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Team:NYMU-Taipei/Experiments/Speedy degrader
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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.
