Signalling

Part:BBa_K2323003

Designed by: Jorge Andres Quintero Serna   Group: iGEM17_Munich   (2017-10-25)


GFP-pdt2E

This GFP (E0040) is tagged with a protein degradation tag (pdt) called pdt2E. This causes the protein to degrade in E.coli with a first-order rate of 0.0046 per min, from the ClpP machinery of E.coli. It is under the control of a pTet promoter (aTc inducible) and the RBS is B0034. The terminator is B0015.

This biobrick is part of a collection of degradation tags, characterized on this page. See also BBa_K2323005, BBa_K2323006, BBa_K2323007, BBa_K2323008.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 724

Plasmid composition

The collection is cloned from BBa_I13522 with overhang PCR using 5'-phosphated primers that contain the tag as an overhang. Primers are indicated with the map (read-only benchling map). Cloning was confirmed with sequencing.

Characterization

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.

Our collection presents a range of degradation kinetics.
16-04-12 subfit ASV.png
16-05-19 fit normAbsFI pdt2.png


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