Team:McGill/Results
Our Results
Synnotch Expression Confirmation by Fluorescence-activated Cell Sorting (FACS)
Figure 1. To prepare the cells for FACs, activation of the circuit was induced by the addition of anti-myc antibody and induction of cellular stress mechanically via repeated up and down pipetting. The cells were then incubated for 12 hours and prepared for analysis. Primary mouse anti-myc antibody and secondary Alexa Fluor 488 anti-mouse was used to detect synnotch expression on our target population of cells. Control cells were untransformed HEK 293 cells. Unstained and secondary only controls were used for each population as a further control. As we can see in figure 1, the five controls all showed similar geometric means while the transformed HEK 293 cells with primary and secondary showed a geometric mean approximately 3-fold higher, indicating successful expression of SynNotch on the surface of our target population.
Synnotch Expression Confirmation by Western Blot
Figure 2. Confirmation by Western Blot of Synnotch Expression. The negative control is EV (empty Vector) and shows no bands around the 75-100kDa region where our target protein is predicted to be. The positive control is a Myc-expressing cell line, to show that the anti-Myc antibody is functioning properly. Indeed strong banding shows that the antibody works and is specific. Our synnotch-expressing cells show a pattern similar to the EV negative control but with an additional band directly below 100kDa, which is compatible with the predicted molecular weight of SynNotch (94kDa).
Basal Expression of GFP by GCIR Transformed Cells
Figure 3. Basal expression of GFP by GCIR1 transformed HEK 293 cells. GFP was detected in its natively fluorescing channel via flow cytometry. HEK 293 cells transformed with empty vectors and transformed with Synnotch and an empty GCIR1 vector showed low baseline GFP. However, both the unstimulated SynNotch GCIR1 double transformants and GCIR1 transformants with empty SynNotch vector show geometric means 8-fold higher than the controls and similar to that of the myc stimulated SynNotch GCIR1 double transformants. There are no significant differences between the fluorescence signitures of these three populations, indicating that the GCIR1 promoter is constitutively active.
Appendix/Supporting Figures
Figure 3. Gating strategy for flow cytometry data. The samples were first gated in order to cut off debris that is smaller than expected cell size. By forming this gating strategy, we were able to select for single cells in our FACS experiments.