Regulatory

Part:BBa_K216005:Experience

Designed by: Edinburgh iGEM 2009   Group: iGEM09_Edinburgh   (2009-09-25)
Revision as of 15:44, 21 October 2009 by Evangelineoh (Talk | contribs) (→‎Applications of BBa_K216005)

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Applications of BBa_K216005

Edinburgh iGEM team, October 2009: To test this part, it was combined with lacZ minigene J33202 for Miller assays (giving part BBa_K216009), and also with GFP for analysis using the promoter characterization kit of Jason Kelly. Results from overnight induction with Miller assay indicated stronger induction by nitrate than nitrite. A time course of induction using the GFP construct also indicated that nitrate gave stronger induction in the first few hours.

Results from Miller's Assay
PyeaR-miller.png



Results from GFP promoter characterization kit
PyeaR response to nitrates
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrates. The most sensitive time period is between 5 ½ hours (320 minutes) 7 ½ hours (440 minutes) after induction. The sensitivity decreases with time, but induction is still at a high level after the peak activity of the promoter. Only two concentrations are shown (0 mM and 10 mM), as we have demonstrated that 10 mM is the sensitivity peak of this promoter (look at last graph).

PyeaR response to nitrites
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrites. The fluorescence levels begin to build up only after about 8 hours; therefore other irrelevant results are omitted from this graph. The sensitivity curve in this case is less steep compared to the one produced due to the presence of nitrates. This indicates that the promoter responds differently to these two nitrogenous compounds. Again, only two concentrations are shown (0 mM and 10 mM), as sensitivity peaks at 10mM.

PyeaR response to nitrates and nitrites after overnight induction
Here we can compare the activity of this promoter to different concentrations of both nitrates and nitrates. We observed that the promoter is activated more readily by nitrates compared to nitrites. Nevertheless, the activation produced by nitrites remains significant. The peak activation concentration for both compounds is 10 mM, after which activation efficiency decreases.

Pyear characterization.jpg

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