Team:Peking/Modeling

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<h3 id="title1">Summary</h3>
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Our modeling group conducted simulations to guide our experiments by combining protein kinetics, thermodynamics, stochastic simulation, and molecular docking together. Specifically, we conducted modeling of our <i>Luminesensor</i>, and the experimental results of the bio-othorgonality part excellently correspond to the model! We also used molecular docking to expand the spectrum of the <i>Luminesensor</i>, which would greatly extend its application, such as multiple-channel communication among cells. In addition, we simulated the process of phototaxis both on the macro level and micro level, using mean-field PDE (partial difference equations) and Stochastic Simulation respectively. In the PDE model, we developed a hexagonal-coordinate simulation environment for dynamic system on a continuous plane, which would be a very useful prototype for future simulation on cellular movement, pattern formation, and other potential systems. Furthermore, we designed the genetic circuit of multi-cellular ring-like pattern formation as a demonstration for cell-cell communication through light; based on parameter analysis, we used our modeling prediction to rationally guide the experiment to form better patterns!
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<h3 id="title2"><i>Luminesensor</i> Modeling</h3>
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  <img src="/wiki/images/3/3a/Peking2012_LuminesensorNodes.png" alt="" style="width:250px;"/>
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Our <i>Luminesensor</i> is a ultra-sensitive fusion protein to sense 450nm to 470nm light and then regulate the gene expression(<a href="/Team:Peking/Project/Luminesensor/Future#FigS">spectrum data here</a>). Although the <i>Luminesensor</i> excels and eclipses similar systems due to its ultra-sensitivity and dynamic range, there are still several imperfect aspects. For example, the response time of the protein can be up to hours<sup><a href="#ref1" title="Zoltowski, B.D., Crane, B.R.(2008). Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. Biochemistry, 47: 7012: 7019">[1]</a></sup> and the contrast of binding efficiency with and without light has much room for improvement. After modeling the DNA binding process of the <i>Luminesensor</i>, we managed to find out four key parameters, two of which mainly control the response time, and the others control the contrast of binding efficiency. According to the feasibility in experiment, we tuned two most facile parameters of each aspect to optimize the performance of the <i>Luminsensor</i>, and later discovered mutation sites related to these two parameters experimentally.<br /><br />
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Furthermore, in order to extend the application of the <i>Luminsensor</i>, we managed to expand the spectrum of the <i>Luminsensor</i> through modeling. Here we use molecular modeling to identify the possiblity of modifications to the conjugating substructure of Flavin Adanine Dinucleotide (FAD) substrate, which acts as the chromophore of VVD.
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<h3 id="title3">Phototaxis Modeling</h3>
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Phototaxis is a light-control bio-system, whose input is the space-distribution of light. By comparison with chemotaxis system, phototaxis has many advantages for application. (See <a href="/Team:Peking/Project/Phototaxis">Project Phototaxis</a>) We have constructed a simple phototaxis system coupling our <i>Luminesensor</i> with the expression level of cheZ protein. In order to confirm the macro light-control to our phototaxis system, we used the Mean-field PDE model. Later we managed to confirm these phenomena by tracing each cells in Stochastic Simulation. We then managed to do the related experiments to prove the effect of light in this simple system.
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On the way to the simulation of the macro population changing process, we developed a hexagonal-coordinate simulation environment for dynamic system on a continuous plane. Since the number of neighbors of a chunk unit in the hexagonal mesh is larger than that of the traditional quadratic mesh, the hexagonal mesh is much closer to an isotropic grid, which would reduce the error caused by anisotropic structure in traditional quadratic mesh. This simulation environment would be a useful prototype for future simulation of 2D dynamic systems, such as cellular movement on plate and pattern formation based on cell-cell communication<sup><a href="#ref2" title="Liu, C.(2011). Sequential Establishment of Stripe Patterns in an Expanding Cell Population. <i>Science</i>, 334: 328">[2]</a></sup>.
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<h3 id="title3">Ring Pattern Formation</h3>
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<h3 id="title1" class="r">Intro</h3>
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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.
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As a hallmark of coordinated cellular behavior, pattern formation typically required cell-cell communication and intracellular signal processing. For more site-specific signaling and pattern formation, light may be more appropriate alternative. Due to the high sensitivity of our <i>Luminesensor</i>, it is possible to construct a ring-like pattern based on light-communication, previously done by AHL. This kind of flexibility enables its application in tissue-engineering, bio-sensing and bio-manufacturing.  
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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.
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The high sensitivity of <i>Luminesensor</i> extends its field applications and enables us to achieve light-communication between E. coli cells. As demonstrations, we constructed and ring-like pattern in E. coli based on light-communication between cells. There are two kinds of E. coli cells on the plate: sender cells and receiver cells. Sender cells in the center of the plate produce blue light (490nm) on the addition of arabinose. Then receiver cells around the senders receive the light and react differently to the different light intensity. A high threshold and a low threshold are created in the system, between which the GFP are activated <sup><a href="#ref1" title="Subhayu Basu et al.(2005), A synthetic multicellular system for programmed pattern formation. Nature, vol.434: 1130: 1134">[3]</a></sup>.  
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Despite the shining promise, several important criteria still impediments the future application of current light-sensitive modules.
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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.
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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.
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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.
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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.  
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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.
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For the detail of our design, please go to <a href="">Design part</a>.
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<h3 id="title5">Reference</h3>
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<ul class="refer"><li id="ref1">
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1. Zoltowski, B.D., Crane, B.R.(2008). Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. <i>Biochemistry</i>, 47: 7012-7019
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2. Liu, C. <i>et al</i> (2011). Sequential Establishment of Stripe Patterns in an Expanding Cell Population. <i>Science</i>, 334: 328
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3. Subhayu Basu et al.(2005), A synthetic multicellular system for programmed pattern formation. <i>Nature</i>, vol.434: 1130: 1134
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</html>{{Template:Peking2012_Color_Epilogue}}

Latest revision as of 19:13, 26 October 2012

Summary

Our modeling group conducted simulations to guide our experiments by combining protein kinetics, thermodynamics, stochastic simulation, and molecular docking together. Specifically, we conducted modeling of our Luminesensor, and the experimental results of the bio-othorgonality part excellently correspond to the model! We also used molecular docking to expand the spectrum of the Luminesensor, which would greatly extend its application, such as multiple-channel communication among cells. In addition, we simulated the process of phototaxis both on the macro level and micro level, using mean-field PDE (partial difference equations) and Stochastic Simulation respectively. In the PDE model, we developed a hexagonal-coordinate simulation environment for dynamic system on a continuous plane, which would be a very useful prototype for future simulation on cellular movement, pattern formation, and other potential systems. Furthermore, we designed the genetic circuit of multi-cellular ring-like pattern formation as a demonstration for cell-cell communication through light; based on parameter analysis, we used our modeling prediction to rationally guide the experiment to form better patterns!

Luminesensor Modeling

Our Luminesensor is a ultra-sensitive fusion protein to sense 450nm to 470nm light and then regulate the gene expression(spectrum data here). Although the Luminesensor excels and eclipses similar systems due to its ultra-sensitivity and dynamic range, there are still several imperfect aspects. For example, the response time of the protein can be up to hours[1] and the contrast of binding efficiency with and without light has much room for improvement. After modeling the DNA binding process of the Luminesensor, we managed to find out four key parameters, two of which mainly control the response time, and the others control the contrast of binding efficiency. According to the feasibility in experiment, we tuned two most facile parameters of each aspect to optimize the performance of the Luminsensor, and later discovered mutation sites related to these two parameters experimentally.

Furthermore, in order to extend the application of the Luminsensor, we managed to expand the spectrum of the Luminsensor through modeling. Here we use molecular modeling to identify the possiblity of modifications to the conjugating substructure of Flavin Adanine Dinucleotide (FAD) substrate, which acts as the chromophore of VVD.

Phototaxis Modeling

Phototaxis is a light-control bio-system, whose input is the space-distribution of light. By comparison with chemotaxis system, phototaxis has many advantages for application. (See Project Phototaxis) We have constructed a simple phototaxis system coupling our Luminesensor with the expression level of cheZ protein. In order to confirm the macro light-control to our phototaxis system, we used the Mean-field PDE model. Later we managed to confirm these phenomena by tracing each cells in Stochastic Simulation. We then managed to do the related experiments to prove the effect of light in this simple system.

On the way to the simulation of the macro population changing process, we developed a hexagonal-coordinate simulation environment for dynamic system on a continuous plane. Since the number of neighbors of a chunk unit in the hexagonal mesh is larger than that of the traditional quadratic mesh, the hexagonal mesh is much closer to an isotropic grid, which would reduce the error caused by anisotropic structure in traditional quadratic mesh. This simulation environment would be a useful prototype for future simulation of 2D dynamic systems, such as cellular movement on plate and pattern formation based on cell-cell communication[2].

Ring Pattern Formation

As a hallmark of coordinated cellular behavior, pattern formation typically required cell-cell communication and intracellular signal processing. For more site-specific signaling and pattern formation, light may be more appropriate alternative. Due to the high sensitivity of our Luminesensor, it is possible to construct a ring-like pattern based on light-communication, previously done by AHL. This kind of flexibility enables its application in tissue-engineering, bio-sensing and bio-manufacturing.

The high sensitivity of Luminesensor extends its field applications and enables us to achieve light-communication between E. coli cells. As demonstrations, we constructed and ring-like pattern in E. coli based on light-communication between cells. There are two kinds of E. coli cells on the plate: sender cells and receiver cells. Sender cells in the center of the plate produce blue light (490nm) on the addition of arabinose. Then receiver cells around the senders receive the light and react differently to the different light intensity. A high threshold and a low threshold are created in the system, between which the GFP are activated [3].

Reference

  • 1. Zoltowski, B.D., Crane, B.R.(2008). Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. Biochemistry, 47: 7012-7019
    2. Liu, C. et al (2011). Sequential Establishment of Stripe Patterns in an Expanding Cell Population. Science, 334: 328
    3. Subhayu Basu et al.(2005), A synthetic multicellular system for programmed pattern formation. Nature, vol.434: 1130: 1134
  • Totop Totop