Team:Peking/Modeling

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Following the successful grand paradigm, we chose carefully our photosensor domain based on the previously described criteria (‘sensitive’, ‘bacteria functional’ and ‘modular’), and found VVD light-sensing domain originated from Neurospora crassa, which dimerize when illuminated by blue light. We then chose the bacteria SOS system repressor LexA’s DNA binding domain as our physiologically functional domain, which only binds it target DNA sequence ‘SOS box’ efficiently when a dimer is formed. We fused these two domains to form our photosensitive fusion protein, which we term ‘luminesensor’. Upon blue light activation, our luminesensor will dimerize and the LexA DNA binding domain will bind to promoters with SOS box, and downstream gene expression will be inhibited. The luminesensor turns out to be a fantastic success. It works efficiently in E.coli with no requirement of extracellular chromophore supply, have a very high signal-noise ratio, and prove to be ultra-sensitive, even responding strongly to the so called ‘bacteria luciferase’ – the vibro fisheri Lux light generating system. Its modular structure may also be modified to create other ‘luminesensor’s to serve other physiological functions.
Following the successful grand paradigm, we chose carefully our photosensor domain based on the previously described criteria (‘sensitive’, ‘bacteria functional’ and ‘modular’), and found VVD light-sensing domain originated from Neurospora crassa, which dimerize when illuminated by blue light. We then chose the bacteria SOS system repressor LexA’s DNA binding domain as our physiologically functional domain, which only binds it target DNA sequence ‘SOS box’ efficiently when a dimer is formed. We fused these two domains to form our photosensitive fusion protein, which we term ‘luminesensor’. Upon blue light activation, our luminesensor will dimerize and the LexA DNA binding domain will bind to promoters with SOS box, and downstream gene expression will be inhibited. The luminesensor turns out to be a fantastic success. It works efficiently in E.coli with no requirement of extracellular chromophore supply, have a very high signal-noise ratio, and prove to be ultra-sensitive, even responding strongly to the so called ‘bacteria luciferase’ – the vibro fisheri Lux light generating system. Its modular structure may also be modified to create other ‘luminesensor’s to serve other physiological functions.
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For the detail of our design, please go to <a>Design part</a>.
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For the detail of our design, please go to <a href="">Design part</a>.
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Revision as of 01:35, 11 September 2012

Intro

During the past decade, numerous light-sensitive optogenetic modules have been designed and characterized, with most of them following the basic design principle of attaching a physiologically functional domain to a photoreceptor domain in order to use light to trigger physiological response.

Equipped with these optogenetic tools, scientists have been able to manipulate cellular processes to achieve some truly fascinating goals, such as tuning the expression of genes through the intensity of light, or making bacteria capable of detecting the edge of a light illuminated pattern, or illuminating specific positions within the cell to trigger pseudopod formation or cell polarization around that site, or even recruiting fluorescent proteins to the membrane to print patterns on the cell.

Despite the shining promise, several important criteria still impediments the future application of current light-sensitive modules.

First of all, in order to become helpful in future applications, a photosensitive module must be truly ‘sensitive’. Some of the existing photosensitive modules work best around light intensity of 10W/m2, which is about the illuminance inside a room on a sunny day; some even require laser beam to activate, whose light intensity can hardly be achieved in natural environment. The requirement of high light intensity to activate has two major defects. Firstly, high-intensity light could be detrimental to cellular function or can even cause cell death; secondly, in future applications, photosensitive modules may be required to respond to, rather than just sunlight, light emitted by biological organisms, such as a firefly’s luminescence or, even more stringent, light emitted by bacteria, which is by no means brighter than the dim light of the moon. Apparently, most of the existing photosensitive modules cannot meet this requirement.

Secondly, in order to be widely applicable, the modules must be functional in a variety of organisms. Most of current photosensitive modules have utilized light-sensitive domains of photoreceptors of eukaryotic origin, which limits their functionability in prokaryotic organisms. Yet comparing to prokaryotic organisms, eukaryotic cells require significantly more stringent culturing conditions and are considerably more susceptible to environmental pollutions. This will indubitably create more trouble for the application of these modules outside the laboratory. Some of current photosensitive modules utilize photoreceptor proteins such as phytochorms which were originated in higher plants. This kind of proteins require chormophores that are not the product or byproduct of any of the metabolic pathways shared by ? most organisms. Thus in order to function normally, these modules require extracellular supply of the specific choromophre they require. This is unthinkable if the modules were to be applied in outside-laboratory useage.

Thirdly, the design of a successful photosensitive module should be instructive to future photosensitive module engineering, which is particularly important in synthetic biology. That is to say, its structure should be modular enough to make possible the reshuffling of the physiolog functional domain, which would be most beneficial to construction of modules with novel physiological functions following the similar designing paradigm. With no much deduction, one can easily postulate that this would require equal amount of modularity in the original photosensor domain based on which the design was constructed.

Aware of the above criteria, our team have been focusing on designing a E.coli ultra-sensitive and modular photosensor capable of responding to even biological luminescence.

Following the successful grand paradigm, we chose carefully our photosensor domain based on the previously described criteria (‘sensitive’, ‘bacteria functional’ and ‘modular’), and found VVD light-sensing domain originated from Neurospora crassa, which dimerize when illuminated by blue light. We then chose the bacteria SOS system repressor LexA’s DNA binding domain as our physiologically functional domain, which only binds it target DNA sequence ‘SOS box’ efficiently when a dimer is formed. We fused these two domains to form our photosensitive fusion protein, which we term ‘luminesensor’. Upon blue light activation, our luminesensor will dimerize and the LexA DNA binding domain will bind to promoters with SOS box, and downstream gene expression will be inhibited. The luminesensor turns out to be a fantastic success. It works efficiently in E.coli with no requirement of extracellular chromophore supply, have a very high signal-noise ratio, and prove to be ultra-sensitive, even responding strongly to the so called ‘bacteria luciferase’ – the vibro fisheri Lux light generating system. Its modular structure may also be modified to create other ‘luminesensor’s to serve other physiological functions.

For the detail of our design, please go to Design part.