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, designed by Strickland et al. and sent to us by Prof. Sosnick from the University of Chicago.

The strategy that has been used to design this hybrid protein was to fuse a light-sensitive domain (Lov in this 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. The latter 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 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.

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 . 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 more 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, which 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: 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, no need of additional equipment to control the spread of a ligand for example. A couple of LEDs with the right wavelength will do the trick!

The main achievements of our team this year are:

• We successfully designed, synthesized and characterized our read-out system n°1 and sent this part to the Registry. More information on the Results page or under Parts submitted to the Registry.
• We designed, synthesized and demonstrated the correct functionality of our read-out system n°2, and sent the DNA to the Registry. More details under Results or Parts submitted to the Registry.
• We successfully cloned the LovTap gene into a BioBrick, and designed, synthesized and characterized our main inducible LovTap BioBrick, which we also sent to the Registry. Check out the Results page or look under Parts submitted to the Registry for more details on how we did all this.
• We validated a set of parameters for the molecular dynamic simulations on both states of the LOV domain.
• We investigated an approach to test the stability of the bound to the chromophore using simple molecular dynamic. That leaves a broad perspective of improvement of the fusion protein stability by testing them in silico.
• We managed to obtain a mutated version of the LovTap gene, which should give a more stable protein than the original one, and sent the part to the Registry. For more information about the experiments that we will conduct, look at the Future directions and check out our poster at the Jamboree.