Team:EPF-Lausanne/Project Abstract

Light-sensitive proteins can be readily found in nature, but despite their tremendous biotechnological potential, they have so far rarely been studied in much detail. In this project, our aim is to demonstrate the ability of light-sensitive fusion proteins to regulate gene expression through light control. For this purpose, we selected the LovTAP hybrid protein, designed by Strickland et al. and made available to us by Prof. Sosnick from the University of Chicago.

The underlying strategy leading to the generation of this hybrid protein was to fuse a light-sensitive domain (Lov in this case) with a regulatory domain (here the Trp repressor) such that the two domains share a continuous helix. This procedure enabled the creation of an allosteric switch whereby the shared helix acts as a rigid lever arm. Upon light induction, a conformational change is transmitted from the light-sensitive to the DNA-binding domain, which ultimately results in an increase of the regulatory domain's affinity for its respective DNA binding site.

Here, for the first time, we demonstrate that the LOVTap fusion protein is functional' in vivo ' based on the fact that we were able to implement small, genetic networks in E. coli which respond to light through the LOVTap protein. In addition, we used molecular dynamics simulations to further analyze the conformational changes between the activated and de-activated state (which is generally very unstable). These efforts identified specific amino acid residues that could be mutated to further improve the stability of the fusion protein and thus its "ON/OFF" characteristics and we are currently testing the latter predictions in vivo.

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 successfully designed, synthesized and characterized 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 light 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. More details under the [validation of the dark state]
• We investigated an approach to test the stability of the bond to the chromophore using simple molecular dynamic. That leaves a broad perspective of improvement of the fusion protein stability by testing them in silico. Please see the [Mutation page in modeling] for more informations.
• 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.