Part:BBa_K1962001

Immunity Protein Im-Ia

This biobrick codes for the Immunity Protein for Colicin Ia (BBa_K1962000). Colicin Ia is a channel-forming bacteriocin that depolarizes the cytoplasmic inner membrane of target bacteria, leading to dissipation of cellular energy. This Immunity Protein is tightly linked to its specific Colicin bacteriocin domain to protect the colicinogenic cell from the cytotoxic activity of the colicin.

••••• 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_K1962001 is immunity against full-length colicin Ia BBa_K1962000. Together they are used as controls that can be used to compare activity of any synthetic toxins that are generated using this part collection.

Usage and Biology

The cell-killing potential of a pore-forming colicin is remarkable in that one molecule kills one cell. Equally remarkable is the protection system that operates at 104 to 107 times the concentration of colicin that would kill a nonimmune cell. This protective mechanism is achieved by a small polypeptide of 11 to 18 kDa, called the immunity protein, encoded by the same plasmid as colicin. The immunity proteins confer upon cells protection against the colicin they produce but not against heterologous colicins with identical modes of action, even those with considerable sequence similarity. Interestingly, immunity proteins are required to protect the cell against the action of exogenous colicin, probably produced by its neighbors, but are not required to protect it from internal colicin, since the polarity of the transmembrane potential is opposite to that required to open the pore. The specificity of colicins with respect to immunity is determined by the C-terminal pore-forming domains.

Cloning Strategy

Our idea was to generate a device that would express Colicin Ia in response to low pH and bile salts so in order to protect the host cells from the toxic activity of Colicin iA we began by cloning the iA-Immunirty protein downstream of a pH sensitive promoter P_asr (BBa_K1231000) and a bile salt sensitive promoter P_acrRA (BBa_K1231001) to generate the composite parts (BBa_K1962015) and (BBa_K1962011), respectively. However, after successfully cloning the iA-Immunity protein we were unable to clone the Colicin iA downstream of the promoter and the immunity.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 46
  Illegal AgeI site found at 253
  
• 1000
  COMPATIBLE WITH RFC[1000]