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.

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.

Pattern Generation

For generating pattern in homogenous cell layers we transfected HEK293T cells with the blue light system and the blue light induced receptor (p14ls_003). Dishes were covered with a photo mask preventing areas in the cell culture from light exposure. However, the blue light system is very sensitive to even low intensities of blue light. Due to scatterd light the receptor was activated in a huge area of the cell culture. Thus patterns were not visible to this time point due to by light scatter activated receptors (Fig.).