Part:BBa_K945002:Experience
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Characterization by Czech Republic 2015
The activity of CUP promoter was characterized by transforming pCUP-GFP plasmids into yeast S. cervisiae and measuring the evolution of fluorescence in time.
5 different concentrations (50 uM, 100 uM, 300 uM, 500uM and 800 uM) of CUP were added to 100 µl yeast culture in SD min medium. 100 uM concentration was recommended as the most efficient in literature. We measured the fluorescence in 20 minutes intervals for 140 minutes using Plate Reader.
The first graph shows evolution of fluorescence in time. We subtracted fluorescence of negative control from the fluorescence of each sample and divided this by difference between sample´s and negative control´s ODs in order to obtain RFU units.
Our measurement confirmed that 100 uM concentration causes quickest activation of the pCUP promoter. The obtained curve is almost linear. 300 uM concentration curve is similar, however the maximal activation of the promoter is slightly lower. Activation when using 500 uM concentration is relatively low. Measurement showed that the 800 uM is fatal for the yeast culture.
Since 100 uM appears to be the convenient concentration for maximal activation of the promoter we used it as a reference and related the activity of other concentration to this concentration. Results are displayed in the second graph.
Characterization by Tec-Monterrey EKAM iGEM 2012
The functionality of this promoter was tested by transforming Pichia pastoris with a construct containing reporter BBa_K945004 under the control of this promoter. The culture was induced with copper (II) sulfate at a concentration of 200 uM and visualized using fluorescence microscopy.
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