Team:EPF-Lausanne/Project Abstract

What we want to do

Light-sensitive proteins can easily be found in nature, but they have never been cloned into other cells. In this project, our aim is to design a fusion protein that would allow genetic regulation through light control.

We are working on cloning strategies that would allow us to fuse a light-sensitive domain (LovTAP in our case) with a regulatory domain (like the Trp operon). By joining these two domains so that they share a continuous helix, an allosteric switch could thus be created, the shared helix acting as a rigid lever arm. The idea is to allow transmission of the conformational change induced by light (on the light-sensitive domain) to the DNA-binding domain. This transmitted conformational change would then result in an increase or decrease of the regulatory domain's affinity for the DNA promoter site.

Because these helical contacts are among the domains, their disruption will cause a global shift in the conformation of the protein. Conversely a photoexcitation (which also changes the conformational ensemble of the protein) will shifts the relative affinity of the shared helix for each domains, thereby allowing a signal sensed by one domain to be allosterically propagated to the other domain.

The overal effect would thus be a genetic expression controlled by light! In other words, we are trying to built a light-controlled DNA-binding protein. There would be many applications to such a "switch" : it could kill bacteria at a certain point, stop their growth, or make them express specific proteins...

To improve the change induced by light (which is generally very unstable), we also plan a modeling part whose aim is to find which residue could be mutated in order to have a stable protein after the switch.

The advantage of such a system is :

• easy to use: we just need to apply light on the system
• precise in space: we can choose exactly the localization of the ray. This one can be as small as a laser beam (a fraction of mm), but can also cover a bigger surface, depending on the needs.
• precise in time: contrary to a ligand-based DNA-binding protein, we can clearly see the advantage of using this method. Indeed, as soon as the light is switched off, the shift will convert back, and the signal will be instantly stopped.
• cheap: need to have a light beam only, there is no need to buy additionnal equipment to control the spread of the ligad for example.
• useful for industry as for academic research, as a new tool for regulating the gene expression for example

Project Abstract

Recent discoveries of photoreceptors in many organisms gave us insights into a possible interest of using light responsive genetic tools in synthetic biology. The final goal of our project is to induce a change in gene expression, more specifically to turn a gene on or off, in a living organism, in response to a light stimulus.

We will use light sensitive DNA binding proteins, or light sensitive proteins that activate DNA binding proteins to transduce a light input into a chosen output, for example reporter genes like GFP, RFP. The genetic circuits allowing us to measure the activity and responsiveness of light sensitive proteins are already designed, whereas the parts and biobricks required to engineer these circuits are still in formation.

If we demonstrate that the light-induced-gene switch tool works in vivo, it would show that easier and faster tools could be used in several fields of biology. It would induce more localized, more precise (time resolution) and drastically faster genetic changes than the current used tools, which will then allow research to evolve even better.

LOV means light, oxygen, and voltage, whereas TAP means tryptophan-activated protein.