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| | <a href="https://2018.igem.org/Team:TJU_China/Human_Practices">Human Practices</a> | | <a href="https://2018.igem.org/Team:TJU_China/Human_Practices">Human Practices</a> |
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| | <div style=" margin-top:28px;z-index:10; border-top: solid #4e72b8 2px;width: 100%; position: fixed;"></div> | | <div style=" margin-top:28px;z-index:10; border-top: solid #4e72b8 2px;width: 100%; position: fixed;"></div> |
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| − | <div class="head">Dynamic Model of Heavy Metal Detection Biosensor</div>
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| − | <div class="subhead">Minghui Yin,Sherry Dongqi Bao
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| − | <br>TianJin University
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| − | <br>October 15,2018</div>
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| − | <div class="title">1 Introduction</div>
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| − | <div class="word">Modeling is a powerful tool in synthetic biology. It provides us with a necessary engineering approach to characterize
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| − | our pathways quantitatively and predict their performance,thus help us test and modify our design.Through the dynamic
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| − | model of heavy-metal detection biosensor,we hope to gain insights into the characteristics of our whole circuit's
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| − | dynamics.
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| − | </div>
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| − | <div class="title">2 Methods</div>
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| − | <div class="subtitle">2.1 Analysis of metabolic pathways</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/0/01/T--TJU_China--y1.png">
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| − | </div>
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| − | <div class="figure">Figure 1: Metabolic pathways related to plasmid#1</div>
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| − | <div class="word">At the beginning, on the plasmid#1, the promoter $P_{arsR}$ isn't bound with ArsR,thus it is active.ArsR and smURFP are
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| − | transcribed and translated under the control of the promoters $P_{arsR_{u}}$ and $P_{arsR_{d}}$,with subscript u
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| − | and d representing upstream and downstream separately.The subscript l of smURFP in the equation means leaky expression
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| − | without the expression of $As^{3+}$.As ArsR is expressed gradually,it will bind with the promoter $P_{arsR}$ and
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| − | make it inactive.[1]</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/a6/T--TJU_China--m1.PNG">
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| − | </div>
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| − | <div class="word">On the plasmid#2,the fusion protein of dCas9 and RNAP(RNA polymerase) are produced after transcription and translation,and
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| − | sgRNA is produced after transcription.
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/2/26/T--TJU_China--m2.png">
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/2/2b/T--TJU_China--2.png">
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| − | </div>
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| − | <div class="figure">Figure 2: Metabolic pathways related to dCas9/RNAP</div>
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| − | <div class="word">dCas9(*RNAP) can bind with its target DNA sequence without cutting, which is at the upstream of the promoter $P_{arsR_{d}}$.Simulataneously,dCas9
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| − | can lead RNAP to bind with the promoter $P_{arsR_{d}}$ and enhance the transcription of smURFP.However,because the
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| − | promoter $P_{arsR_{d}}$ has already bound with ArsR,as a result,RNAP can't bind with the promoter $P_{arsR_{d}}$.
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| − | can’t bind with the promoter $P_{arsR_{d}}$.</div>
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| − | <div class="word">However,at the presence of $As^{3+}$,it can bind with ArsR,then dissociate ArsR and $P_{arsR_{d}}$ , which makes the
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| − | combination of RNAP and $P_{arsR_{d}}$ possible.</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/4/4d/T--TJU_China--m3.png">
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| − | </div>
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| − | <div class="word">We then take degradation into account: </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/a1/T--TJU_China--m4.png">
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/3/32/T--TJU_China--m5.png">
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| − | </div>
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| − | <div class="subtitle">2.2 Analysis of ODEs</div>
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| − | <div class="word">Applying mass action kinetic laws,we obtain the following set of differentiak equations.The several complexes involved:Ars$R^*$$P_{arsR}$,$As^{3+}$,${dCas9}^*$RNAP,${dCas9}^*$RNAP:sgRNA,${dCas9}^*$RNAP:${sgRNA}^*P_{arsR}$,
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| − | are respectively abbreviated as $cplx_1$,$cplx_2$,$cplx_3$,$cplx_4$,$cplx_5$.</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/e/e4/T--TJU_China--m6.png">
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/4/45/T--TJU_China--m7.png">
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/b/b7/T--TJU_China--m8.png">
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/ad/T--TJU_China--m9.png">
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| − | </div>
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| − | <div class="subtitle">2.3 Simulation</div>
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| − | <div class="word">Our simulation is based on two softwares: MATLAB (SimBiology Toolbox) and COPASI.
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| − | <br> SimBiology Toolbox provides functions for modeling,simulating and analyzing biochemical pathways by the powerful
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| − | computing engine of MATLAB.</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/a5/T--TJU_China--s3.png">
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| − | </div>
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| − | <div class="figure">Figure 3:Reaction map generated from the reaction sets above by SimBiology Toolbox</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/0/04/T--TJU_China--11.png">
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| − | </div>
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| − | <div class="figure">Figure 4:Simulation of smURFP production as a function of time by MATLAB Through the figure, we can see that the smURFP
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| − | can gradually increase and reach a steady state after a period in the presence of arsenic ions.</div>
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| − | <div class="subtitle">2.4 Sensitivity</div>
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| − | <div class="word">A good biosystem should have certain stability towards fluctuations in parameters.A good model should reflect this,and
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| − | hence a test for robustness can be essential to the model.
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| − | <br> Robustness analysis can also pinpoint which reactions/parameters that are important for obtaining a specific biological
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| − | behavior.A simple measure for sensitivity is to measure the relative change of a system feaure due to a change in
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| − | a parameter.As for our model,the feature can be the equilibrium concentration of the smURFP(C) for which the sensitivity(S)
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| − | to a parameter k is:
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/1/11/T--TJU_China--m10.png">
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| − | </div>
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| − | <div class="word">After analysis, we found that the concentration of smURFP is relatively sensitive to parameters such as ktx3,ktl3,ktx4,kb4,kb6,kd2,kd5,
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| − | kd6,kd7,kd8,kd11, etc. Among these parameters, except for the parameters that directly affect the production and
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| − | degradation of smURFP,the rest of them are all related to dCas9-RNAP:sgRNA. It shows that our model reflects the
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| − | critical role of dCas9-RNAP:sgRNA,which initially confirms our hypothesis:dCas0-RNAP can enhance transcription to
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| − | increase the concentration of smURFP. However, due to the lack of previous modeling studies on dCas9-RNAP,some kinetic
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| − | parameters may not be very accurate,and due to time limitation,we have not implemented experiments to measure related
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| − | parameters,which may lead to some deviations in our model.
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| − | <br> The sensitivity of each parameter is shown in the figures below.</div>
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| | | | |
| − | <div> | + | <div id="banner"> |
| − | <div class="doublepic"> | + | <div class="pic"> |
| − | <img src="https://static.igem.org/mediawiki/2018/f/f1/T--TJU_China--tx1.png"> | + | <a href="#" style="display:block; "> |
| − | </div>
| + | <img src="https://static.igem.org/mediawiki/2018/ 6/ 62/T--TJU_China-- home1. jpg" /> |
| − | <div class="doublepic">
| + | </a> |
| − | <img src="https://static.igem.org/mediawiki/2018/e/e0/T--TJU_China--tl1.png"> | + | <a href="#"> |
| − | </div>
| + | <img src="https://static.igem.org/mediawiki/2018/1/19/T--TJU_China--home2.jpg" /> |
| − | </div>
| + | </a> |
| − | <div class="figure">(a)sensitivity of ktx1 (b)sensitivity of ktl1</div>
| + | <a href="#"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/ 0/ 07/T--TJU_China-- home3. jpg" /> |
| | + | </a> |
| | + | <a href="#"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/c/c0/T--TJU_China--home4.jpg" /> |
| | + | </a> |
| | + | <a href="#"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/9/9c/T--TJU_China--home5.jpg" /> |
| | + | </a> |
| | + | <a href="#"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/4/49/T--TJU_China--home6.jpg" /> |
| | + | </a> |
| | + | <a href="#"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/e/ed/T--TJU_China--home7.jpg" /> |
| | + | </a> |
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| − | <div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/8/8a/T--TJU_China--tx2.png">
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| | </div> | | </div> |
| − | <div class="doublepic"> | + | <div class="btn_background"></div> |
| − | <img src="https://static.igem.org/mediawiki/2018/4/41/T--TJU_China--tl2.png"> | + | <div class="btn"> |
| − | </div>
| + | <ul> |
| − | </div>
| + | <li class="one"></li> |
| − | <div class="figure">(c)sensitivity of ktx2 (d)sensitivity of ktl2</div>
| + | <li></li> |
| | + | <li></li> |
| | + | <li></li> |
| | + | <li></li> |
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| − | <div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/1/1a/T--TJU_China--tx3.png">
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| − | </div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/3/30/T--TJU_China--tl3.png">
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| − | </div>
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| − | </div>
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| − | <div class="figure">(e)sensitivity of ktx3 (f)sensitivity of ktl3</div>
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| | | | |
| − | <div>
| + | </ul> |
| − | <div class="doublepic">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/0/03/T--TJU_China--tx4.png"> | + | |
| | </div> | | </div> |
| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/c/c9/T--TJU_China--b1.png">
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| − | </div>
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| − | </div>
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| − | <div class="figure">(g)sensitivity of ktx4 (h)sensitivity of kb1</div>
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| | | | |
| − | <div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/6/69/T--TJU_China--b2.png">
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| − | </div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/3/33/T--TJU_China--b3.png">
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| − | </div>
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| | </div> | | </div> |
| − | <div class="figure">(i)sensitivity of kb2 (j)sensitivity of kb3</div>
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| | | | |
| − | <div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/a9/T--TJU_China--b4.png">
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| − | </div>
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| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/2/2b/T--TJU_China--b5.png">
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| − | </div>
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| − | </div>
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| − | <div class="figure">(a)sensitivity of kb4 (b)sensitivity of kb5</div>
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| | | | |
| − | <div> | + | <div class="home_abstract_logo"></div> |
| − | | + | |
| − | <img src="https://static.igem.org/mediawiki/2018/e/e9/T--TJU_China--b6.png">
| + | <div class=" home_abstract"> |
| | + | <div class="home_abstract_head"> |
| | + | ABSTRACT |
| | </div> | | </div> |
| − | <div class="doublepic"> | + | <div class="home_abstract_word"> |
| − | <img src="https://static.igem.org/mediawiki/2018/5/5b/T--TJU_China--d1.png"> | + | This year, the CRISPR-Cas family is the protagonist in our story series. The old member, dCas9, |
| | + | is the enhancer for the heavy-metal detection based on E. coli, while the newbie, Cas12a, is a worker for the |
| | + | high-throughput cancer-related SNP detection chip. We have also built a "highway" for tracking and delivering |
| | + | the Cas9/sgRNA complex in mammalian cells, and we try to apply it to manipulate the mitochondrial genome. |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="figure">(c)sensitivity of kb6 (d)sensitivity of kd1</div>
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| | | | |
| − | <div> | + | <div class="home_medal"> |
| − | | + | <a href="https://2018.igem.org/Team:TJU_China/Medal"> |
| − | <img src="https://static.igem.org/mediawiki/2018/5/56/T--TJU_China--d2.png">
| + | <img class="home_medal_pic" src="https://static.igem.org/mediawiki/2018/7/70/T--TJU_China--medal.png"> </a> |
| − | </div>
| + | |
| − | <div class="doublepic">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/0/0a/T--TJU_China--d3.png">
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| − | <div class="figure">(e)sensitivity of kd2 (f)sensitivity of kd3</div> | + | <div class="home_achievements_logo"></div> |
| − | | + | <div class="home_achievements"> |
| − | <div> | + | <div class="home_achievements_head"> |
| − | <div class="doublepic"> | + | ACHIEVEMENTS |
| − | <img src="https://static.igem.org/mediawiki/2018/4/44/T--TJU_China--d4.png"> | + | |
| | </div> | | </div> |
| − | <div class="doublepic">
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| − | <img src="https://static.igem.org/mediawiki/2018/2/2f/T--TJU_China--d5.png">
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| − | </div>
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| − | </div>
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| − | <div class="figure">(g)sensitivity of kd4 (h)sensitivity of kd5</div>
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| | | | |
| − | <div>
| + | <div class="home_achievements_word"> |
| − | <div class="doublepic"> | + | <div> |
| − | <img src="https://static.igem.org/mediawiki/2018/e/e4/T--TJU_China--d6.png"> | + | Click the medals to see how we met |
| − | </div>
| + | <br> the iGEM medal requirements for 2018!</div> |
| − | <div class="doublepic">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/3/35/T--TJU_China--d7.png">
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="figure">(a)sensitivity of kd6 (b)sensitivity of kd7</div>
| |
| | | | |
| − | <div> | + | <div class="home_contact"> |
| − | <div class="doublepic"> | + | <div class="home_contact_icon1"> |
| − | <img src="https://static.igem.org/mediawiki/2018/c/c5/T--TJU_China--d8.png"> | + | <img style="max-width:100%;height:auto;" src="https://static.igem.org/mediawiki/2018/e/e7/T--TJU_China--tju_logo.png"> |
| | </div> | | </div> |
| − | <div class="doublepic"> | + | <div class="home_contact_word1"> |
| − | <img src="https://static.igem.org/mediawiki/2018/4/44/T--TJU_China--d9.png"> | + | <div>天津大学</div> |
| | + | <div>TianJin University</div> |
| | </div> | | </div> |
| − | </div>
| + | <div class=" home_contact_icon2"> |
| − | <div class=" figure" >(c)sensitivity of kd8 (d)sensitivity of kd9</div> | + | <img style="max-width:100%;height:auto;" src="https://static.igem.org/mediawiki/2018/d/d1/T--TJU_China--life_science_logo.png"> |
| − | | + | |
| − | <div>
| + | |
| − | <div class="doublepic">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/3/3d/T--TJU_China--d10.png">
| + | |
| | </div> | | </div> |
| − | <div class="doublepic"> | + | <div class="home_contact_word2"> |
| − | <img src="https://static.igem.org/mediawiki/2018/7/7a/T--TJU_China--d11.png"> | + | <div>天津大学生命科学学院</div> |
| | + | <div>School of life Sciences,</div> |
| | + | <div>TianJin University</div> |
| | </div> | | </div> |
| − | </div>
| |
| − | <div class="figure">(e)sensitivity of kd10 (f)sensitivity of kd11</div>
| |
| − | <div class="figure">Note:The ordinate axis represents the sensitivity S,and the abscissa axis is the parameter k for which we want to evaluate
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| − | the sensitivity.</div>
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| − | <div class="subtitle">2.5 Application of the model</div>
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| − | <div class="word">Since the goal of our project is to increase the sensitivity of biosensors by introducing a complex of dCas9-RNAP and
| |
| − | sgRNA, and one of the purposes of our model is to explore whether this complex is effective.So we assume a reasonable
| |
| − | and large enough concentration value for this complex. We use the concentration of Glyceraldehyde-3-phosphate dehydrogenase
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| − | A as the assumed concentration.Glyceraldehyde-3-phosphate dehydrogenase A(gapA) is a crucial enzyme in the glycolytic
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| − | pathway,and the gene encoding this enzyme is a housekeeping gene in E.coli cells with high expression levels.We find
| |
| − | in the literature that the protein mass of gapA is 48645 fg/cell,and its molecular weight is 35492 Da.[4] The amount
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| − | of abundance of Glyceraldehyde-3-phosphate dehydrogenase A protein per cell can be calculated as follows:
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| − | </div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/c/c3/T--TJU_China--m11.png">
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| − | </div>
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| − | <div class="word">As for the size of E.coli,we found relevant data from the literature,as the figure below shows.[5]</div>
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| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/a/a7/T--TJU_China--dc.png">
| |
| − | </div>
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| − | <div class="figure">Figure 8:Size of E.coli </div>
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| − | <div class="word">The volume of E.coli can be calculated as follows:</div>
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| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/2/2a/T--TJU_China--m12.png">
| |
| − | </div>
| |
| − | <div class="word">Then the concentration of Glyceraldehyde-3-phosphate dehydrogenase A protein in the cell can be determined:</div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/3/3a/T--TJU_China--m13.png">
| |
| − | </div>
| |
| − | <div class="word">With this concentration,we can get very nice results:</div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/d/d1/T--TJU_China--23.png">
| |
| − | </div>
| |
| − | <div class="figure">Figure 9:smURFP production with enough dCas9-RNAP:sgRNA</div>
| |
| − | <div class="word">Compared to the diagram without introducing dCas9-RNAP:sgRNA:</div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/0/0b/T--TJU_China--21.png">
| |
| − | </div>
| |
| − | <div class="figure">Figure 10:smURFP production within a reasonable time frame</div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/6/6c/T--TJU_China--22.png">
| |
| − | </div>
| |
| − | <div class="figure">Figure 11:smURFP production reached equilibrium but it takes a long time</div>
| |
| − | <div class="word">From these three figures, we can conclude that dCas9-RNAP:sgRNA does have the effect of promoting transcription and increasing
| |
| − | fluorescence intensity,thereby increasing sensitivity,as long as its concentration is sufficient.This result enhances
| |
| − | the confidence of the experimental group,and they need to try to improve the expression of dCas9-RNAP:sgRNA in E.coli
| |
| − | without having to doubt its role.
| |
| − | </div>
| |
| − | <div class="head">References</div>
| |
| − | <div class="word">[1] LA Pola-Lopez et al."Novel arsenic biosensor "POLA" obtained by a genetically modified E.coli bioreporter cell" .In:Sensors
| |
| − | and Actuators B:Chemical254(2018),pp.1061-1068.
| |
| − | <br>[2] Yves Berset et al."Mechanistic Modeling of Genetic Circults for ArsR Arsenic Regulation".In:ACS synthetic biology
| |
| − | 6.5(2017),pp.862-874.
| |
| − | <br>[3] Eyal Karzbrun et al."Coares-grained dynamics of protein synthesis in a cell-free system".In:Phtsical review letters
| |
| − | 106.4(2011),p.048104.
| |
| − | <br> [4] Yasushi Ishihama et al."Exponentially modified protein abundance index(emPAI) for estimation of absolute protein
| |
| − | amount in proteomics by the number of sequenced peptides per protein".In:Molecular E Cellular Proteomics 4.9(2005),pp.1265-1272.
| |
| − | <br>[5] Nili Crossman,Eliora Z Ron,and Conrad L Woldringh."Changes in cell dimensions during amino acid starvation of
| |
| − | Escherichia coli."In:Journal of bacteriology 152.1(1982),pp.35-41.
| |
| − | </div>
| |
| | | | |
| | + | <div class="home_copyright">@IGEM 2018 TJU_China.All Rights Reserved.丨Contact us:syq47xx@sina.cn丨(Designed by Peicheng Li)</div> |
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| − |
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| − | <div class="head">Construction of Free Energy Model</div>
| |
| − | <div class="subhead">Zheng Hu,Sherry Dongqi Bao
| |
| − | <br>TianJin University
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| − | <br>October 10,2018</div>
| |
| − | <div class="title">1 Introduction</div>
| |
| − | <div class="word">Nowadays,the analysis of cleavage possibility can be devided into two type,i,e.meta-empirical and empirical.For the first
| |
| − | one, people develop the various score function based on experiment data to evaluate if a sgDNA is good or bad.Correspondingly,the
| |
| − | other group chooce set up a theoretical model based on kinetic theory.But because using many approximations,it has
| |
| − | drawbacks inevitably.
| |
| − | <br>Our model aims to investigate the off-target problem in gene editing by the CRISPR-Cas system,therefore finding efficient
| |
| − | ways to enhance the reliability of gene editing.The foundations of thsi model are mostly simple probability theory
| |
| − | and dynamic deduction,which make our model both convincing and pellucid.
| |
| − | <br>Currently,people have constructed a similar model as illustrated in the following figure1.There are four common rules
| |
| − | when Cas nuclease cleaves the DNA[1].
| |
| − | </div>
| |
| − | <div class="pic">
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| − | <img src="https://static.igem.org/mediawiki/2018/4/4f/T--TJU_China--z1.png">
| |
| − | </div>
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| − | <div class="figure">Figure q:schematic diagram</div>
| |
| − | <div class="word">(1)Seed region:single mismatch(es) within a PAM proximal seed region can completely disrupt interference.
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| − | <br> (2)Mismatch spread:when mismatches are outside the seed region,off-targets with spread out mismatches are targeted
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| − | most strongly.
| |
| − | <br> (3)Differential binding versus differential cleavage:binding is more tolerant of mismatched than cleavage.
| |
| − | <br>(4)Specificity-efficiency decoupling:weakened protein-DNA interatctions can improve target selectivity while still
| |
| − | maintaining efficiency.
| |
| − | <br>Based on these four rules,probability theory is applied in to explain it.As we know,there are always only two results
| |
| − | in an experiment,which are successful cleavage and unsuccessful cleavage.In math view,it can be one-hot encoded,and
| |
| − | they are corresponding to 1 and 0.
| |
| − | </div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/d/d9/T--TJU_China--z2.png">
| |
| − | </div>
| |
| − | <div class="figure">Figure 2</div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/4/44/T--TJU_China--z3.png">
| |
| − | </div>
| |
| − | <div class="figure">Figure 3</div>
| |
| − | <div class="word">However,giving a 0/1 prediction is hard and unreliable.To solve this problem, one choice is to consider it as a cluster
| |
| − | problem;however,it is easier to find a continuous quantitative function rather than to find a suitable cluster distance
| |
| − | function.Sonaturally,finding an approximate probability distribution is a good choice.
| |
| − | <br> In many target design toolkits,they use a score function with several param eters which can generate a score to
| |
| − | evaluate whether the target is good or bad. Here we consider the score function has the similar ability to probability,which
| |
| − | is a description of ”better” or ”worse” while can’t affirm whether successful cleavage willappear.For our case,our
| |
| − | goal is to find a function indicating which target is BETTER.
| |
| − | <br> Considering the difference between model prediction and experimental data,our model consists of two aspects,which
| |
| − | are kinetic inference and an updating module.
| |
| − | </div>
| |
| − | <div class="title">2 Methods</div>
| |
| − | <div class="subtitle">2.1 Knietic module</div>
| |
| − | <div class="word">Figure 2 shows that the whole binding-cleavage process begins with the bind ing between PAM andprotein.Therefore,it corresponds
| |
| − | to rule1 mentioned before.And as the reaction proceeds,every step of it is reversible,and its irre versibility mainly
| |
| − | depends on the binding energy of two DNA bases. The boundary probability Pclv;N,representing the probability of matching
| |
| − | at the Nth position(the last position of sgRNA) of nucleotide base,is given by:
| |
| − | </div>
| |
| − | <div class="pic">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/9/92/T--TJU_China--m14.png">
| |
| | </div> | | </div> |
| | | | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/d/df/T--TJU_China--z4.png"></div> | + | <script src="https://2018.igem.org/Template:TJU_China/jquery-3.0.0.min_js?action=raw&ctype=text/javascript"></script> |
| − | <div class="figure">Figure 4</div>
| + | <script src="https://2018.igem.org/Template:TJU_China/home_js?action=raw&ctype=text/javascript"></script> |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/f/f7/T--TJU_China--z5.png"></div>
| + | |
| − | <div class="figure">Figure 5</div>
| + | |
| − | <div class="word">Where k is the reaction rate constant; f represents the forward reactions;b represents the backward reaction.And </div>
| + | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/e/ec/T--TJU_China--zm1.PNG"></div>
| + | |
| − | <div class="word">So for a complete match:</div>
| + | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/0/00/T--TJU_China--zm2.png"></div>
| + | |
| − | <div class="word">Consider the rate constant $K_f(i)$ and #k_b(i)$:</div>
| + | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/b/b2/T--TJU_China--zm3.png"></div>
| + | |
| − | <div class="word">where $F_i$ means free energy of each metastable state,$T_{i,i+1}$means the highest free energy point on the reaction path from position i | + | |
| − | to position i+1.Therefore,$T_{i,i+1}$-$F_i$ is the activation energy of forward reaction and $T_{i,i+1}$-$F_i$ is activation energy of the backward reaction.
| + | |
| − | </div>
| + | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/0/02/T--TJU_China--zm4.png"></div>
| + | |
| − | <div class="word">We define</div>
| + | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/f/fe/T--TJU_China--zm5.png"></div>
| + | |
| − | <div class="word">So</div>
| + | |
| − | <div class="pic"><img src="https://static.igem.org/mediawiki/2018/9/93/T--TJU_China--zm6.png"></div>
| + | |
| − | <script src="https://2018.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML"></script>
| + | |
| − | <script type="text/x-mathjax-config">
| + | |
| − | MathJax.Hub.Config({tex2jax: {inlineMath: [['$','$'], ['\\(','\\)']]}});
| + | |
| − | </script>
| + | |
| | </body> | | </body> |
| − | <!-- <div>
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| − | <div class="pic"><img src=""></div>
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| − | <div class="word"></div>
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| − | <div class="figure">Figure </div>
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| − | <div class="title"></div>
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| − | <div class="subtitle"></div>
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| − |
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| − |
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| − |
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| − | $P_{arsR_{d}}$
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| − |
| |
| − | $As^{3+}$
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| − | <div class="equation"> \(P_{J23104} \xrightarrow {k_{tx1}} P_{J23104} + mRNA_{ArsR}\)</div> <div class="number">(1)</div>
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| − | </div> -->
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| | </html> | | </html> |
