Team:EPF-Lausanne/Project Abstract

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<a href="https://2009.igem.org/Team:EPF-Lausanne/LOVTAP"
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<a href="https://2009.igem.org/Team:EPF-Lausanne/Strategy"
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<a href="https://2009.igem.org/Team:EPF-Lausanne/LOVTAP_Results"
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<font size="12" color="#007CBC">Project Overview</font>  
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<font size="12" color="#007CBC">Abstract</font>  
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== What we want to do ==
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== Abstract ==
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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.
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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 [https://2009.igem.org/Team:EPF-Lausanne/References Strickland et al.] and made available to us by Prof. Sosnick from the University of Chicago.
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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 the DNA promoter site.
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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.
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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) 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.  
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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''.
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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...
 
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To improve the change induced by light (which is generally very unstable), we 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.
 
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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
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*'''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.
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*'''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.
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*'''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).  
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*'''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).  
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*'''reversible''' because it acts as a simple switch
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*'''reversible''': it acts as a simple switch
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*'''fast''': there is no intermediate/additional reaction that has to take place, so the response to the light stimulus is straightforward and immediate.
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*'''fast''': there is no ''intermediate/additional reaction'' that has to take place, so the response to the light stimulus is straightforward and immediate.
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*'''cheap''': you only need a light source, it is not necessary to buy additional equipment to control the spread of a ligand for example.
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*'''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!
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==Possible applications==
 
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Our system could be very useful for '''industry''' as well as for '''academic research''', as a new tool for regulating gene expression.
 
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<br>If we focus on the applications in industry:
 
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* '''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!
 
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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.
 
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* '''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.
 
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==Results Highlights==
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The main achievements of our team this year are:
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* We successfully designed, synthesized and characterized our '''read-out system n°1''' and sent this part to the Registry. More information on the [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP_Results#Characterization_of_Read-Out_n.C2.B01 Results page] or under [https://2009.igem.org/Team:EPF-Lausanne/Parts Parts submitted to the Registry].
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* We successfully designed, synthesized and characterized our '''read-out system n°2''', and sent the DNA to the Registry. More details under [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP_Results#Characterization_of_Read-Out_n.C2.B02 Results] or [https://2009.igem.org/Team:EPF-Lausanne/Parts Parts submitted to the Registry].
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* 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 [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP_Results#Characterization_of_the_entire_system Results page] or look under [https://2009.igem.org/Team:EPF-Lausanne/Parts Parts submitted to the Registry] for more details on how we did all this.
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* We validated a set of parameters for the molecular dynamic simulations on both states of the LOV domain. More details under the [[https://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Results/EDS#Validation_of_dark_state_simulation validation of the dark state]]
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* 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 [[https://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Results/Mutations#Motivation Mutation page in modeling]] for more informations.
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* 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 [https://2009.igem.org/Team:EPF-Lausanne/Future_directions Future directions] and check out our poster at the Jamboree.
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Latest revision as of 21:44, 21 October 2009

Contents







                                                               



Abstract




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.




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!




Results Highlights

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.