Part:BBa_K2757004

pbuE riboswitch eCFP construct

Adenine riboswitch linked with eCFP.

Usage and Biology

The PbuE riboswitch is used to control the expression of the purine eflux pump - PbuE. it is a transcriptionally activating RNA structure with an adenine binding aptamer. it is believed the 5' region is involved in the secondary structure changing which leads to the unfolding of a terminator loop in the presence of the ligand.

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]

Characterisation

The riboswitch parts were characterised by inserting a unidirectional Tet promoter before the upstream folding region and after it. K12 cells transformed with these constructs were then grown in LB at 37^oC for 3 hours. This would allow us to assess this importance of this region in the sensitivity to the binding of adenine. We decided to use a plate reader assay which would measure the fluorescence of eCFP at 480nm. Adenine has a solubility in water at pH 7 of 7.4uM which is insufficient to accurately measure the mass of solute for a stock solution. In preliminary experiments, adenine was dissolved in 100% DMSO to a stock solution of 15uM. The DMSO concentration was shown to be high enough to inhibit cell growth. We believe that this will have altered the sensitivity of the riboswitch and invalidated our results. To overcome this issue, we changed our inducer from adenine to adenine sulfate dihydrate which is soluble in water to 15uM. We repeated the assay using this new stock, however there was no significant change when LB was used. M9 minimal medium gave the graph below, showing sensitivity for all concentrations in the soluble range.