Team:Peking/Modeling/Luminesensor

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  <h3 id="title1">Summary</h3>
  <h3 id="title1">Summary</h3>
  <p>
  <p>
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The <i>Luminesensor</i> is a fusion protein that we have built to sense light with wavelengths 450nm or 470nm and then regulate the gene expression.(<a href="/Team:Peking/Project/Luminesensor/Future#FigS">spectrum data here</a>) Although 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<sup><a href="#ref1" title="Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. B. D. Zoltowski etc. Biochemistry">[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 <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 <i>Luminsensor</i>, and later figured out mutation sites related to these two parameters experimentally.
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We have managed to conduct a series of modeling simulations to guide the optimization of our <i>Luminesensor</i>, combining protein kinetics, thermodynamics, and stochastic simulation with molecular docking together. The modeling results also shows the system still works well even if considering noise and endogenous competition.
  </p>
  </p>
</div>
</div>
<div class="PKU_context floatR">
<div class="PKU_context floatR">
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  <h3 id="title2">Network System with ODE Method</h3>
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  <h3 id="title2">Kinetic Network</h3>
  <p>
  <p>
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Firstly we established a reaction network for the DNA binding process of <i>Luminesensor</i>. To quantify this system, we used <a href="/Team:Peking/Modeling/Background/ODE">ODE (Oridinary Differential Equations)</a> model at the beginning. Previous works by other scientists indicate that the Vivid (VVD) protein, a sensing domain of <i>Luminesensor</i>, dimerizes in the presence of light,<sup><a href="#ref1" title="Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. B. D. Zoltowski etc. Biochemistry">[1]</a></sup> and the LexA protein, a binding domain of <i>Luminesensor</i>, binds at specific sequences on DNA predominantly when coupled<sup><a href="#ref2" title="LexA Repressor Forms Stable Dimers in Solution. R. Mohana-Borges etc. THE JOURNAL OF BIOLOGICAL CHEMISTRY">[2]</a></sup> and subsequently represses the interest gene. By combining with the mechanisms of the two functional domains of Luminesensor, we concluded with the following network:
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Firstly we established a reaction network for the DNA binding process of the <i>Luminesensor</i> (For details see <a href="/Team:Peking/Project/Luminesensor/Design#title4" title="">Project Luminesensor Design</a>). Previous works by other scientists indicate that the Vivid (VVD) protein, the sensing domain of the <i>Luminesensor</i>, dimerizes in the presence of light,<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 LexA protein, the binding domain of the <i>Luminesensor</i>, binds at specific sequences on DNA predominantly when coupled<sup><a href="#ref2" title="Mohana-Borges, R.,et al.(2000). The LexA repressor forms stable dimers in solution. The role of specific DNA in tightening protein-protein interactions. J. Biol. Chem., 275: 4708: 4712">[2]</a></sup> and subsequently represses the interest gene. By combining with the mechanisms of the two functional domains of the <i>Luminesensor</i>, we concluded with the following kinetic network (Figure 1):
  </p>
  </p>
  <div class="floatC">
  <div class="floatC">
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[fig 1: Reaction Network (Cartoon Style)]
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  <img src="/wiki/images/3/3a/Peking2012_LuminesensorNodes.png" alt="Network System Fig" style="width:500px;"/>
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   <p class="description">Fig 1. </p>
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   <p class="description" style="text-align:center;width:350px;">Figure 1. Kinetic Network of our Luminesensor</p>
  </div>
  </div>
  <p>where</p><ul><li>
  <p>where</p><ul><li>
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L denotes Luminesensor,</li><li>
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L : <i>Luminesensor</i>,</li><li>
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L<sub>G</sub> denotes the ground state --- with its VVD N-cap locked,</li><li>
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L<sub>G</sub> : the ground state --- with its VVD N-cap locked,</li><li>
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L<sub>A</sub> denotes the active state --- with its VVD N-cap released,</li><li>
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L<sub>A</sub> : the active state --- with its VVD N-cap released,</li><li>
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D<sub>L</sub> denotes the specific DNA binding site to Luminesensor.</li></ul>
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D<sub>L</sub> : the specific DNA binding site to <i>Luminesensor</i>.</li></ul>
  <p>
  <p>
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Since no multi-intermediate reactions hidden in the network above, all reactions above can be regarded as elementary reactions. We list all the differential equations as following:
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Since no multi-intermediate reactions are hidden in the network above, all reactions can be regarded as elementary reactions.
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</p>
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<div class="floatC">
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[fig 2: Ordinary Equations]
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  <p class="description">Fig 2. </p>
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</div>
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<p>with parameters listed following:</p>
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<div class="floatC">
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  <table>
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  <tr>
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    <td>Parameter</td><td>Value</td><td>Unit</td><td>Description</td><td>Source</td>
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-
  </tr><tr>
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    <td>k<sub>1</sub></td><td>3.x10<sup>-4</sup></td><td>s<sup>-1</sup></td><td>vivid decay rate constant</td><td></td>
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  </tr><tr>
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    <td>k<sub>2</sub></td><td>5.6x10<sup>-5</sup></td><td>s<sup>-1</sup></td><td>vivid dissociation rate constant</td><td><a href="#ref3" title="Mechanism-based tuning of a LOV domain photoreceptor, Brian D. Zoltowski, etc. NATURE CHEMICAL BIOLOGY">[3]</a></td>
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  </tr><tr>
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    <td>k<sub>3</sub></td><td>8.x10<sup>-4</sup></td><td>s<sup>-1</sup></td><td>monomer LexA releasing rate constant from specific binding site</td><td></td>
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  </tr><tr>
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    <td>k<sub>4</sub></td><td>1.x10<sup>-3</sup></td><td>s<sup>-1</sup></td><td>binded monomer LexA dissociation rate constant</td><td></td>
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  </tr><tr>
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    <td>k<sub>5</sub></td><td>1.x10<sup>-4</sup></td><td>s<sup>-1</sup></td><td>dimered LexA releasing rate constant from specific binding site</td><td></td>
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  </tr><tr>
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    <td>K<sub>1</sub>(Dark)</td><td>0</td><td>1</td><td>equilibrium excitation constant on dark</td><td></td>
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  </tr><tr>
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    <td>K<sub>1</sub>(Light)</td><td>1.x10<sup>+3</sup></td><td>1</td><td>equilibrium excitation constant on light</td><td></td>
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  </tr><tr>
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    <td>K<sub>2</sub></td><td>7.7x10<sup>-5</sup></td><td>(n mol/L)<sup>-1</sup></td><td>vivid association equilibrium constant</td><td><a href="#ref4" title="Protein Vivid Generates a Rapidly Exchanging Dimer, Brian D. Zoltowski, etc. BIOCHEMISTRY">[4]</a></td>
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  </tr><tr>
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    <td>K<sub>3</sub></td><td>1.x10<sup>-3</sup></td><td>(n mol/L)<sup>-1</sup></td><td>monomer LexA binding equilibrium constant with specific binding site</td><td><a href="#ref2" title="LexA Repressor Forms Stable Dimers in Solution, R.Mohana-Borges, etc. THE JOURNAL OF BIOLOGICAL CHEMISTRY">[2]</a></td>
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  </tr><tr>
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    <td>K<sub>4</sub></td><td>K<sub>2</sub>xK<sub>5</sub>/K<sub>3</sub></td><td>(n mol/L)<sup>-1</sup></td><td>binded monomer LexA association equilibrium constant</td><td>Thermal Principle</td>
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  </tr><tr>
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    <td>K<sub>5</sub></td><td>1.</td><td>(n mol/L)<sup>-1</sup></td><td>dimered LexA binding equilibrium constant</td><td><a href="#ref2" title="LexA Repressor Forms Stable Dimers in Solution, R.Mohana-Borges, etc. THE JOURNAL OF BIOLOGICAL CHEMISTRY">[2]</a></td>
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  </tr><tr>
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    <td>[L<sub>G</sub>]<sub>0</sub></td><td>1000</td><td>n mol/L</td><td>initial concentration of Luminesensor in ground state</td><td></td>
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  </tr><tr>
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    <td>[L<sub>A</sub>]<sub>0</sub></td><td>0</td><td>n mol/L</td><td>initial concentration of Luminesensor in active state</td><td></td>
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  </tr><tr>
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    <td>[L<sub>A</sub><sup>2</sup>]<sub>0</sub></td><td>0</td><td>n mol/L</td><td>initial concentration of dimered Luminesensor</td><td></td>
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  </tr><tr>
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    <td>[D<sub>L</sub>]<sub>0</sub></td><td>100</td><td>n mol/L</td><td>initial concentration of free specific binding site on DNA</td><td>high-copy plasmid</td>
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  </tr><tr>
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    <td>[L<sub>G</sub>D<sub>L</sub>]<sub>0</sub></td><td>0</td><td>n mol/L</td><td>initial concentration of dimered Luminesensor binded Luminesensor in ground state</td><td></td>
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  </tr><tr>
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    <td>[L<sub>A</sub>D<sub>L</sub>]<sub>0</sub></td><td>0</td><td>n mol/L</td><td>initial concentration of dimered Luminesensor binded Luminesensor in active state</td><td></td>
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-
  </tr><tr>
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    <td>[L<sub>A</sub><sup>2</sup>D<sub>L</sub>]<sub>0</sub></td><td>0</td><td>n mol/L</td><td>initial concentration of binded and dimered Luminesensor</td><td></td>
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  </tr>
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  </table>
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  <p class="description">Tab 1. Reaction Parameters</p>
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</div>
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<p>and then simulated the dynamic behavior of the components, with our results shown beneath:</p>
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<div class="floatC">
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[fig 3: ODE Simulation]
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  <p class="description">Fig 3. Simulation of Origin System</p>
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</div>
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<div>
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  <p>where the binding rate</p><p class="center">
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r<sub>b</sub> = 1 - [D<sub>L</sub>]/[D<sub>T</sub>]
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<br />(D<sub>T</sub> means the total concentration of specific binding site on DNA)
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  </p><p>indicates the repressing degree, and the dimerizing rate</p><p class="center">
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r<sub>d</sub> = 2[L<sub>A</sub><sup>2</sup>X]/[L<sub>T</sub>]
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<br />(L<sub>T</sub> means the total concentration of Luminesensor, X denotes D<sub>L</sub> or nothing)
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  </p><p>indicates the dimerizing degree, and the activating rate</p><p class="center">
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r<sub>a</sub> = ([L<sub>A</sub>X] + 2[L<sub>A</sub><sup>2</sup>X])/[L<sub>T</sub>]
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-
  </p>
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</div>
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<div>
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  <p>
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From the figure above, we discovered that the activation and decay of Luminesensor are the pioneers of the progress, and the activating rate is the most thoroughly switched variable in the variation of the lighting. The promoters on DNA are repressed even though the Luminesensors are not thoroughly dimered.
+
-
  </p>
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</div>
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</div>
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<div class="PKU_context floatR">
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<h3 id="title3">Stochastic Simulation</h3>
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<p>
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In order to check the working stability of Luminesensor, we simulated this reaction network with <a href="/Team:Peking/Modeling/Background/Stochastic">stochastic model</a>. By estimating the volume of a cell, we convert the concentration of a component into the number of molecules by 1 n mol/L : 1. The results are shown beneath:
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</p>
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<div class="floatC">
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[fig 4: Stochastic Result]
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  <p class="description">Fig 4. Stochastic Simulation</p>
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</div>
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<p>
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The noise does not influence this system according to the figure above. Luminesensor is thus expected to work theoretically. Besides, the average value of stochastic simulation is coupled with the result of ODE model, which in turn proves the self-consistency of our ODE model.
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</p>
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</div>
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<div class="PKU_context floatR">
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<h3 id="title4">Parameter Analysis & Optimization</h3>
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<p>
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After modeling the origin system, we rationally optimized it. We tuned the parameters up and down one by one, and finally discovered four parameters which predominantly influence this system. k1 (Vivid decay rate constant) and k3 (rate constant of monomer LexA releasing from specific binding site) determines the time scale while K2 (Vivid association equilibrium constant) and K5 (dimered LexA binding equilibrium constant) determine the contrast between dark and light. Here shows the simulation data:
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</p>
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<div class="floatC">
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[fig 5: Parameter Tuning]
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  <p class="description">Fig 5. Parameter Tuning, the left show the simulation by ODE method and the right show the stochastic simulation. (a) the origin system. (b) tuning k1 up by 10 times while K1 remains unchanged. (c) tuning k3 up by 10 times while K3 remains unchanged. (d) tuning K2 up by 10 times. (e) tuning K5 up by 10 times.</p>
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</div>
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<p>
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As for contrast optimizing, we measured the thoroughly-repressing time (no free specific binding site) in simulation when light to quantify the interest. The thoroughly-repressing rate is (thoroughly-repressing time / total sample time) shown at right of Figure 5. The higher thoroughly-repressing rate is, the better the Luminesensor represses. Within the two chosen parameters, K2 (vivid association equilibrium constant) is related to the association mechanism of vivid protein and K5 (dimered LexA binding equilibrium constant) is related to the cooperative binding mechanism.
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</p><p>
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As for speed optimizing, k1 (vivid decay rate constant) is related to the activation mechanism of vivid protein and k3 (monomer LexA releasing rate constant from specific binding site) is related to the binding mechanism and thus to the LexA and sequence. If we change the binding affinity of the sequence, then K3 (monomer LexA binding equilibrium constant with specific binding site) is hard to make constant, whose variance is predicted to ruin the contrast of this system, as we saw from simulation. Therefore, we chose to mutate Vivid protein to get a faster Luminesensor which has a high k1 (vivid decay rate constant).
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</p><p>
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After searching recent papers, we decided to mutate M135I in Vivid dimerization domain to enhance K2 (vivid association equilibrium constant)<sup><a href="#ref?" title="">[?]</a></sup> and I74V, amino acids surrounding Cys108, to enhance k1 (vivid decay rate constant)<sup><a href="#ref3" title="Mechanism-based tuning of a LOV domain photoreceptor, Brian D. Zoltowski, etc. NATURE CHEMICAL BIOLOGY">[3]</a></sup>. As will be mentioned later, we also mutated LexA to LexA408 and its corresponding promoter, which substantially increase K5 (dimered LexA binding equilibrium constant).<sup><a href="#ref5" title="A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli, M. Dmitrova. etc. Springer-Verlag Mol Gen Genet">[5]</a></sup> Actually, the main reason that we preferred LexA408 to wild LexA is due to the bio-orthogonality.
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</p>
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</div>
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<div class="PKU_context floatR">
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<h3 id="title5">Orthogonal Test in silico</h3>
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<p>
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To modularize the genetic system, our Luminesensor is expected to be bio-orthogonal with the endogenous LexA in bacteria. Endogenous LexA, from lactin-SOS system in bacteria, may cause unexpected crosstalk. To remove this obstacle, we use LexA408 as the binding domain instead of the wild LexA. LexA408 and LexA are bio-orthogonal with each other since the sequence of the binding sites are different between them.
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</p><p>
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By adding several nodes into the network, we constructed modeling for orthogonality test:
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</p>
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<div class="floatC">
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[fig 6: Reaction Network for Orthogonal Test]
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  <p class="description">Fig 6. Reaction Network for Orthogonal Test</p>
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</div>
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<p>where</p><ul><li>
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L denotes Luminesensor</li><li>
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I denotes the inner wild LexA</li><li>
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D<sub>L</sub> denotes the specific DNA binding site to Luminesensor</li><li>
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D<sub>I</sub> denotes the specific DNA binding site to endogenous LexA</li></ul>
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<p>The parameters are estimated as following:</p>
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<div class="floatC">
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  <table>
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  <tr>
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    <td>Parameter</td><td>Value</td><td>Unit</td><td>Description</td><td>Source</td>
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  </tr><tr>
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    <td>k<sub>6</sub></td><td>1.x10<sup>-4</sup></td><td>s<sup>-1</sup></td><td>dimered LexA releasing rate constant from non-specific binding site</td><td></td>
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  </tr><tr>
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    <td>K<sub>6</sub></td><td>1.x10<sup>-2</sup></td><td>(n mol/L)<sup>-1</sup></td><td>dimered non-specific binding equilibrium constant</td><td><a href="#ref9">[9]</a></td>
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  </tr>
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  </table>
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[table 2: Reaction Parameters for Orthogonal Test]
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  <p class="description">Tab 2. Reaction Parameters for Orthogonal Test</p>
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</div>
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<div class="floatC">
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[fig 7: Orthogonal Test Result]
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  <p class="description">Fig 7. Orthogonal Test Result, the left show the simulation by ODE method and the right show the stochastic simulation. (a) the origin system. (b) competition induced. (c) competition induced and tuning K6 up by 10 times.
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-
  </p>
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</div>
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<p>
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The result shows that the endurance of K6/K5 is up to around 1%. Our Luminesensor can be used in bacteria with little interference from endogenous LexA.
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</p>
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</div>
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<div class="PKU_context floatR">
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<h3 id="title6">Conclusion</h3>
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<p>
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The modeling above points out a way to optimize our <i>Luminesensor</i> -- the two critical mutation. It also shows the system still works well even if considering noise and inner competition.
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  </p>
  </p>
</div>
</div>
<div class="PKU_context floatR">
<div class="PKU_context floatR">
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  <h3 id="title7">Referrence</h3>
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  <h3 id="title3">Reference</h3>
  <p></p>
  <p></p>
  <ul class="refer"><li id="ref1">
  <ul class="refer"><li id="ref1">
-
[1] Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. B. D. Zoltowski etc. Biochemistry
+
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
-
</li><li id="ref2">
+
  </li><li id = "ref2">
-
[2] LexA Repressor Forms Stable Dimers in Solution. R. Mohana-Borges etc. THE JOURNAL OF BIOLOGICAL CHEMISTRY
+
2. Mohana-Borges, R., Pacheco, A.B., Sousa, F.J., Foguel, D., Almeida, D.F., and Silva, J.L. (2000). LexA repressor forms stable dimers in solution. The role of specific DNA in tightening protein-protein interactions. <i>J. Biol. Chem.</i>, 275: 4708: 4712
-
</li><li id="ref3">
+
-
[3] Mechanism-based tuning of a LOV domain photoreceptor, Brian D. Zoltowski, etc. NATURE CHEMICAL BIOLOGY
+
-
</li><li id="ref4">
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[4] Protein Vivid Generates a Rapidly Exchanging Dimer, Brian D. Zoltowski, etc. BIOCHEMISTRY
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-
</li><li id="ref5">
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[5] A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli, M. Dmitrova. etc. Springer-Verlag Mol Gen Genet
+
  </li></ul>
  </li></ul>
</div>
</div>
</html>{{Template:Peking2012_Color_Epilogue}}
</html>{{Template:Peking2012_Color_Epilogue}}

Latest revision as of 03:39, 25 October 2012

Summary

We have managed to conduct a series of modeling simulations to guide the optimization of our Luminesensor, combining protein kinetics, thermodynamics, and stochastic simulation with molecular docking together. The modeling results also shows the system still works well even if considering noise and endogenous competition.

Kinetic Network

Firstly we established a reaction network for the DNA binding process of the Luminesensor (For details see Project Luminesensor Design). Previous works by other scientists indicate that the Vivid (VVD) protein, the sensing domain of the Luminesensor, dimerizes in the presence of light,[1] and the LexA protein, the binding domain of the Luminesensor, binds at specific sequences on DNA predominantly when coupled[2] and subsequently represses the interest gene. By combining with the mechanisms of the two functional domains of the Luminesensor, we concluded with the following kinetic network (Figure 1):

Network System Fig

Figure 1. Kinetic Network of our Luminesensor

where

  • L : Luminesensor,
  • LG : the ground state --- with its VVD N-cap locked,
  • LA : the active state --- with its VVD N-cap released,
  • DL : the specific DNA binding site to Luminesensor.

Since no multi-intermediate reactions are hidden in the network above, all reactions can be regarded as elementary reactions.

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. Mohana-Borges, R., Pacheco, A.B., Sousa, F.J., Foguel, D., Almeida, D.F., and Silva, J.L. (2000). LexA repressor forms stable dimers in solution. The role of specific DNA in tightening protein-protein interactions. J. Biol. Chem., 275: 4708: 4712
  • Totop Totop

Retrieved from "http://2012.igem.org/Team:Peking/Modeling/Luminesensor"