Team:EPF-Lausanne/Project Abstract

Light-sensitive proteins can easily be found in nature, but they have not been studied a lot yet. In this project, our aim is to design and work with a fusion protein that would allow genetic regulation through light control. The primarily designed protein that we will start with is the LovTAP hybrid protein. It has been designed by Strickland et al. and generously sent to us by Prof. Sosnick from the University of Chicago, department of Biochemistry and Molecular Biology.

The strategy that has been used to design this hybrid protein was to fuse a light-sensitive domain (Lov in our case) with a regulatory domain (like the Trp repressor). 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 its DNA binding site.

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

The overall effect would thus be a genetic expression controlled by light! In other words, we are trying to build a light-controlled DNA-binding protein, more specifically to prove that the one created by Sosnick et al. can work and be controlled in vivo. 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 will also conduct a modelling part, whose aim is to find which residues could be mutated in order to have a stable protein after the light induction. This would allow a better "ON/OFF" protein with less interferences.

The advantages of such a system are :

• easy to use: we just need to shine light onto the system
• precise in space: we can choose the exact localization of the ray. This one can be as thin as a laser beam (a fraction of a mm), but can also cover a larger surface, depending on the need.
• 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 almost instantly stopped (provided that the switch is reversible of course).
• reversible because it acts as a simple switch
• fast: there is no intermediate/additional reaction that has to take place, so the response to the light stimulus is straightforward and immediate.
• cheap: you only need a light source, it is not necessary to buy additional equipment to control the spread of a ligand for example.

Our system could be very useful for industry as well as for academic research, as a new tool for regulating gene expression.
If we focus on the applications in industry:

• in bioreactors, used in biochemical engineering. Currently, one main issue is that molecules added in bioreactors to perform/catalyse a particular reaction cannot be removed once in the medium, or only very tediously, involving long and expensive filtration procedures. A major advantage of our system is that it is easily reversible: just switch the light on or off!

Another major issue for bioreactors is keeping them sterile: in our case this wouldn't be a problem as the light source could simply be placed behind a transparent, sterile plastic wall, for example.

• in academic research : If we look further into the future, the light switch could be applied to larger model organisms (not only single cells) for example to switch genes on or off (the "off" state would be analogous to the case where you knock-out the gene). It would be more efficient than the techniques currently used (example: the Cre-Lox system) because of the advantages listed above, namely it would allow a fine control over the target gene, a reversible action, and above all an immediate response.