 : CcdB activity assay. Successive shown colonies are 10-fold dilutions: 10⁰, 10^-2, 10^-3, 10^-4, 10^-6. Cells were transformed with pBAD33/CcdB, pKK233/CcdA, or both vectors and plated
on LB medium with glucose (1 %) and IPTG (0.1 M) or arabinose (1 %) and IPTG (0.1 M).

This assay shows that induction of CcdB by IPTG inhibits cell growth while cells not expressing CcdB or expressing CcdA (induced by arabinose) alongside CcdB are able to grow. In summary, we confirmed that the CcdB biobrick is toxic for E.coli and that this toxicity can be inhibited by the expression of the CcdA antitoxin.