Part:BBa_K1893017

Arabinose inducible GFP (pBAD+GFP)

A GFP reporter under control of a pBAD promoter, used to characterise the activation range of the pBAD promoter

Characterisation data

Without any arabinose in the cell, araC forms a homodimer, binding to operator sites upstream of the pBAD promoter, generating an inhibitory loop in the DNA that prevents access by the RNAP holoenzyme. In the absence of glucose, and the presence of L-arabinose, the dimer dissociates from the DNA, allowing transcription of GFP.

We added varying concentrations of arabinose (optimum concentration ranges were obtained from 2010 iGEM team Slovenia characterisation), and recorded fluorescence in a BMG plate reader.

Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. (BBa_K1893017).Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation

Figure 2: Transfer function of normalised GFP fluorescence against concentration of L-arabinose

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1342
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1281
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1116
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2017
  Illegal SapI site found at 1098