Part:BBa_K1962004

Immunity Protein Im-E3

This biobrick encodes for the Immunity protein specific for Colicin E3. This Immunity Protein inhibits the 16S RNA hydrolyzing activity of Colicin E3 by binding with high affinity to the C-terminal catalytic domain of E3. The sequence was obtained from Uniprot and then codon optimised for E. coli K-12 and synthesised by IDT as a gBlock gene fragment.

••••• 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_K1962004 is an immunity protein against colicin E3, which is not currently in The Registry. This part will be useful for teams wishing to clone colicin E3 in the future.

Usage and Biology

Our idea was to generate a device that would express Colicin E3 in the presence of low pH and bile salts. In order to protect the host cells from the toxic activity of Colicin E3 we began by cloning the E3-Immunity protein downstream of a pH sensitive promoter Pasr (BBa_K1231000) and a bile salt sensitive promoter PacrRA (BBa_K1231001) to generate the composite parts (BBa_K1962016) and (BBa_K1962012), respectively. Unfortunately we were unable to clone Colicin E3 downstream of the E3-Immunity proteins.

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
  COMPATIBLE WITH RFC[1000]