Part:BBa_K2442102:Design

Minimal pBAD + I13500 GFP

Assembly Compatibility:

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

  Illegal BamHI site found at 241
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 76
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 973
  Illegal SapI site found at 58
  

Design Notes

In absence of L-arabinose the AraC dimer binds to pBAD operator half-sites O₂ and I₁ and represses transcription by excluding RNA polymerase from binding to pBAD or PC. Binding of L-arabinose causes a conformational change in the protein such that the DNA-binding domains of the dimer bind to adjacent I₁ and I₂ half-sites, resulting in transcription activation of downstream genes, in this case GFP (Fig. 1). GFP was included in this part to characterise the activity of the pBAD promoter and to enable mutant screening in our AraC mutagenesis project (visit our [http://2017.igem.org/Team:Glasgow/araC wiki] for details).

Figure 1: Regulation of the L-arabinose operon by arabinose. In the absence of L-arabinose, AraC dimer binds to O₂ and I₁ half sites causing DNA looping, which prevents RNA polymerase from accessing P_BAD. Upon binding of L–arabinose, the AraC dimer binds I₁ and I₂ half sites instead, allowing transcription of the polycistronic araBAD mRNA. araC is transcribed in opposite direction from araBAD, and is under control of the PC promoter. RNA polymerase and AraC compete for binding at O₁ and P_C. Adapted from Shleif (2000)

Source

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