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Team:Freiburg/Content/Project/The light system
From 2014.igem.org
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<h2>Optogenetics in mammalian gene expression</h2> | <h2>Optogenetics in mammalian gene expression</h2> | ||
<p>Optogenetics describe the use of genetically encoded, light-sensing proteins to modulate cellular function or even organismal behavior with high spatio-temporal resolution. In the past few years a large number of versatile light-controlled tools and devices has been developed in all kind of species, reaching from bacteria (Levskaya et al., 2005) to mammalian cells (Muller and Weber, 2013). The introduction of microbial melanopsin into neurons (Nagel et al 2003) was the first optogenetic approach in mammalian cells and constitutes a milestone in neuronal research (Boyen et al, 2005). More recently, light-regulated tools have been successfully applied to control divers signaling processes and gene expression in mammalian cells (Pathak et al 2013; Müller et al, 2014?).</p> | <p>Optogenetics describe the use of genetically encoded, light-sensing proteins to modulate cellular function or even organismal behavior with high spatio-temporal resolution. In the past few years a large number of versatile light-controlled tools and devices has been developed in all kind of species, reaching from bacteria (Levskaya et al., 2005) to mammalian cells (Muller and Weber, 2013). The introduction of microbial melanopsin into neurons (Nagel et al 2003) was the first optogenetic approach in mammalian cells and constitutes a milestone in neuronal research (Boyen et al, 2005). More recently, light-regulated tools have been successfully applied to control divers signaling processes and gene expression in mammalian cells (Pathak et al 2013; Müller et al, 2014?).</p> | ||
| - | <p>Light inducible gene expression systems follow the yeast two hybrid principle, using photoreceptors, which allow organisms to respond to environmental light conditions, of numerous different species and as building blocks. In synthetically designed systems transcriptional activation by light is mediated by recruitment of a transcriptional activation domain to the DNA site. Therefore, either photoreceptors and their light-depenent specific interaction partners are fused to a transactivation domain or to a DNA binding domain, respectively, or light-induced homo-dimerization of some photoreceptors is exploited to reconstitute a DNA binding domain. | + | <p>Light inducible gene expression systems follow the yeast two hybrid principle, using photoreceptors, which allow organisms to respond to environmental light conditions, of numerous different species and as building blocks. In synthetically designed systems transcriptional activation by light is mediated by recruitment of a transcriptional activation domain to the DNA site. Therefore, either photoreceptors and their light-depenent specific interaction partners are fused to a transactivation domain or to a DNA binding domain, respectively, or light-induced homo-dimerization of some photoreceptors is exploited to reconstitute a DNA binding domain. To date, spatio-temporal control over mammalian gene expression is provided by optogenetic gene switches, which are responsive to three distinct light spectra – blue, red and UV light (Weber review).</p> |
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<h2>The AcCELLerator light systems</h2> | <h2>The AcCELLerator light systems</h2> | ||
| - | <p>In order to assess an appropriate light system for our purpose, we considered benefits and disadvantages of all three light systems regarding different aspects, such as toxic side effects, handling and induction rate. | + | <p>In order to assess an appropriate light system for our purpose, we considered benefits and disadvantages of all three light systems regarding different aspects, such as toxic side effects, handling and induction rate. UVB light inducible gene switches show high induction rates. However, UV light has a strong toxic effect on cells, and was therefore excluded. (weber müller review)In contrast, red light induction has minimal toxic effects and provides high tissue penetration. Still, there are limitations in terms of handling, since cells have to be protected from ambient light and the requirement of addition of PCB (toggle switch paper).Blue light responsive expression systems do not depend on exogenous additon of a chromophore, but are likewise sensitive to unintentional activation by room light.Since drawbacks regarding the handling can be overcome by working under safe green light conditions, we considered both, blue and red light-inducible systems as suited for our application.</p> |
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<p> </p> | <p> </p> | ||
<p>LOV2 based blue light responsive system</p> | <p>LOV2 based blue light responsive system</p> | ||
Revision as of 03:44, 11 October 2014
The Light System
Optogenetics in mammalian gene expression
Optogenetics describe the use of genetically encoded, light-sensing proteins to modulate cellular function or even organismal behavior with high spatio-temporal resolution. In the past few years a large number of versatile light-controlled tools and devices has been developed in all kind of species, reaching from bacteria (Levskaya et al., 2005) to mammalian cells (Muller and Weber, 2013). The introduction of microbial melanopsin into neurons (Nagel et al 2003) was the first optogenetic approach in mammalian cells and constitutes a milestone in neuronal research (Boyen et al, 2005). More recently, light-regulated tools have been successfully applied to control divers signaling processes and gene expression in mammalian cells (Pathak et al 2013; Müller et al, 2014?).
Light inducible gene expression systems follow the yeast two hybrid principle, using photoreceptors, which allow organisms to respond to environmental light conditions, of numerous different species and as building blocks. In synthetically designed systems transcriptional activation by light is mediated by recruitment of a transcriptional activation domain to the DNA site. Therefore, either photoreceptors and their light-depenent specific interaction partners are fused to a transactivation domain or to a DNA binding domain, respectively, or light-induced homo-dimerization of some photoreceptors is exploited to reconstitute a DNA binding domain. To date, spatio-temporal control over mammalian gene expression is provided by optogenetic gene switches, which are responsive to three distinct light spectra – blue, red and UV light (Weber review).
The AcCELLerator light systems
In order to assess an appropriate light system for our purpose, we considered benefits and disadvantages of all three light systems regarding different aspects, such as toxic side effects, handling and induction rate. UVB light inducible gene switches show high induction rates. However, UV light has a strong toxic effect on cells, and was therefore excluded. (weber müller review)In contrast, red light induction has minimal toxic effects and provides high tissue penetration. Still, there are limitations in terms of handling, since cells have to be protected from ambient light and the requirement of addition of PCB (toggle switch paper).Blue light responsive expression systems do not depend on exogenous additon of a chromophore, but are likewise sensitive to unintentional activation by room light.Since drawbacks regarding the handling can be overcome by working under safe green light conditions, we considered both, blue and red light-inducible systems as suited for our application.
LOV2 based blue light responsive system
Red/far red light responsive system
