Team:EPF-Lausanne/Project Abstract

From 2009.igem.org

(Difference between revisions)
(What we want to do)
(Advantages)
Line 30: Line 30:
The advantages of such a system are :
The advantages of such a system are :
* '''easy to use''': we just need to shine light onto the system
* '''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 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).  
+
*'''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
+
*'''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.
+
*'''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. A couple of LEDs with the right wavelength will do the trick!
+
*'''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!
</div>
</div>
<br>
<br>
</div><div CLASS="epfl09bouchon"></div>
</div><div CLASS="epfl09bouchon"></div>

Revision as of 17:43, 20 October 2009

Contents


Project Overview



What we want to do

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 proteinthat 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.




Advantages

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!