We created a library of GFP-containing constructs fused to different degradation tags which are targeted by the ClpP machinery of E. coli. The degradation tags for BBa_K2323003, BBa_K2323006, BBa_K232300, BBa_K2323008, and BBa_K2323009 were pdt2E, ASV, LAA, LVA, and pdt2B, respectively.
Further, the constructs were put under control of a pTet promoter to make them inducable by aTc.
After induction of protein expression in cell culure and stopping the translation with Chloramphenicol, the reaction rate of degradation for the different tags was measured (Figure 1).
Since these tags promote degradation at different rates which are described here and in more detail on the respective registry page, this degradation tag library can be used by future teams for creation and fine-tuning of bacterial systems.
The constructs were cloned in pSB1A2 and transformed in DH5αZ1, which expresses TetR constitutively, so that the expression would be turned off. An overnight culture from the glycerol stocks of GFP+derivatives was grown in LB medium with antibiotics (Carb here). The next day, the cultures were diluted to OD 0.1 and induced with x1 anhydrotetracycline aTc (214nM). This induced the GFP expression. The cells were grown at 37°C for 2.5 hours.
The cells were then softly pelleted (2000rcf, 5min) and resuspended in fresh M9 Medium. This step was repeated a second time. This transfer into a minimal medium stops cell growth. The cells were then diluted at OD 0.45 with M9 medium and x1 chloramphenicol (25µg/mL) was added. Chloramphenicol stops protein synthesis by inhibiting the ribosome.
3x300µL of each culture were pipetting in the 96-well plate of a plate reader (Fluostar, BMG Labtech). The absorbance at 600nm and the fluorescence in the GFP channel (exc: 485nm, em: 520nm) were measured. The plate was shaken so that the cells do not deposit.
For analysis, we divided the fluorescence intensity by the absorbance. We then normalized this data to the highest value (which corresponds to the maximal amount of GFP), and plotted the percentage of remaining GFP over time.