Team:NAU-CHINA/testh

Fig.2. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP. Co-culture the 293T cells expressing GFP on the cell surface with the cells transferred with Lag16-synNotch-TEV for 1h to extract protein for western bolt detection. (A) Fluorescence microscope observation of the cells transfected with plasmids containing the gene of cell surface-expressed GFP. (B) Image results developed in Western blot shows that Lag16-SynNotch-TEV affected by surface-expressed GFP can be resolved into FLAG-TEV and V5-mNotch. (C) Gray scale analysis of western blot image shows the relative level of the Flag tagged LaG16-synNotch-TEV affected by cell surface-expressed GFP. Data are mean ±S.E. (n=3).

• • , p < 0.01;

N.S., no significance. The above two experiments show that the modified synNotch can be located on the surface of cell membrane normally and release intracellular domain after receiving external signals. The replacement of intracellular domain with TEV has no effect on the function of synNotch. Eukaryotic verification of TEV activation transcription system based on modified tetR As mentioned earlier, inducible promoters using transcription activator factors that cannot inhibit transcription often have some leakage due to background expression.

However, our system hopes to realize the absolute function of 0/1 switch, and the background expression is what we do not expect. Therefore, we need to find a transcription activation system with very low background expression.
Coincidentally, the previous team iGEM 2017 Oxford was making similar attempt. They have designed TEV activation transcription system based on the modified tetR. Although they have not fully proved that the system can work effectively due to time constraints, we believe that their theoretical basis for designing the system is reasonable. Therefore, we attempt to verify their system with eukaryotic cells.