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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 2ml 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.
Reporting Assay
- 'Result'
- In our ssrA degradation test, we use fluorescence protein(FP) as our demonstration. The half-life of the FP should be longer than the one with LVA tag. However, the graphs show that the half life of YFP is 236 mins and 181 mins(Fig. 1 & Fig.2), and the half-time of the RFPLVA is 450 mins.(Fig. 3) It seems that our data do not support the theory.
- In Fig. 4, we can see that E. coli stop growing in ABT medium. The OD600 remains the same versus time. Therefore, we can infer that the amount of the E. coli remain almost the same in our fluorescence measurement.
- In the condition of same amount of the E. coli, our data ought to fit the theory. In fact, it does not. The data are doubtful and we think our experiment data were not enough to get the confident results.
