The "SWITCH" Project

The Problem

Producing complex therapeutic proteins requires biosynthesis in mammalian cells. Such proteins can sometimes have a certain level of toxicity for the cells and limit their productivity if they are produced constantly and are accumulating. To avoid this, the pharmaceutical industry use 'rewired' cells that synthesize toxic proteins only when a special molecule is added to the bioreactor. This solution has two disadvantages. First, cell rewiring affects several pathways and decreases cell productivity. Second, the 'special' molecule will mix with the final product and a purification will be needed to get rid of it.

The pharmaceutical industry needs an easier way to induce the production of a specific compound in mammalian cell.

This is where our "SWITCH" project steps in.

The Simple Switch

We want to design a light-sensitive switch, so that the toxic protein will only be synthesized by the cells in presence of blue light, allowing them to grow happily in the dark. We've based our switch on an existing chimeric protein, LovTAP. This protein was originally intended to act as a light-induced repressor in bacteria. The EPFL 2009 iGEM team proposed to fuse it with VP16, a viral activator, in order to convert it into a light-induced activator in mammalian cells. This way, it will be enough to add a LovTAP binding site in front of the gene of interest and a LovTAP encoding gene to induce protein expression without using any additional chemical or disturbing too many pathways.

This year we will try to realize this idea by transfecting CHO (Chinese hamster ovary) cells with two plasmids: one coding for the LovTAP-VP16 fusion protein and another with a read-out ( red fluorescence ) protein preceded by a binding site for LovTAP-VP16. If everything goes as expected, LovTAP-VP16 will only bind the plasmid and activate the production of read-out when exposed to light.

The Complex Switch

In addition to LovTAP switch, we will be realizing a more complex, melanopsin-based light switch, as described by Fussenegger et al. In this switch, light-sensitive melanopsin triggers a cascade involving calcium ion channels that eventually leads to the transcription of the gene of interest. The switch involves more complex signalling cascades, but it also uses only one natural protein and might function better then the simple switch. Fussenegger's team did this using HEK (human embryo kidney) cells, and we will also try to make it work on CHO cells, so that we can determine which switch works better in the CHO cells.