Difference between revisions of "Team:ZJU-China/Project/voc"

 
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                               <li><a href="https://2017.igem.org/Team:ZJU-China/Project/conclusion">Conclusions</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Project/conclusion">Conclusions</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Notebook">Notebook</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Notebook">Notebook</a></li>
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                              <li><a href="https://2017.igem.org/Team:ZJU-China/Protocols">Protocols</a></li>
 
                           </ul>
 
                           </ul>
 
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         <p class="bs-docs-section">
 
         <p class="bs-docs-section">
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        <h1 id="voc" class="page-header ArticleHead GreenAH">VOC sensor</h1>
 +
        <h2 id="overview" class="H2Head">Overview</h2>
  
 +
        <p class="PP">In our project, we are exploring a new way to sense the health condition of  crops which will further send this information (healthy or not) to both human and our <em>T.atroviride</em>. In this way, we can take actions on time and achieve the goal of prevention some phytopathogens in advance. Meanwhile, this method should be conveniently used in variety range of plants.</p>
 +
        <p class="PP">We finally build up an e-nose (electronic nose) system to sense the VOC (volatile organic compounds) emitted by the plants. Afterwards, using machine learning method, we achieved a high accuracy rate, which is over 85%, when sensing the tobaccos infected by <em>P.nicotianae</em>. That is to say: our device successfully distinguishes whether the tobaccos are ill by "smelling them".</p>
  
         <h1 id="bbp" class="ArticleHead GreenAH">Best Basic Part</h1>
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         <h2 id="background" class="H2Head">Background</h2>
        <p class="PP">For this year’s iGEM competition, our team has chosen to present Calcineurin-dependent response element (CDRE) (BBa_K2207021)and Mature serine protein from <em>Paecilomyces lilacinus</em> (BBa_K2207023) for the award of the basic part.</p>
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         <p class="PP">Plants will release a variety of volatile organic compounds (VOC) to resist some infected phytopathogens, which can lead to a change of VOC composition around the plants[1]. Firstly, we verified the VOC differences between healthy tobaccos and tobaccos infected by  <em>P.nicotianae</em> by GC-MS.</p>
        <h2 class="H2Head" id="cdre">CDRE</h2>
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         <p class="PP">CDRE,calcineurin-dependent response element,is a segment of DNA sequence which can be regulated by specific transcription factor. This TF is activated by calcineurin in the present of calcium ion. To create a promoter can be up-regulated by calcium influx,we replaced the upstream activating sequence(UAS) of CYC1 promoter with 4 CDREs. This promoter is designed for Saccharomyces cerevisiae and it depends on S.cerevisiae's endogenous calcineurin and calmodulin<sup>[1][2]</sup>. We choose mRFP as the report gene. We cultured the yeast in calcium-inducing medium and uninduced medium which contains a relatively low concentration of calcium ion (1xYPD medium).</p>
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        <p class="PP">Obviously, the transgenic yeast cultured in the calcium-inducing medium(200mM Ca<sup>2+</sup>) is turning red while the control shows no significant changes. Then we detected the fluorescence of this two cultures. To eliminate the influence of the concentration of the yeast, we calculated the value of Fluorescent Intensity/OD600 to evaluate these two groups.</p>
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        <p class="PP">The value indicated that the fluorescence intensity of the calcium-induced group has improved about 147%. It’s not quite a huge change and the uninduced group has already shown a great intensity of fluorescence. Then we detected the intracellular calcium ion concentration with Fluo 4-AM whose fluorescent intensity can represent the relative concentration of calcium ion.</p>
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         <div class="imgdiv col-md-6 col-sm-6"><img class="textimg" style="height: 300px !important; width:auto !important;" src="https://static.igem.org/mediawiki/2017/1/19/ZJU_China_MWF_Rplot05.jpeg"></div>
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         <div class="imgdiv"><img class="textimg" style="width: 90% !important;" src="https://static.igem.org/mediawiki/2017/8/8e/ZJU_China_VOCsensor_new1.png"></div>
         <div class="imgdiv col-md-6 col-sm-6"><img class="textimg" style="height: 300px !important; width:auto !important;"  src="https://static.igem.org/mediawiki/2017/e/ea/ZJU_China_MWF_Rplot03.jpeg"></div>
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         <p class="capture">Fig.1 GC-MS analysis of VOCs emitted by healthy tobaccos and tobaccos infected by <em>P.nicotianae</em></p>
         <p class="capture">Fig.1 (a) Relative fluorescent intensity of two groups. (b) Relative calcium content in two groups</p>
+
         <p class="capture">(Thanks Hunan Tobaccos Bureau for providing this data).</p>
  
         <p class="PP"> As the result shows, 8% change of the intracellular ion concentration can contribute to 147% higher expression level of the downstream gene. It seems that our CDRE promoter is much more sensitive than we expected.</p>
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         <p class="PP">We decide to use ten highly sensitive CMOS(Complementary Metal Oxide Semiconductor) gas detector to catch the VOC information, each detector is sensitive to one kind or type of VOCs. E-nose can’t sense a specific VOC like mass spectrometers did. instead, it catches the overall characteristics of VOCs as "fingerprint".</p>
  
 +
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/1/1c/ZJU_China_VOCsensor_2.jpg"></div>
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        <p class="capture">Fig.2 A photo of our gas sensor chamber.</p>
  
         <h2 class="H2Head" id="msp">Mature serine protein</h2>
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         <p class="PP">Our device is controlled by Arduino Single Chip Microcomputer(SCM) in order to achieve automatic measurement, and we standardize the testing steps to ensure the data between every groups is comparable.</p>
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        <!--<div class="imgdiv col-md-6 col-sm-6"><img style="height: 330px !important; width: auto !important;" class="textimg" src="https://static.igem.org/mediawiki/2017/9/9f/ZJU_China_VOCSensor_s1.jpg"></div>-->
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        <!--<div class="imgdiv col-md-6 col-sm-6"><img style="height: 330px !important; width: auto !important;" class="textimg" src="https://static.igem.org/mediawiki/2017/b/bc/ZJU_China_VOCSensor_s2.jpg"></div>-->
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        <div class="imgdiv col-md-4 col-sm-8"><img class="textimg" style="height: 310px !important; width: auto !important;" src="https://static.igem.org/mediawiki/2017/e/ec/ZJU_China_VOCsensor_3.png"></div>
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        <div class="imgdiv col-md-8 col-sm-8"><img class="textimg" style="height: 320px !important; width: auto !important;" src="https://static.igem.org/mediawiki/2017/9/90/ZJU_China_VOCsensor_30.png"></div>
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        <p class="capture">Fig.3 The response curve of gas sensors (from ill tobacco)and the stability curve (40min in air)</p>
  
         <p class="PP">Root-knot nematodes (Meloidogyne spp.), which are one of the most destructive nematodes, cause the loss of crop about 10%, serious as high as 75%. Like the insect cuticlehe, nematode eggshell consists mainly of proteins and chitins. The egg-parasitic fungus <em>P.lilacinum</em> secretes protease and chitinase to hydrolyze the nematode eggshell, so that the root knot nematodes cannot grow normally<sup>[3]</sup>. Among them, serine protases plays an important role in hydrolyzing the eggshell of root-knot nematodes.</p>
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         <p class="PP">We use high purity air to blow out the gas out of the box into the gas sensor chamber.</p>
        <p class="PP">The gene which can express serine protease in yeast was synthesized by Genscript. Before synthesizing this gene, we did codon optimization based on the codon preference of yeast and added a flag-tag to the N-terminal of the serine protease.After extracting the whole proteins of the yeast which transferred plasmid successfully, we performed western-blot and checked the serine protein was expressed in the yeast. (Result is as follow) The band was very shallow, in other words, the concentration of the serine protease was very low.</p>
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         <div class="imgdiv"><img class="textimg" style="width: 80% !important;" src="https://static.igem.org/mediawiki/2017/d/d9/ZJU_China_ZC3in1.jpg"></div>
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/f/fe/ZJU_China_temp_1.png"></div>
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         <p class="capture">Fig.4 An illustration of our standardized measuring steps</p>
        <p class="capture">Fig.2 Western-blot result</p>
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        <p class="PP">The band with red circle was the band of the serine protease and the marker was a protein marker of aidlab.</p>
+
        <p class="PP">In order to test whether the serine protease could work normally in the yeast, we performed the enzyme activity detection using BAEE solution. We did two sets of experiments: one added PMSF, which was a inhibitor of serine protease, and the other did not. And then, reading the OD253 of these two solutions.(You can know more details about the detection from the protocol) Obviously, the OD253 of the former one is higher than the later one and the values of OD253 increased with time in a period of time ; therefore, we made the conclusion that the yeast produced the serine protase successfully and effectively.</p>
+
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/8/83/ZJU_China_temp_2.png"></div>
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        <p class="capture">Fig.3 The values of OD253 increased with time</p>
+
        <p class="PP">The values of OD253 increased with time and this solution did not add the PMSF.</p>
+
         <div class="imgdiv"><img class="textimg" style="width: 40% !important;" src="https://static.igem.org/mediawiki/2017/9/9c/ZJU_China_temp_3.png"></div>
+
         <p class="capture">Fig.4 A box-plot of the OD253</p>
+
        <p class="PP">These were the OD253 of the solutions after a period of reaction.The red one was the solution that did not add the PMSF; the blue one was the solution that added. Obviously, the OD253 of the former one is higher than the later one, so that, we could say that the yeast produced the serine protase successfully and effectively.</p>
+
  
         <p class="PP">This year we submitted several well-working basic parts to the BioBrick Registry.</p>
+
         <p class="PP"><a class="cite" href="https://2017.igem.org/Team:ZJU-China/Hardware/Device">Click here</a> to see more information about our device.</p>
        <p class="PP" style="text-align: center !important;"><strong>Table of the Basic Parts we submitted to the BioBrick Registry</strong></p>
+
        <p class="PP">We build median filter algorithm in SCM to remove some outliers. Then, we preprocess the raw data after getting them: First, we identify and remove the base line value; Then, we read the response value on 1min, 2 min, the integral average value and maximum value of the response curve.</p>
        <table class="table">
+
        <p class="PP">We have measured 17 groups of healthy tobaccos and 18 groups of infected ones. Click to download our <a class="cite" href="https://static.igem.org/mediawiki/2017/2/25/ZJU_China_Hardware_rawdata.xlsx">raw data</a> and <a class="cite" href="https://static.igem.org/mediawiki/2017/f/fb/ZJU_China_Hardware_preprocess.xlsx">preprocessed data</a>.</p>
            <tr><th class="yellowTable">Name</th><th class="yellowTable">Type</th><th class="yellowTable">Description</th><th class="yellowTable">Designer</th><th class="yellowTable">Length(bp)</th><th class="yellowTable">Submitted</th></tr>
+
            <tr><th class="grayTable">BBa_K2207003</th><td>Composite Parts</td><td>Trichoderma HR System I SOD-Tubulin dobule promoter</td><td>Yihe Zhang</td><td>3155</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207004</th><td>Composite Parts</td><td>Trichoderma HR System II Ech42-H3 double promoter</td><td>Yihe Zhang</td><td>4845</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207005</th><td>Composite Parts</td><td>Trichoderma HR System III L1-ADH1-RP27-L2</td><td>Yihe Zhang</td><td>903</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207006</th><td>Composite Parts</td><td>Trichoderma HR System IV L3-ADH1-RP27-L4</td><td>Yihe Zhang</td><td>903</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207007</th><td>Composite Parts</td><td>Trichoderma HR System V Homologous Binding SiteA</td><td>Yihe Zhang</td><td>1676</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207008</th><td>Composite Parts</td><td>Trichoderma HR System VI Homologous Binding SiteB</td><td>Yihe Zhang</td><td>1650</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207009</th><td>Coding Sequences</td><td>PhlF transcription factor</td><td>Qianjin Jiang</td><td>627</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207010</th><td>Other</td><td>Phl Operator</td><td>Yuxing Chen</td><td>825</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207021</th><td>Promoters</td><td>Calcineurin-dependent response element (CDRE)</td><td>Shisheng Li</td><td>474</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207023</th><td>Coding Sequences</td><td>Mature serine protein from Paecilomyces lilacinus</td><td>Zifan Xie</td><td>882</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207024</th><td>Promoters</td><td>T7-mutant1</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207025</th><td>Promoters</td><td>T7-mutant2</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207026</th><td>Promoters</td><td>T7-mutant3</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207027</th><td>Promoters</td><td>T7-mutant4</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207028</th><td>Promoters</td><td>T7-mutant5</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207029</th><td>Promoters</td><td>T7-mutant6</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207030</th><td>Promoters</td><td>T7-mutant7</td><td>Yuxing Chen</td><td>42</td><td>✔</td></tr>
+
            <tr><th class="grayTable">BBa_K2207031</th><td>Coding Sequences</td><td>PhlE efflux pump</td><td>Junming Qian</td><td>1272</td><td>✔</td></tr>
+
        </table>
+
        <p class="capture">Table.OD600 reference point</p>
+
  
 +
        <div class="imgdiv"><img class="textimg" style="width: 80% !important;" src="https://static.igem.org/mediawiki/2017/d/de/ZJU_China_VOCsensor_5.png"></div>
 +
        <p class="capture">Fig.5 The integral average value of 35 response curve.</p>
  
        <h2 class="H2Head" id="ref">Reference<hr></h2>
+
         <p class="PP">After preprocessing the data, we apply some algorithms to do the classification work. We employ Decision Tree, Multi-Layer Perception algorithm, and Leaner Model. Finally, we achieve more than 85% accuracy rate of distinguishing whether the tobaccos are infected by <em>P.nicotianae</em>.</p>
         <p class="PP ref">[1] Cyert M S. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[J]. Biochemical and biophysical research communications, 2003, 311(4): 1143-1150.</p>
+
         <p class="PP">Moreover, based on the result of the modeling, 4 CMOS detectors are enough to make a judgement for the health condition of tobaccos, which means we can further reduce the cost of our device.</p>
         <p class="PP ref">[2] Cyert M S. Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae[J]. Annual review of genetics, 2001, 35(1): 647-672.</p>
+
         <p class="PP"><a class="cite" href="https://2017.igem.org/Team:ZJU-China/Model">Click here</a> to see the details of our modeling process.</p>
         <p class="PP ref">[3] Brand D, Roussos S, Pandey A, et al. Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita.[J]. Applied Biochemistry & Biotechnology, 2004, 118(1-3):81-88.</p>
+
  
         <br><br><br>
+
         <h2 id="conclusion" class="H2Head">Conclusions</h2>
 +
        <p class="PP">Our method achieves more than 85% accuracy rate on distinguish the health state of tobaccos, and we find we only need four detectors for our devices to make such judgments. Moreover, the VOC data can be sent to PC port, we also build a web app to achieve real time monitoring. In the future, we plan to make the web app possible to show the healthy condition of plants in real time.</p>
 +
        <p class="PP">On the other hand, the sensitivity of our device is still limited when facing real world conditions. It is hard to fit different  conditions in different field. Therefore, we come up with a plan to solve this problem, <a class="cite" href="https://2017.igem.org/Team:ZJU-China/Hardware/Improvements">Click here</a> to see our improvements.</p>
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             <ul class="nav bs-docs-sidenav shorterli">
  
                 <li><a href="#cdre">CDRE</a></li>
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                 <li><a href="#overview">Overview</a></li>
                 <li><a href="#msp">Mature serine protein</a></li>
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                 <li><a href="#background">Background</a></li>
                 <li><a href="#ref">Reference</a></li>
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                 <!--<li><a href="#exp">Experimental Design</a></li>-->
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                <!--<li><a href="#fullauto">Sample and Test</a></li>-->
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                <li><a href="#conclusion">Conclusions</a></li>
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Latest revision as of 03:55, 2 November 2017

"

VOC sensor

Overview

In our project, we are exploring a new way to sense the health condition of crops which will further send this information (healthy or not) to both human and our T.atroviride. In this way, we can take actions on time and achieve the goal of prevention some phytopathogens in advance. Meanwhile, this method should be conveniently used in variety range of plants.

We finally build up an e-nose (electronic nose) system to sense the VOC (volatile organic compounds) emitted by the plants. Afterwards, using machine learning method, we achieved a high accuracy rate, which is over 85%, when sensing the tobaccos infected by P.nicotianae. That is to say: our device successfully distinguishes whether the tobaccos are ill by "smelling them".

Background

Plants will release a variety of volatile organic compounds (VOC) to resist some infected phytopathogens, which can lead to a change of VOC composition around the plants[1]. Firstly, we verified the VOC differences between healthy tobaccos and tobaccos infected by P.nicotianae by GC-MS.

Fig.1 GC-MS analysis of VOCs emitted by healthy tobaccos and tobaccos infected by P.nicotianae

(Thanks Hunan Tobaccos Bureau for providing this data).

We decide to use ten highly sensitive CMOS(Complementary Metal Oxide Semiconductor) gas detector to catch the VOC information, each detector is sensitive to one kind or type of VOCs. E-nose can’t sense a specific VOC like mass spectrometers did. instead, it catches the overall characteristics of VOCs as "fingerprint".

Fig.2 A photo of our gas sensor chamber.

Our device is controlled by Arduino Single Chip Microcomputer(SCM) in order to achieve automatic measurement, and we standardize the testing steps to ensure the data between every groups is comparable.

Fig.3 The response curve of gas sensors (from ill tobacco)and the stability curve (40min in air)

We use high purity air to blow out the gas out of the box into the gas sensor chamber.

Fig.4 An illustration of our standardized measuring steps

Click here to see more information about our device.

We build median filter algorithm in SCM to remove some outliers. Then, we preprocess the raw data after getting them: First, we identify and remove the base line value; Then, we read the response value on 1min, 2 min, the integral average value and maximum value of the response curve.

We have measured 17 groups of healthy tobaccos and 18 groups of infected ones. Click to download our raw data and preprocessed data.

Fig.5 The integral average value of 35 response curve.

After preprocessing the data, we apply some algorithms to do the classification work. We employ Decision Tree, Multi-Layer Perception algorithm, and Leaner Model. Finally, we achieve more than 85% accuracy rate of distinguishing whether the tobaccos are infected by P.nicotianae.

Moreover, based on the result of the modeling, 4 CMOS detectors are enough to make a judgement for the health condition of tobaccos, which means we can further reduce the cost of our device.

Click here to see the details of our modeling process.

Conclusions

Our method achieves more than 85% accuracy rate on distinguish the health state of tobaccos, and we find we only need four detectors for our devices to make such judgments. Moreover, the VOC data can be sent to PC port, we also build a web app to achieve real time monitoring. In the future, we plan to make the web app possible to show the healthy condition of plants in real time.

On the other hand, the sensitivity of our device is still limited when facing real world conditions. It is hard to fit different conditions in different field. Therefore, we come up with a plan to solve this problem, Click here to see our improvements.