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).

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]