The problem

Producing complex therapeutic proteins requires biosynthesis using mammalian cells to obtain the desired product. This product sometimes has some level of toxicity for the cells, limiting the productivity.

Our solution

We want to transfect a light switch along with the gene of interest. This means that the product will only be synthesized by the cells in the presence of blue light, allowing them to grow happily in the dark. The switch consists in an already 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 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 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.

Plan B

In addition, we will be realizing the light switch described by Fussenegger et al. In this light switch, light-sensitive melanopsin triggers a cascade involving calcium ion channels that eventually leads to the transcription of the gene of interest. Fussenegger's team did this using HEK (human embryo kidney) cells, and we will also try to make it work on CHO cells.

Project Details

LovTAP

LovTAP is a chimeric protein, made from the light-sensitive domain (LOV) and the TrpR DNA-binding domain (TAP). LOV stands for Light, Oxygen, Voltage, to which LOV-domain is sensitive. TrpR is acterial transcription factor trp repressor and can bind it's site on DNA as a dimer. In the cell, two LovTAP proteins dimerize, so that the two LovTAP domains are bound together and TrpR domains face the same direction. When the light shines on the LovTAP domain, it changes conformation, so that [put an image to show how it works].

For more information, you can have a look at the EPFL 2009 iGEM team wiki or at the incredible [http://addrefhere simulation] done by Diego and voiced by David.

You can find the annotated protein sequence of LovTAP [http://addref here ]

Melanopsin

The Experiments

Part 3

Results