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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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