Difference between revisions of "Team:JiangnanU China/Model"

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        #HQ_page h1, h2, h3, h4, h5 {
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2019.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2019.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2019.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1> Modeling</h1>
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<p>Mathematical models and computer simulations provide a great way to describe the function and operation of BioBrick Parts and Devices. Synthetic Biology is an engineering discipline, and part of engineering is simulation and modeling to determine the behavior of your design before you build it. Designing and simulating can be iterated many times in a computer before moving to the lab. This award is for teams who build a model of their system and use it to inform system design or simulate expected behavior in conjunction with experiments in the wetlab.</p>
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<h3> Gold Medal Criterion #3</h3>
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Convince the judges that your project's design and/or implementation is based on insight you have gained from modeling. This could be either a new model you develop or the implementation of a model from a previous team. You must thoroughly document your model's contribution to your project on your team's wiki, including assumptions, relevant data, model results, and a clear explanation of your model that anyone can understand.
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        }
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<br><br>
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The model should impact your project design in a meaningful way. Modeling may include, but is not limited to, deterministic, exploratory, molecular dynamic, and stochastic models. Teams may also explore the physical modeling of a single component within a system or utilize mathematical modeling for predicting function of a more complex device.
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<h3>Best Model Special Prize</h3>
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To compete for the <a href="https://2019.igem.org/Judging/Awards">Best Model prize</a>, please describe your work on this page  and also fill out the description on the <a href="https://2019.igem.org/Judging/Judging_Form">judging form</a>. Please note you can compete for both the Gold Medal criterion #3 and the Best Model prize with this page.
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<br><br>
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You must also delete the message box on the top of this page to be eligible for the Best Model Prize.
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            <div class="centers">
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                <div class="fb_72">
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                    <b>Model</b>
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                </div>
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            </div>
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        </div>
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    </div>
 
</div>
 
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            <div class="fb_72"><b>Introduction</b></div>
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            <br/>
 +
            <div class="fm_22">
 +
                1. The stakeholders informing us that benefits matter most in application, we realized that it's
 +
                essential to make our anti-phage strains grow as robust as original strain under the current
 +
                fermentation conditions. However, it's difficult to tell which strain grows most suitable or which
 +
                device has the least impact on cell growth from the growth curve graph of all those strains (Figure 1).
 +
                Therefore, we established a mathematical model to evaluate the growth properties of strains containing
 +
                different parts for the most suitable device in future application.
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            </div>
  
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            <div class="split_small"></div>
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            <div align="center">
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            <img src="https://static.igem.org/mediawiki/2019/f/f9/T--JiangnanU_China--model_1.png" width="80%"
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            <div class="fm_22">
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<div class="highlight decoration_A_full">
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                2. The phage-induced promoters are vital in our genetic circuit, which would response the phage
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<h3> Inspiration </h3>
+
                stimulation and start transcription of <i>antP</i> and P-1 (antimicrobial peptide) against the phage
−
<p>
+
                infection
−
Here are a few examples from previous teams:
+
                (Figure 2). And the phage-induced promoters we used were selected from <i>E. coli</i>, so there might be
−
</p>
+
                some
−
<ul>
+
                potential problems such as leakage and inclusion body due to the inappropriate promoter strength. Thus
−
<li><a href="https://2018.igem.org/Team:GreatBay_China/Model">2018 GreatBay China</a></li>
+
                we developed a quantitative design method for phage-induced promoters based on strength prediction using
−
<li><a href="https://2018.igem.org/Team:Leiden/Model">2018 Leiden</a></li>
+
                artificial neural network, which allows us to choose or design promoters with desired strength without
−
<li><a href="https://2016.igem.org/Team:Manchester/Model">2016 Manchester</a></li>
+
                extra experiments.
−
<li><a href="https://2016.igem.org/Team:TU_Delft/Model">2016 TU Delft</li>
+
            </div>
−
<li><a href="https://2014.igem.org/Team:ETH_Zurich/modeling/overview">2014 ETH Zurich</a></li>
+
 
−
<li><a href="https://2014.igem.org/Team:Waterloo/Math_Book">2014 Waterloo</a></li>
+
            <div class="split_small"></div>
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</ul>
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            <img src="https://static.igem.org/mediawiki/2019/e/e0/T--JiangnanU_China--model_2.png" width="100%"
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</div>
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                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="fb_48">
 +
                <b>Assumption</b>
 +
            </div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                (1) The promoter strengths in the training set are the same using various vectors.
 +
                <br/>
 +
                (2) The original strain growth properties were most suitable for fermentation industries.
 +
                <br/>
 +
                (3) The impact of parts in cell growth are relatively the same under different situation.
 +
            </div>
 +
 
 +
            <div class="split"></div>
 +
            <div class="fb_72"><b>GRA_EWM Model</b></div>
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Symbol Description</b></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/7/72/T--JiangnanU_China--model_21.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Weights Calculation of The Growth Curve[1]</b></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/a/a0/T--JiangnanU_China--model_12.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div align="center">
 +
            <img src="https://static.igem.org/mediawiki/2019/d/da/T--JiangnanU_China--model_5.png" width="65%"
 +
                height="auto" align:center></div>
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Analysis of The Weight Distribution</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                As showed in Figure 3, the 4OD600 measured after 4 to 10 hours were more useful. However, the
 +
                weight
 +
                given by the experts might consider plateau phase as an important period for industry fermentation.
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Grey Relational Analysis for Picking The Most Suitable Strain[2]</b></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/b/b9/T--JiangnanU_China--model_13.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div align="center">
 +
            <img src="https://static.igem.org/mediawiki/2019/1/1b/T--JiangnanU_China--model_7.png" width="70%"
 +
                height="auto" align:center></div>
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Conclusion</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                Figure 4 demonstrates that the impact of the distinguishing coefficient on the result of GRA is very
 +
                significant. In particular, for all tested distinguishing coefficients, part <i>gntR</i> always ranks
 +
                first,
 +
                which means it is the most suitable part for future application.
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_72"><b>BP-ANN Model</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                Accurate and controllable regulatory elements like promoters are indispensable tools to quantitatively
 +
                regulate gene expression for rational pathway engineering, which means a promoter with proper strength
 +
                might be an easy solution to leakage and inclusion body. In order to select or design promoters with
 +
                desired strength without experiments, we developed a quantitative strength prediction method[3] using
 +
                artificial neural network (ANN) with Neural Network Toolbox under Matlab environment.
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Construction of Early Phage-induced Promoter Strength Library</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                The endogenous promoters in <i>E.coli</i> BL21(DE3) we selected to respond to phage infection might
 +
                start
 +
                transcription without phage stimulation, so we selected 19 promoters with distributed strength from T4
 +
                phage mentioned in previous study as the phage-induced promoter library. [4-5](attachment: T4PE.docx)
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/d/de/T--JiangnanU_China--model_15.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="fm_22">
 +
                The relative promoter strength is determined as the quotient of the β-lactamase divided by the
 +
                6-phospho-β-galactosidase activity. This ratio then is normalized to that obtained from a clone
 +
                harboring P46.7.
 +
                Reference in all promoter strength measurements), resulting in the pKWIII unit.
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Computational platform construction</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                Matlab2019b (Mathworks Inc., http://www.mathworks.com/) ran on a personal laptop with Windows 10
 +
                64-bit(Microsoft Inc., http://www.microsoft.com/) operation system. Neural Network Toolbox within Matlab
 +
                served as the basic tool for artificial neural network (ANN) model construction, data fitting and
 +
                prediction. All programs used in this work were designed and run upon Neural Network Toolbox and Matlab
 +
                environment.
 +
            </div>
 +
 
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Construction And Training of ANN Predicting Models</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                The initial BP-ANN model was built by Neural Network Toolbox. The model contains four layers, including
 +
                an input layer, an output layer and two hidden layer. Neuron numbers of two hidden layer and a output
 +
                layer were 26, 5, 1 respectively. The initial weight for all neuron connections were randomly assigned
 +
                by Matlab functions. And other parameters were set as followed:
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/f/f7/T--JiangnanU_China--model_14.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="fm_22">
 +
                net = newff(minmax(p),[26,5,1],{'tansig', 'tansig', 'purelin'},'trainlm');
 +
                net.trainParam.epochs = 15000;
 +
                net.trainParam.mc = 0.98;
 +
                net.trainParam.goal = 1e-6;
 +
                net.trainParam.lr = 0.01;
 +
                And the original sequence data were translated to digital data and served as the input matrix according
 +
                to the following rules via python program (attachment: seqDigtal.py):
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/3/35/T--JiangnanU_China--model_9.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="fm_22">
 +
                Among all generated models, NET_26_5 (attachment: NET_26_5.mat) shows the highest correlation
 +
                coefficient values of 0.93336 for test set prediction, and its correlation coefficient values for
 +
                fitting the training data set reaches up to 0.99104. (Figure 3)
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div align="center">
 +
            <img src="https://static.igem.org/mediawiki/2019/6/65/T--JiangnanU_China--model_10.png" width="70%"
 +
                height="auto" align:center></div>
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="fm_22">
 +
                And all the promoter prediction strengths were showed in Figure 4. (attachment:
 +
                pStrengthVsStrength.xlsx)
 +
            </div>
 +
 
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/5/5d/T--JiangnanU_China--model_16.png" width="100%"
 +
                height="auto" alt="">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div align="center">
 +
            <img src="https://static.igem.org/mediawiki/2019/3/32/T--JiangnanU_China--model_11.png" width="60%"
 +
                height="auto" align:center></div>
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48"><b>Conclusion</b></div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                We could predict the designed promoter strength based on this BP-ANN model, which could save a lot time
 +
                for us to test millions of designed promoters.
 +
            </div>
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                [1]. LI X, WANG K, LIU L, et al. Application of the Entropy Weight and TOPSIS Method in Safety
 +
                Evaluation of Coal Mines [J]. Procedia Engineering, 2011, 26(4): 2085-2091.
 +
                <br/>
 +
                [2]. KUO Y, YANG T, HUANG G W. The use of grey relational analysis in solving multiple attribute
 +
                decision-making problems [J]. Computers & Industrial Engineering, 2008, 55(1): 80-93.
 +
                <br/>
 +
[3] Meng H, Wang J, Xiong Z, Xu F, Zhao G, Wang Y: Quantitative design of regulatory elements based on
 +
                high-precision strength prediction using artificial neural network. PLoS One 2013, 8(4):e60288.
 +
<br/>
 +
                [4] Liebig HD, Ruger W: Bacteriophage T4 early promoter regions. Consensus sequences of promoters and
 +
                ribosome-binding sites. J Mol Biol 1989, 208(4):517-536.
 +
<br/>
 +
                [5] Wilkens K, Ruger W: Characterization of bacteriophage T4 early promoters in vivo with a new promoter
 +
                probe vector. Plasmid 1996, 35(2):108-120.
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                <br/>
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Latest revision as of 22:49, 21 October 2019

JiangNan

Introduction

1. The stakeholders informing us that benefits matter most in application, we realized that it's essential to make our anti-phage strains grow as robust as original strain under the current fermentation conditions. However, it's difficult to tell which strain grows most suitable or which device has the least impact on cell growth from the growth curve graph of all those strains (Figure 1). Therefore, we established a mathematical model to evaluate the growth properties of strains containing different parts for the most suitable device in future application.
2. The phage-induced promoters are vital in our genetic circuit, which would response the phage stimulation and start transcription of antP and P-1 (antimicrobial peptide) against the phage infection (Figure 2). And the phage-induced promoters we used were selected from E. coli, so there might be some potential problems such as leakage and inclusion body due to the inappropriate promoter strength. Thus we developed a quantitative design method for phage-induced promoters based on strength prediction using artificial neural network, which allows us to choose or design promoters with desired strength without extra experiments.
Assumption

(1) The promoter strengths in the training set are the same using various vectors.
(2) The original strain growth properties were most suitable for fermentation industries.
(3) The impact of parts in cell growth are relatively the same under different situation.
GRA_EWM Model
Symbol Description
Weights Calculation of The Growth Curve[1]
Analysis of The Weight Distribution

As showed in Figure 3, the 4OD600 measured after 4 to 10 hours were more useful. However, the weight given by the experts might consider plateau phase as an important period for industry fermentation.
Grey Relational Analysis for Picking The Most Suitable Strain[2]
Conclusion

Figure 4 demonstrates that the impact of the distinguishing coefficient on the result of GRA is very significant. In particular, for all tested distinguishing coefficients, part gntR always ranks first, which means it is the most suitable part for future application.
BP-ANN Model

Accurate and controllable regulatory elements like promoters are indispensable tools to quantitatively regulate gene expression for rational pathway engineering, which means a promoter with proper strength might be an easy solution to leakage and inclusion body. In order to select or design promoters with desired strength without experiments, we developed a quantitative strength prediction method[3] using artificial neural network (ANN) with Neural Network Toolbox under Matlab environment.
Construction of Early Phage-induced Promoter Strength Library

The endogenous promoters in E.coli BL21(DE3) we selected to respond to phage infection might start transcription without phage stimulation, so we selected 19 promoters with distributed strength from T4 phage mentioned in previous study as the phage-induced promoter library. [4-5](attachment: T4PE.docx)
The relative promoter strength is determined as the quotient of the β-lactamase divided by the 6-phospho-β-galactosidase activity. This ratio then is normalized to that obtained from a clone harboring P46.7. Reference in all promoter strength measurements), resulting in the pKWIII unit.
Computational platform construction

Matlab2019b (Mathworks Inc., http://www.mathworks.com/) ran on a personal laptop with Windows 10 64-bit(Microsoft Inc., http://www.microsoft.com/) operation system. Neural Network Toolbox within Matlab served as the basic tool for artificial neural network (ANN) model construction, data fitting and prediction. All programs used in this work were designed and run upon Neural Network Toolbox and Matlab environment.
Construction And Training of ANN Predicting Models

The initial BP-ANN model was built by Neural Network Toolbox. The model contains four layers, including an input layer, an output layer and two hidden layer. Neuron numbers of two hidden layer and a output layer were 26, 5, 1 respectively. The initial weight for all neuron connections were randomly assigned by Matlab functions. And other parameters were set as followed:
net = newff(minmax(p),[26,5,1],{'tansig', 'tansig', 'purelin'},'trainlm'); net.trainParam.epochs = 15000; net.trainParam.mc = 0.98; net.trainParam.goal = 1e-6; net.trainParam.lr = 0.01; And the original sequence data were translated to digital data and served as the input matrix according to the following rules via python program (attachment: seqDigtal.py):
Among all generated models, NET_26_5 (attachment: NET_26_5.mat) shows the highest correlation coefficient values of 0.93336 for test set prediction, and its correlation coefficient values for fitting the training data set reaches up to 0.99104. (Figure 3)
And all the promoter prediction strengths were showed in Figure 4. (attachment: pStrengthVsStrength.xlsx)
Conclusion

We could predict the designed promoter strength based on this BP-ANN model, which could save a lot time for us to test millions of designed promoters.
[1]. LI X, WANG K, LIU L, et al. Application of the Entropy Weight and TOPSIS Method in Safety Evaluation of Coal Mines [J]. Procedia Engineering, 2011, 26(4): 2085-2091.
[2]. KUO Y, YANG T, HUANG G W. The use of grey relational analysis in solving multiple attribute decision-making problems [J]. Computers & Industrial Engineering, 2008, 55(1): 80-93.
[3] Meng H, Wang J, Xiong Z, Xu F, Zhao G, Wang Y: Quantitative design of regulatory elements based on high-precision strength prediction using artificial neural network. PLoS One 2013, 8(4):e60288.
[4] Liebig HD, Ruger W: Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites. J Mol Biol 1989, 208(4):517-536.
[5] Wilkens K, Ruger W: Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector. Plasmid 1996, 35(2):108-120.
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