Part:BBa_K1962011

A device for expression of Im-Ia in response to bile salts

This is a composite part which consists of a bile salt responsive promoter (BBa_K318514), a RBS (BBa_B0030) and the biobrick encoding the Immunity Protein to Colicin Ia (BBa_K1962001). For best results use in conjunction with the RamA transcriptional activator (BBa_K1962009). The promoter activity has been chracterised using a GFP reporter (BBa_K1962010).

••••• 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_K1962011 is producing an immunity protein in response to bile salts (use in conjunction with BBa_K1962009. It is ready for addition of new toxin genes.

Usage and Biology

The immunity proteins for colicin Ia and E3 were cloned in front of the acrRA bile salt promoter BBa_K1962011 and BBa_K1962012 respectively. A HA tag was fused onto the C-terminal of the immunity proteins to allow visualisation of expression via western blotting (Fig 1).

Figure 1. Western blot of colicin Ia and E3 immunities with bile salt sensitive pacrRA: The col Ia and E3 immunities were amplified with a HA tag to detect expression via a western blot. Empty pSB1C3 and acrRA promoter was blotted alongside as a control. The constructs with ramA were also blotted to see if this has any effect on acrRA function. No expression of acrRA col Ia immunity was detected however we were able to see expression of the E3 immunity in the acrRA col E3 immunity with and without ramA.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 471
  Illegal XhoI site found at 785
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 910
  Illegal AgeI site found at 1117
  
• 1000
  COMPATIBLE WITH RFC[1000]