The Light System

Light inducible systems allow spatial and temporal control over gene expression. Our goal is the expression of the mCAT-1 receptor through a light inducible system to allow spatiotemporal control of the virus entry into the target cells. We used two different systems: expression is induced by light of the blue and red wavelength range. To test the performance of the light systems, we used an assay where the cells express secreted embryonic alkaline phosphatase (SEAP). The amount of produced SEAP can be measured by its enzymatic activity. It breaks down the substrate para-nitrophenylphosphate to the yellow para-nitrophenol, which can be measured with a spectrophotometer.

Function of the Light System

Because the proper function of the light system is important for our project, we tested two different light systems. The red light system is based on the protein phytochrome B (PhyB) from Arabidopsis thaliana and is activated by light around 660 nm, whereas the blue light system contains a protein from Avena sativa (AsLOV2) which requires 465 nm light for activation.

To test the function of the red light system, we used Chinese hamster ovary cells (CHO) that are known to work better for this purpose than other cell lines. For the blue light system, we tested CHO cells and human embryonic kidney cells (HEK-293T). As reporter the gene SEAP was transfected together with the genes for the light systems (red light system: PKM006, PKM078; blue light system: PKM292, PKM297, PKM084). A SEAP-assay was performed after illumination. Measuring SEAP concentrations in the supernatant lead us two two important conclusions: First, the blue light system works more efficient than the red light system; second, the blue light system works better with HEK-293T cells than with CHO cells (Fig. 1).

The blue light system is very effective in activating an existing reporter, like the gene for seap, brought into the cell before illumination. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time spans for illumination. Our results indicate that five hours of illumination lead to the highest activation level of SEAP expression (Fig.2).

Another important finding was the specificity of the blue light system. As we always have a dark control in our experiments, we determined almost no activation of the SEAP reporter in these samples leading to a very low background level in our system.

SEAP as Reporter

In order to test the capability of the blue light system for pattern generation, we transfected HEK-293T cells with the appropriate plasmids (PKM292 and PKM297) using seap as a reporter gene (PKM084). Cells were transfected in suspension and seeded on 96 well plates afterwards. Single wells of these plates were covered with a photo mask that prevents illumination with blue light. Since, all other wells on the plate were exposed to the blue light, SEAP expression was induced in these wells. SEAP that was secreted into the culture medium leads to colour changing from red to yellow after addition of pNPP (chromogenic substrate) by its phosphorylation.

First we generated a coarse pattern (fig.) realizing that by scattered light also covered wells close to illuminated wells were exposed to light. This was leading to an unclear pattern. However, we managed to give an idea of the desired pattern (Fig.3, A-B).

Since, it was essential to completely prevent light exposure on covered plates we tested 96 well plates with optical separated wells. As shown in fig. we could generate patterns with sharp contours (Fig.3, C-D).

Colour changing of pNPP in cell culture medium could be measured at 405 nm in a plate reader. Calculating the gradient of light absorption we also generated patterns using a data matrix (Excel) by setting a distinct threshold, thus making pattern recognition easier.

For generating QR codes with our method 384W plates were used. Since we had a collaboration with the iGEM Team Aachen 2014, they made a photo mask for a QR code for us fitting on these plates (Fig.4) Collaborations.