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+ | figcaption { | ||
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MathJax.Hub.Config({ | MathJax.Hub.Config({ | ||
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</head> | </head> | ||
− | <body | + | <body> |
− | + | <ul id="left-nav"> | |
<li> | <li> | ||
<a>PROJECT</a> | <a>PROJECT</a> | ||
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<li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsFeedback">Feedback</a></li> | <li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsFeedback">Feedback</a></li> | ||
<li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsOutput">Output</a></li> | <li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsOutput">Output</a></li> | ||
− | <li><a href="https://2018.igem.org/Team:BIT-China/ | + | <li><a href="https://2018.igem.org/Team:BIT-China/Results">Results</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<ul> | <ul> | ||
<li><a href="https://2018.igem.org/Team:BIT-China/Model">Overview</a></li> | <li><a href="https://2018.igem.org/Team:BIT-China/Model">Overview</a></li> | ||
− | <li><a href="https://2018.igem.org/Team:BIT-China/FluorescentProbesModel">Fluorescent | + | <li><a href="https://2018.igem.org/Team:BIT-China/FluorescentProbesModel">Fluorescent Probe Model </a></li> |
<li><a href="https://2018.igem.org/Team:BIT-China/H2O2DecompositionModel">H<sub>2</sub>O<sub>2</sub> | <li><a href="https://2018.igem.org/Team:BIT-China/H2O2DecompositionModel">H<sub>2</sub>O<sub>2</sub> | ||
Decomposition Model</a></li> | Decomposition Model</a></li> | ||
<li><a href="https://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel">roGFP2-Orp1 | <li><a href="https://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel">roGFP2-Orp1 | ||
− | Michaelis | + | Michaelis equation Model</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<div class="MD-title"> | <div class="MD-title"> | ||
<a style="border-bottom-style: solid;text-decoration: none;color:#131313;">H<sub>2</sub>O<sub>2</sub> | <a style="border-bottom-style: solid;text-decoration: none;color:#131313;">H<sub>2</sub>O<sub>2</sub> | ||
− | + | DECOMPOSITION MODEL</a> | |
</div> | </div> | ||
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up a model to simulate | up a model to simulate | ||
the intracellular H<sub>2</sub>O<sub>2</sub>. At the same time, this model will provide a base for | the intracellular H<sub>2</sub>O<sub>2</sub>. At the same time, this model will provide a base for | ||
− | roGFP2-Orp1 | + | roGFP2-Orp1 Michealis |
equation model. | equation model. | ||
</p> | </p> | ||
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some natural reduction molecular (like Glutathione) directly react with H<sub>2</sub>O<sub>2</sub>. | some natural reduction molecular (like Glutathione) directly react with H<sub>2</sub>O<sub>2</sub>. | ||
And slow degradation | And slow degradation | ||
− | is led by enzymatic degradation.( | + | is led by enzymatic degradation.<sup onmouseover="tooltip.pop(this, '#tip-1', {position:1, offsetX:-20, effect:'slide',hideDelay: 10})" |
+ | style="color:#38679a;">[1]</sup> | ||
</p> | </p> | ||
<figure class="MD-Fig MD-margin-toContentP"> | <figure class="MD-Fig MD-margin-toContentP"> | ||
− | <img src="https://static.igem.org/mediawiki/2018/ | + | <img src="https://static.igem.org/mediawiki/2018/9/92/T--BIT-China--modelingh202decompositiondig1chaizong.png"> |
− | <figcaption>Fig.1 The kinetic model for | + | <figcaption>Fig.1 The kinetic model for H<sub>2</sub>O<sub>2</sub> degradation</figcaption> |
</figure> | </figure> | ||
</div> | </div> | ||
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<p class="MD-content-p"> | <p class="MD-content-p"> | ||
− | The ODE model accounted for | + | The ODE model accounted for intracellular and extracellular H<sub>2</sub>O<sub>2</sub> levels, and the |
consumption of a finite | consumption of a finite | ||
antioxidant capacity using three rates: | antioxidant capacity using three rates: | ||
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<ul class="MD-content-p"> | <ul class="MD-content-p"> | ||
<li> | <li> | ||
− | 1) The membrane permeability (k1).( | + | 1) The membrane permeability (k1).<sup onmouseover="tooltip.pop(this, '#tip-2', {position:1, offsetX:-20, effect:'slide',hideDelay: 10})" |
+ | style="color:#38679a;">[2]</sup><sup onmouseover="tooltip.pop(this, '#tip-3', {position:1, offsetX:-20, effect:'slide',hideDelay: 10})" | ||
+ | style="color:#38679a;">[3]</sup><sup onmouseover="tooltip.pop(this, '#tip-4', {position:1, offsetX:-20, effect:'slide',hideDelay: 10})" | ||
+ | style="color:#38679a;">[4]</sup> | ||
</li> | </li> | ||
<li> | <li> | ||
− | 2) Intracellular slow degradation rate (k2).( | + | 2) Intracellular slow degradation rate (k2).<sup onmouseover="tooltip.pop(this, '#tip-5', {position:1, offsetX:-20, effect:'slide',hideDelay: 10})" |
+ | style="color:#38679a;">[5]</sup> | ||
</li> | </li> | ||
<li> | <li> | ||
3) Intracellular fast degradation rate (k3) dependent upon a finite capacity (g), e.g. NADPH | 3) Intracellular fast degradation rate (k3) dependent upon a finite capacity (g), e.g. NADPH | ||
− | levels in naive cells.( | + | levels in naive cells.<sup onmouseover="tooltip.pop(this, '#tip-6', {position:1, offsetX:-20, effect:'slide',hideDelay: 10})" |
+ | style="color:#38679a;">[6]</sup> | ||
</li> | </li> | ||
</ul> | </ul> | ||
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To verify the feasibility of the model and to confirm this dynamic process, we did some experiments | To verify the feasibility of the model and to confirm this dynamic process, we did some experiments | ||
using our system's output, roGFP2-Orp1. The results show that the fluorescence ratio value | using our system's output, roGFP2-Orp1. The results show that the fluorescence ratio value | ||
− | I405/I488, an oxidant condition indicator, decreased by time when S. cerevisiae exposed to | + | I405/I488, an oxidant condition indicator, decreased by time when <i>S. cerevisiae</i> exposed to |
different concentrations of H<sub>2</sub>O<sub>2</sub>. This ratio's decreasing stands for the | different concentrations of H<sub>2</sub>O<sub>2</sub>. This ratio's decreasing stands for the | ||
increasing of | increasing of | ||
− | intercellular | + | intercellular H<sub>2</sub>O<sub>2</sub> concentration. It also stopped at a stable concentration at last, which's |
correspond to our model's result. | correspond to our model's result. | ||
</p> | </p> | ||
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</div> | </div> | ||
</div> | </div> | ||
+ | <div style="display:none;font-family: 'helveticaregular';"> | ||
+ | <div id="tip-1"> | ||
+ | <div class="column" style="width:230px;height: 100px;"> | ||
+ | <a>Altıntaş A, Davidsen K, Garde C, et al. High-resolution kinetics and modeling of hydrogen peroxide degradation in live cells.[J]. Free Radical Biology & Medicine, 2016, 101:143-153. | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="tip-2"> | ||
+ | <div class="column" style="width:230px;height: 100px;"> | ||
+ | <a>Branco M R, Marinho H S, Cyrne L, et al. Decrease of H<sub>2</sub>O<sub>2</sub> plasma membrane permeability during adaptation to H<sub>2</sub>O<sub>2</sub> in Saccharomyces cerevisiae.[J] . Journal of Biological Chemistry, 2004, 279(8):6501 | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div id="tip-3"> | ||
+ | <div class="column" style="width:230px;height: 100px;"> | ||
+ | <a>Henzler T, Steudle E. Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H(2)O(2) across water channels.[J]. Journal of Experimental Botany, 2000, 51(353):2053-2066. | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="tip-4"> | ||
+ | <div class="column" style="width:230px;height: 100px;"> | ||
+ | <a>Bienert G P, Schjoerring J K, Jahn T P. Membrane transport of hydrogen peroxide[J]. Biochimica Et Biophysica Acta, 2006, 1758(8):994-1003. | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="tip-5"> | ||
+ | <div class="column" style="width:230px;height: 100px;"> | ||
+ | <a>Branco M R, Marinho H S, Cyrne L, et al. Decrease of H<sub>2</sub>O<sub>2</sub> plasma membrane permeability during adaptation to H<sub>2</sub>O<sub>2</sub> in Saccharomyces cerevisiae.[J]. Journal of Biological Chemistry, 2004, 279(8):6501. | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="tip-6"> | ||
+ | <div class="column" style="width:230px;height: 100px;"> | ||
+ | <a>Altıntaş A, Davidsen K, Garde C, et al. High-resolution kinetics and modeling of hydrogen peroxide degradation in live cells.[J]. Free Radical Biology & Medicine, 2016, 101:143-153. | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
<!-- footer start --> | <!-- footer start --> | ||
<div class="footer-all" style="margin-top:65px;"> | <div class="footer-all" style="margin-top:65px;"> |
Latest revision as of 00:44, 18 October 2018
In our experiment, we found it hard to measure the intracellular H2O2 concentration, so we controlled the external H2O2 concentration in culture to confirm our system' function. Although the H2O2 can entry the cell quickly through simple diffusion, we needed to build up a model to simulate the intracellular H2O2. At the same time, this model will provide a base for roGFP2-Orp1 Michealis equation model.
Firstly, in order to describe the process in cells more accurately, three reactions in this process are considered: diffusion, fast degradation and slow degradation. The fast degradation is caused by some natural reduction molecular (like Glutathione) directly react with H2O2. And slow degradation is led by enzymatic degradation.[1]
$$\frac{\mathrm{d_{\mathit{p}_{e}}} }{\mathrm{d} t}=k_{1}(p_{i}-p_{e})v$$
$$\frac{\mathrm{d_{\mathit{p}_{i}}} }{\mathrm{d}t}=k_{1}(p_{e}-p_{i})-(k_{2}+k_{3}\frac{g}{k_{g}+g})p_{i}$$
$$\frac{\mathrm{d_{\mathit{g}}} }{\mathrm{d} t}=-k_{3}(\frac{g}{k_{g}+g})p_{i}$$
The ODE model accounted for intracellular and extracellular H2O2 levels, and the consumption of a finite antioxidant capacity using three rates:
- 1) The membrane permeability (k1).[2][3][4]
- 2) Intracellular slow degradation rate (k2).[5]
- 3) Intracellular fast degradation rate (k3) dependent upon a finite capacity (g), e.g. NADPH levels in naive cells.[6]
We reviewed the above literature and combined the results of the wet experiment, as shown in Fig.3, to determine the parameters required for the model simulation, listed in the table below.
Table 1. Description of Variables Used in The Kinetic Model.
Variable | Explanation | Value |
---|
k1 | Membrane Permeability | 11 |
k2 | Slow Degradation Rate | 8 |
k3 | Fast Degradation Rate | 55 |
G | Finite Capacity Substance | 60 |
pi | Intracellular/Cytosolic [H2O2] | / |
pe | Extracellular [H2O2] | / |
V | Ratio of Cell Volume to Media | 0.005 |
We used MATLAB to simulate the kinetics of extracellular and intracellular H2O2 changes over time. (Fig. 2)
To verify the feasibility of the model and to confirm this dynamic process, we did some experiments using our system's output, roGFP2-Orp1. The results show that the fluorescence ratio value I405/I488, an oxidant condition indicator, decreased by time when S. cerevisiae exposed to different concentrations of H2O2. This ratio's decreasing stands for the increasing of intercellular H2O2 concentration. It also stopped at a stable concentration at last, which's correspond to our model's result.
In this model, we successfully simulated the kinetics of extracellular and intracellular H2O2 changes over time. Furthermore, the dynamic process of fluorescence ratio value by time was detected by using roGFP2-Orp1 green fluorescent protein in wet experiment, and the feasibility of the model was further verified.