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Team:Freiburg/Content/Results/The combination
From 2014.igem.org
The Combination
We, the iGEM Team Freiburg 2014, combined the spatial resolution of the light system with the specificity of our viral vector generating patterns in a homogenous cell culture.
Light Induced Receptor
Since our viral vector is specific for the mCAT-1 receptor that is under normal conditions only expressed in murine cells, we linked this specific receptor to the blue light system or the red light system and transfected non-murine cells with this receptor combinations. Mammalian cells which contain the inducible receptor expressed mCAT-1 after illumination. Cells expressing the receptor are visible by co-expression of mCherry. They were infected with our viral vector leading to EGFP expression. Cells of the same cell culture that were not illuminated were not able to express the receptor leading to no infection with our viral vector.
Red light induced receptor.
CHO cells were transfected with the red light system (PKM022) and the light induced receptor (p14rz_002). The receptor was labeled with mCherry; (left) after illumination with red light, (middle) incubation in the dark, (right) positive control. Cells were stained with DAPI. Labjournal
Blue light induced receptor.
HEK cells were transfected with the blue light system (PKM292 and PKM297) and the light induced receptor (p14ls_003). The receptor was labeled with mCherry. Cells were infected with MuLV EGFP afterwards; (left) incubation in the dark, (middle) after illumination with blue light, (right) cells expressing the light induced receptor were infected with MuLV. Labjournal
As we know that the transient mCAT-1 receptor that was not linked to a light system needs 24 hours for expression in HEK293 cells, we determined the expression time of the receptor that was induced by blue light after illumination for five hours. We used a receptor that was labeled with both, mCherry and an HA-tag (p14ls_003), for analysis with Western blot and fluorescence microscopy. The results show that the receptor had an expression peak at 24 hours after beginning of illumination.
Kinetik of the blue light induced receptor.
HEK cells were transfected with the blue light system (PKM292 and PKM297) and the light induced receptor (p14ls_003, mCherry labeled receptor). Pictures were taken after 12h, 15h, 18h and 24h. Labjournal
