Difference between revisions of "Part:BBa K945002:Experience"

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===Characterization by Czech Republic 2015===
 
===Characterization by Czech Republic 2015===
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The activity of pCUP1 promoter was characterized by transforming pCUP1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time.  
  
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[[File:K945002_Timeresponse.png|500px|left|border]]
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5 different concentrations (50 μM, 100 μM, 300 μM, 500 μM and 800 μM) of CUP were added to 100 µl yeast culture in SD min medium. 100 μM concentration was recommended as the most efficient in literature. We measured the fluorescence in 20 minutes intervals for 140 minutes using a plate reader.
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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.
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Our measurement confirmed that 100 μM concentration causes quickest activation of the pCUP promoter. The obtained curve is almost linear. 300 μM concentration curve is similar, however the maximal activation of the promoter is slightly lower. Activation when using 500 μM concentration is relatively low. Measurement showed that the 800 μM is fatal for the yeast culture.
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[[File:K945002_timeactivity.png|500px|left]]
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Since 100 μM 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.
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===Characterization by Tec-Monterrey EKAM iGEM 2012===
 
===Characterization by Tec-Monterrey EKAM iGEM 2012===

Latest revision as of 18:58, 18 September 2015

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Characterization by Czech Republic 2015

The activity of pCUP1 promoter was characterized by transforming pCUP1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time.

K945002 Timeresponse.png

5 different concentrations (50 μM, 100 μM, 300 μM, 500 μM and 800 μM) of CUP were added to 100 µl yeast culture in SD min medium. 100 μM concentration was recommended as the most efficient in literature. We measured the fluorescence in 20 minutes intervals for 140 minutes using a 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 μM concentration causes quickest activation of the pCUP promoter. The obtained curve is almost linear. 300 μM concentration curve is similar, however the maximal activation of the promoter is slightly lower. Activation when using 500 μM concentration is relatively low. Measurement showed that the 800 μM is fatal for the yeast culture.

K945002 timeactivity.png

Since 100 μM 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.

Control: Pichia pastoris visualized under the microscope.
Control: Pichia pastoris visualized with fluorescence microscopy (Zeiss filter set 18).
Transformant visualized under the microscope.
Transformant visualized with fluorescence microscopy (Zeiss filter set 18).

Applications of BBa_K945002

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