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Speedy reporter

Speedy switch

Speedy protein 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.

Reporting Assay

   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 236mins and 181mins(Fig. 1 & Fig.2) and, the half-time of the RFPLVA is 450mins.(Fig. 3) It seems that our data does not support the theory. 
   

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