Part:BBa_P1010

ccdB cell death gene

The ccd operon having a constitutive promoter and the ccdB coding region. ccdB protein is lethal in normal cloning strains. This part is used to aid the process of moving a BioBrick part to a new plasmid. A target plasmid carrying P1010 is cut and mixed with the cut insert. Plasmids that recombine are selected against because they kill the cell that picks them up. P1010 is only provided as plasmid or in DB3.1 which is ccdB resistant.

Usage and Biology

This is the part that is present inside the BioBricks insertion site in many of the pSB series of plasmids. It is sufficient for positive selection even in pSB2* series plasmids in the absence of inducer (~4-5 copies per cell).

ccdB

CcdB, a natural analogue of the quinolone antibiotics (e.g., ciprofloxacin, enoxacin, etc.), binds to the DNA gyrase subunit A, the product of the gyrA gene, turning it into a cellular poison (Bahassi, et al. 1999 J. Biol. Chem. 274: 10936-10944). E.coli strains that carry the gyrA462 mutation (e.g., DB3.1) are resistant to CcdB poisoning.

Frame shift mutation<b>

During assembly work at the Registry, we have found that point mutations (particularly frame shifts) occur at a high enough rate that several colonies will survive during a normal transformation. -Randy

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 664
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 114