Team:NYMU-Taipei/Experiments/Speedy degrader

From 2010.igem.org

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1.Selected genes to be reported are incubated overnight in an LB liquid culture at 37oC and 180-200rpm. This makes sure they are fresh in the morning. Positive and negative controls are also needed.  
1.Selected genes to be reported are incubated overnight in an LB liquid culture at 37oC and 180-200rpm. This makes sure they are fresh in the morning. Positive and negative controls are also needed.  
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2.Overnight liquid culture is diluted to OD600 of 0.1, Theophylline is added at concentrations ranging from 0.01mM to 20mM, and the mix incubated for 2-2.5 hours.
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2.The liquids cultured overnight are diluted into an OD600 of 1 and incubated for 2 hours.
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3.Measurement of OD at 2 hours: For each used well in the 96-well plate:
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Take 200uL from the liquid (make sure you pipette this step well) and put it in a cuvette to read the OD600.
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Note down the OD600 ["OD at 2 hours"], then take the liquid in the cuvette and put it in the right place in the 96-well plate.  
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4.Measurement of fluorescence:  
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3. After 2 hours, we took out the liquids and centrifuge them for 30 seconds at 13.2k rpm and then discard the supernatant. We then resuspended the pellets in ____ABT medium.
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Continuous measurement of fluorescence with the excitation/emission wavelengths 488/511nm for 2 hours, with one fluorescence data point every 2 minutes.
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4.Take 200uL from the liquids to measure OD600, doing three replicates, noting down OD value as well.
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5.Measurement of fluorescence:  
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Continuous measurement of fluorescence with the excitation/emission wavelengths depending on different FP for one hour, with one fluorescence data point every 2 minutes.
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6.After measuring the fluorescence, we recorded OD value again to confirm that the E. coli has stopped growing in the ABT medium.
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5.Measurement of OD at 4 hours: For each used well in the 96-well plate:
 
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Take the liquid from the well and put it in the cuvette to measure the OD ["OD at 4 hours"].
 
*The optimizing data:
*The optimizing data:
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We took the data from OD600 of each sample, which should have an exponential growth curve, and took the ln of each value. After taking the logarithm of the data, we created a linear curve. Since we have the two end points of the OD 600 of each sample, we use this linear curve to modulate  OD value of each sample at each specific time point. This value was then recalculated back into its original curve using exponents.
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Check if the ODs remained the same to make sure the E. coli does not grow in ABT medium. We took fluorescence averages for all the replicates and sketched a curve and fitted a trend line. Exponential curves were fitted and the half-life of the FP calculated. However, the actual data do not fit exponential curve quite well.
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Our fluorescent data was normalized by taking the fluorescence of our sample at each time point and subtracting the fluorescence of the negative control in the same OD value at the same time point.
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Finally, plot the nomalized fluorescence versus time in minutes scale.
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Revision as of 20:57, 27 October 2010


Method

  • Protocol:

1.Selected genes to be reported are incubated overnight in an LB liquid culture at 37oC and 180-200rpm. This makes sure they are fresh in the morning. Positive and negative controls are also needed.

2.The liquids cultured overnight are diluted into an OD600 of 1 and incubated for 2 hours.

3. After 2 hours, we took out the liquids and centrifuge them for 30 seconds at 13.2k rpm and then discard the supernatant. We then resuspended the pellets in ____ABT medium.

4.Take 200uL from the liquids to measure OD600, doing three replicates, noting down OD value as well.

5.Measurement of fluorescence: Continuous measurement of fluorescence with the excitation/emission wavelengths depending on different FP for one hour, with one fluorescence data point every 2 minutes.

6.After measuring the fluorescence, we recorded OD value again to confirm that the E. coli has stopped growing in the ABT medium.


  • The optimizing data:

Check if the ODs remained the same to make sure the E. coli does not grow in ABT medium. We took fluorescence averages for all the replicates and sketched a curve and fitted a trend line. Exponential curves were fitted and the half-life of the FP calculated. However, the actual data do not fit exponential curve quite well.



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