Part:BBa_K1962006

Truncated Colicin E9 Lacking Bacteriocin Active Domain

Colicins are anti-bacterial proteins produced by some strains of E. coli that typically have three domains: a translocation domain; a receptor binding domain; and a cytotoxic domain. This biobrick encodes a truncated version of Colicin E9 which lacks the C-terminal bacterocin domain, which is this case is a DNase.

The part has an engineered multiple cloning site at the 3' end, preceding the RFC[10] suffix and regulation double stop codons, containing the following in-frame restriction sites: BamHI, KpnI, SalI, BglII and NheI. The presence of this multiple cloning site will allow for different toxic domains, or other polypeptides, to be fused at the C-terminus of the truncated colicin and thereby generating a synthetic colicin or novel fusion protein in a rapid and facile manner.

This Biobrick was used to generate two synthetic colicins (BBa_K1962007 and BBa_K1962008).

••••• Parts Collection 2016

This is part of a Part Collection of 18 BioBricks designed by Dundee iGEM 2016. This collection will be useful to teams working with toxins as we have submitted new toxins to the registry. Working with bacterial toxins is difficult due to the risk of toxicity to the chassis, so the corresponding immunity for our toxins were also submitted. We have also submitted these toxins lacking their cytotoxic domains replacing it with a multiple cloning site which will allow for different toxic domains to be fused at the C-terminus and thereby generating a synthetic toxin. In addition, there are three well-characterised promoters that can be used to initiate gene expression at various points in the digestive tract, to enable devices to function within a human or animal. Finally, a lysis cassette was constructed to lyse or burst cells, thus releasing the toxins and destroying the GM bacteria to prevent its release to the environment. This BBa_K1962006 is truncated colicin E9. This part will be useful for teams wishing to clone novel toxins in the future.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1381
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1375
  Illegal BamHI site found at 1357
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]