Part:BBa_K1962016

A device for expression of Im-E3 in response to pH

This is a composite part for the regulated expression of the Immunity Protein for netralization of the Colicin E3, which is a specific RNase. The composite part was assembled from a pH responsive promoter (asr BBa_K1231000), an RBS (BBa_B0030) and the biobrick encoding the Immunity Protein against E3 (Im-E3) (BBa_K1962004).

••••• 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_K1962015 is producing an immunity protein in response to pH. It is ready for addition of new toxin genes.

Usage and Biology

We cloned the E3-Immunity protein (BBa_K1962016) 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.

The immunity proteins for colicin Ia and E3 were cloned in front of the asr pH sensitive promoter. The immunity proteins were amplified with a HA tag fused onto the C-terminal of the immunity proteins to allow for visualisation of expression via western blotting. As shown in Fig 1 we were unable to detect expression of E3-Immunity protein.

Figure 1. Western blot of colicin Ia and E3 immunities with pasr: The col Ia and E3 immunities were amplified with a HA tag to detect expression via a western blot. The asr promoter was blotted alongside as a control. No expression of the immunities in front of the asr promoter was detected.

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]