Difference between revisions of "Team:Peking/Project/CRISPR"

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                     <div class="navbar-collapse collapse"  style="padding-top:5px;padding-bottom:0">
 
                     <div class="navbar-collapse collapse"  style="padding-top:5px;padding-bottom:0">
 
                         <!-- Start Navigation List -->
 
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                        <ul class="nav navbar-nav navbar-right " style="padding-bottom:15px;height:88px;padding-top:10px">
 
                             <li>
 
                             <li>
 
                                 <a href="https://2015.igem.org/Team:Peking/JudgingCriteria">Achievements</a>
 
                                 <a href="https://2015.igem.org/Team:Peking/JudgingCriteria">Achievements</a>
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                                     <li><a href="https://2015.igem.org/Team:Peking/Design">Overview</a>
 
                                     <li><a href="https://2015.igem.org/Team:Peking/Design">Overview</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="http://2015.igem.rg/Team:Peking/Design/Sequence-specific visualization">Sequence-specific Visualization</a>
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                                     <li><a href="#">CRIPSR</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="https://2015.igem.org/Team:Peking/Design/Isothermal">Iso-thermal Amplification</a>
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                                     <li><a href="#">Isothermal PCR</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="https://2015.igem.org/Team:Peking/Design/Array">Array-based diagnosis</a>
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                                     <li><a href="#">B. subtillis</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="https://2015.igem.org/Team:Peking/Device">Hardware</a>
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                                     <li><a href="#">Device</a>
                                    </li>
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                                    <li><a href="https://2015.igem.org/Team:Peking/Design/Speculation">Speculation</a>
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                                     </li>
 
                                     </li>
 
                                 </ul>
 
                                 </ul>
 
                             </li>
 
                             </li>
 
                             <li>
 
                             <li>
                                 <a href="https://2015.igem.org/Team:Peking/Modeling">Modeling</a>
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                                 <a href="https://2015.igem.org/Team:Peking/Modeling">Modelling</a>
 
                                 <ul class="dropdown">
 
                                 <ul class="dropdown">
                                     <li><a href="https://2015.igem.org/Team:Peking/Modeling">Array Design</a>
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                                     <li><a href="#">Link 1</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="https://2015.igem.org/Team:Peking/Modeling/Analysis">Analysis algorithm</a>
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                                     <li><a href="#">Link 2</a>
 
                                     </li>
 
                                     </li>
 
                                 </ul>
 
                                 </ul>
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                                     <li><a href="https://2015.igem.org/Team:Peking/Practices">Overview</a>
 
                                     <li><a href="https://2015.igem.org/Team:Peking/Practices">Overview</a>
 
                                     </li>
 
                                     </li>
                                     <li><a class="active" href="https://2015.igem.org/Team:Peking/Practices/Background">Facts about TB</a>
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                                     <li><a href="#">TB Facts</a>
 
                                     </li>  
 
                                     </li>  
                                     <li><a href="https://2015.igem.org/Team:Peking/Practices/Consultation">Consultation and Interview</a>
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                                     <li><a href="https://2015.igem.org/Team:Peking/Pratices/Consultation">Consultation and Interview</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="https://2015.igem.org/Team:Peking/Practices/Engagement">Public Engagement</a>
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                                     <li><a class="active" href="https://2015.igem.org/Team:Peking/Pratices/Engagement">Public Engagement</a>
 
                                     </li>
 
                                     </li>
                                     <li><a href="https://2015.igem.org/Team:Peking/Practices/Outreach">Ethics and Economics Issue</a>
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                                     <li><a href="https://2015.igem.org/Team:Peking/Pratices/Outreach">Ethics and Economics Issue</a>
 
                                     </li>
 
                                     </li>
 
                                 </ul>
 
                                 </ul>
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         <div class="row">
 
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           <div class="col-md-6">
 
           <div class="col-md-6">
             <h2 style="font-size:20px; margin-bottom:5px; padding-bottom:0"><b>Practices</b></h2>
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             <h2 style="font-size:20px; margin-bottom:5px; padding-bottom:0"><b>Project</b></h2>
 
             <p style="margin-top:0px;font-size:14px">Study how our work affects the world, and how the world affects our work.</p>
 
             <p style="margin-top:0px;font-size:14px">Study how our work affects the world, and how the world affects our work.</p>
 
           </div>
 
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             <ul class="breadcrumbs">
 
             <ul class="breadcrumbs">
 
               <li><a href="https://2015.igem.org/Team:Peking">Home</a></li>
 
               <li><a href="https://2015.igem.org/Team:Peking">Home</a></li>
               <li>Practices</li>
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               <li>Project</li>
 
             </ul>
 
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               <h4 style="font-size:18px">Practices<span class="head-line"></span></h4>
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               <h4 style="font-size:18px">Project<span class="head-line"></span></h4>
 
                 <ul>
 
                 <ul>
 
                 <li>
 
                 <li>
                   <a href="https://2015.igem.org/Team:Peking/Practices">Overview</a>
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                   <a href="https://2015.igem.org/Team:Peking/Design">Overview</a>
 
                 </li>
 
                 </li>
 
                 <li>
 
                 <li>
                   <a href="#" style="color:#00afd1">TB Facts</a>
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                   <a href="https://2015.igem.org/Team:Peking/Practices/Background" >Paired dCas9 Reporter System</a>
 
                 </li>
 
                 </li>
 
                   <li>
 
                   <li>
                   <a href="https://2015.igem.org/Team:Peking/Practices/Consultation">Consultation and Interview</a>
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                   <a href="https://2015.igem.org/Team:Peking/Practices/Consultation">Iso-Thermal Amplification</a>
 
                 </li>
 
                 </li>
 
                 <li>
 
                 <li>
                   <a href="https://2015.igem.org/Team:Peking/Practices/Engagement">Public Engagement</a>
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                   <a style="color:#00afd1" href="https://2015.igem.org/Team:Peking/Practices/Engagement">Array-Based Diagnosis</a>
 
                 </li>
 
                 </li>
 
                 <li>
 
                 <li>
                   <a href="https://2015.igem.org/Team:Peking/Practices/Outreach">Ethic and Economic Issue</a>
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                   <a href="https://2015.igem.org/Team:Peking/Practices/Outreach">Hardware</a>
 +
                </li>
 +
                <li>
 +
                  <a href="https://2015.igem.org/Team:Peking/Practices/Outreach">Speculation</a>
 
                 </li>
 
                 </li>
 
               </ul>
 
               </ul>
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                 </li>
 
                 </li>
 
                 <li>
 
                 <li>
                   <a href="#" style="color:#00afd1">TB Facts</a>
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                   <a href="https://2015.igem.org/Team:Peking/Practices/Background" >TB Facts</a>
 
                 </li>
 
                 </li>
 
                   <li>
 
                   <li>
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                 <li>
 
                 <li>
                   <a href="https://2015.igem.org/Team:Peking/Practices/Engagement">Public Engagement</a>
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                   <a style="color:#00afd1" href="https://2015.igem.org/Team:Peking/Practices/Engagement">Public Engagement</a>
 
                 </li>
 
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           </div>
 
           </div>
 
           <!--End sidebar-->
 
           <!--End sidebar-->
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  <!-- Page Content -->
 
           <div class="col-md-9 page-content">
 
           <div class="col-md-9 page-content">
 
             <div id="practices">
 
             <div id="practices">
 
             <!-- Classic Heading -->
 
             <!-- Classic Heading -->
 
               <div id="Background">
 
               <div id="Background">
             
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                <div>
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                  <h3 class="classic-title" style="margin-top:50px"><span>Questionnaire</span></h3>
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                  <div style="margin-top:30px; margin-bottom:0">
 +
                   
 +
                    <p><a href="https://static.igem.org/mediawiki/2015/0/0b/Questionnaire.pdf">The Questionnaire about acknowledgment of Tuberculosis and Synthetic Biology.</a>             
 +
                    </p>
 +
                  </div>
 +
                  <h3 class="classic-title"  style="margin-top:50px"><span>Questionnaire Report</span></h3>
  
                 <h3 class="classic-title" style="margin-top:50px"><span>Introduction</span></h3>
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              <div style="margin-top:30px; margin-bottom:0">
 +
                 <p class="text-center"><big><big><b>Investigation Report on the Cognition of Tuberculosis and Synthetic Biology</b></big></big></p>
 +
                <p class="text-center"><br><big><b>Abstract</b></big><br/></p>
 +
                  <p><b>Objective</b><br> To investigate the current awareness rate of tuberculosis and synthetic biology knowledge among Chinese people so as to provide the evidence of tuberculosis health education and synthetic biology education in Chinese people. </p>
 +
                  <p><b>Methods</b><br> 194 tourists around the Weiming Lake of Peking University from August 16th to August 18<sup>th</sup> were selected by random sampling. The anonymous questionnaire was of our own design. </p>
 +
                  <p><b>Results</b></big><br/> The awareness rates of the sources, routes of transmission, clinical symptoms, severity and treatment effect of tuberculosis are high, which accounted for 70%-90% among 194 tourists. However, the awareness rates of the diagnosis methods, the specific treatment methods and the national prevention and control policy of tuberculosis are less than 40%. As for the part of cognitive attitude of synthetic biology, only 6.2% of the tourists have a deeper understanding of synthetic biology, while 33.3% of the respondents have never heard of synthetic biology. The awareness rates of the selected terms about the field of synthetic biology are generally lower than 35%, of which 58.1% and 52.8% of the tourists have access to relevant knowledge through the Internet and television, respectively. 76% of the tourists agree that applying synthetic biology methods to clinical diagnosis is feasible, and 86% of the visitors are willing to use the relevant technology products if possible, the main reasons of which are rapidness, individuation and the reliable results of the new detection technology. To the contrary, the tourists who are not willing to use that detection technology mainly worry about the potential side effects, and they believe that traditional detection methods are mature enough. </p>
 +
                <p><b>Conclusion</b><br/>Chinese people have certain knowledge of tuberculosis, but not systematic or comprehensive; they have a low awareness rate of synthetic biology, which means its popularization needs to be strengthened.</p>
 +
                <p><b>Keywords</b> &nbsp;Tuberculosis; Synthetic Biology; Cognition</p>
  
 +
                <p class="text-center"><big><b><br>Objects and Methods</b></big><br/><br></p>
 +
                  <p><b>Objects</b><br>194 tourists around the Weiming Lake of Peking University from August 16th to August 18<sup>th</sup></p>
 +
                  <p><b>Methods</b><br>Respondents were selected by random sampling in our investigation. The anonymous questionnaire was of our own design. A total of 200 questionnaires were distributed, and 194 questionnaires were collected, the effective recovery rate was 97% (Table 1).</p>
 +
                  <p><b>Statistical analysis:</b><br>Using EpiData3.1 to import the questionnaire data based on the methods of data entry, thus to establish a database; then SPSS20.0 was used to statistical analysis.</p>
 +
 +
                <p><big><b>Results</b></big></p>
 +
                <p><b>General demographic situation</b></p>
 +
                <p><b>Gender and age structure</b></p>
 +
                <p>A total of 194 questionnaires were collected in this investigation, of which the proportion of men was 48.7%; for female, it was 50.8%. Most of the population was among the 15-50 years old age group, accounting for 82.3%. Therefore, the composition of gender and age of the population in the survey was different from the national population in some extent (Table 1). </p>
 +
                <p><b>Region</b></p>
 +
                <p>Among the 194 respondents, 139 were from urban, accounting for 72.4%, and 53 were from rural, accounting for 27.6%.</p>
 +
                <p><b>Education level and economic level.</b></p>
 +
                <p>In the survey population, the educational background of the majority was undergraduate degree or above, accounting for 61.5%. The economic levels of most respondents are not bad because 71.4% of whose family income were more than 2000 yuan.</p>
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              </div>
 
               <div style="margin-top:30px; margin-bottom:0">
 
               <div style="margin-top:30px; margin-bottom:0">
                 <p><br><big><b>Background</b></big><br/></p>
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                 <p class="text-center"><small><b>Table.1  The General Demographic Situation</b></small></p>
              <p>Tuberculosis (TB) ranks as the 2nd leading cause of death among various infectious diseases [1]. The fact that earlier diagnosis and treatment indicate better prognosis attaches great importance to the diagnostic services for TB patients. The current methods include tuberculin skin test, blood test and sputum culture. However, tuberculin skin test is not sensitive, blood test is risky and sputum culture needs a waiting period as long as 1-2 months which will delay timely treatment. Methods based on nucleic acid detection (NAD) are highly sensitive, safe and rapid; however, they are not widely applied to clinical practice. Why? </p>
+
                <table class="table" style="color:#666;font-size:14px">
+
<thead>
              <P>After consulting some front-line practitioners of TB control, we found that NAD has high false positive rate because non-specific amplification frequently occurs and current methods of NAD are not able to read out sequence-specific information; this makes them barely reliable. What' more, NAD requires expensive and clumsy instruments which are not accessible to rural areas where TB is epidemic (For more detailed information, see Human Practice). Therefore, Peking iGEM 2015 is devoted to building a new reporter system that enables NAD to be specific, reliable and less instrument-dependent.</P>
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  <tr>
              <p><br><big><b>CRISPR</b></big><br/></p>
+
<th rowspan="1">Aspects</th>
              <P>To improve the specificity, we need a system that can read out sequence information directly. The first thing coming to our mind is the clustered regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, which is originally from prokaryotic immune system [2]. Cas9 is a sequence-specific, DNA cleavage protein guided by single-guide RNA (sgRNA); they form a complex and can be easily programmed to recognize any target sequence [3]; in previous studies it was engineered to be catalytically dead (dCas9), thus to form a programmable DNA binding complex [4]. But how can we convert the invisible sequence information into easily measurable signal?
+
<th colspan="1">Options</th>
               </P>
+
<th colspan="1">Percentage</th>
              <p><br><big><b>Split luciferase</b></big><br/></p>
+
<th colspan="1">Ratio</th>
              <P>Split enzymes [5] were selected as the candidates to convert the presence of target DNA sequence into measureable signal. Each protein fragment by itself is inactive; when the fragments are reassembled, the enzymatic activity of the original protein would be reconstituted, thus providing easily measurable readout through enzymatic reaction. Among diverse split enzymes, we chose split luciferase which produces bioluminescence signal (Figure 1).</P>
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</tr>
              <img src="https://static.igem.org/mediawiki/2015/d/d3/Peking-CRISPR-Figure1.png">
+
</thead>
              <p class="text-center"><small><b>Figure 1. Illustration of the working mechanism of split luciferase. A functional luciferase is split into two inactive fragments (Nluc, orange and Cluc, yellow). Each fragment is fused to an interacting domain (gray) that tends to dimerize. The interacting domains cause non-covalent complementation of Nluc and Cluc to reconstitute the enzymatic activity, thus to emit measurable bioluminescence.</b></small></p>
+
<tbody>
              </div>
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<tr>
 +
<th rowspan="5">1.Age</th>
 +
<th>Below 15  </th>
 +
<th>13.5</th>
 +
<th>26/192</th>
 +
</tr>
 +
<tr>
 +
<th>15-30</th>
 +
<th>35.9</th>
 +
<th>69/192</th>
 +
</tr>
 +
<tr>
 +
<th>30-50</th>
 +
<th>46.4</th>
 +
<th>89/192</th>
 +
</tr>
 +
<tr>
 +
<th>50-70</th>
 +
<th>4.2</th>
 +
<th>8/192</th>
 +
</tr>
 +
<tr>
 +
<th>Above 70</th>
 +
<th>0.0</th>
 +
<th>0/192</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="2">2.Gender</th>
 +
<th>Male</th>
 +
<th>50.8</th>
 +
<th>98/193</th>
 +
</tr>
 +
<tr>
 +
<th>Female</th>
 +
<th>48.7</th>
 +
<th>94/193</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="2">3.Census register</th>
 +
<th>Rural</th>
 +
<th>27.6</th>
 +
<th>53/192</th>
 +
</tr>
 +
<tr>
 +
<th>Urban</th>
 +
<th>72.4</th>
 +
<th>139/192</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="5">4.Degree of education</th>
 +
<th>Untutored</th>
 +
<th>0.5</th>
 +
<th>1/192</th>
 +
</tr>
 +
<tr>
 +
<th>Grade school</th>
 +
<th>6.3</th>
 +
<th>12/192</th>
 +
</tr>
 +
</tr>
 +
<tr>
 +
<th>Junior high school</th>
 +
<th>13.0</th>
 +
<th>25/192</th>
 +
</tr>
 +
<tr>
 +
<th>High school</th>
 +
<th>18.8</th>
 +
<th>36/192</th>
 +
</tr>
 +
<tr>
 +
<th>Undergraduate or graduate</th>
 +
<th>61.5</th>
 +
<th>18/192</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="5">5. Month-earning of the family</th>
 +
<th>Below 500 RMB</th>
 +
<th>1.0</th>
 +
<th>2/192</th>
 +
</tr>
 +
<tr>
 +
<th>500-1999 RMB</th>
 +
<th>10.4</th>
 +
<th>20/192</th>
 +
</tr>
 +
<tr>
 +
<th>2000-4999 RMB</th>
 +
<th>40.1</th>
 +
<th>77/192</th>
 +
</tr>
 +
<tr>
 +
<th>Above 5000 RMB</th>
 +
<th>31.3</th>
 +
<th>60/192</th>
 +
</tr>
 +
<tr>
 +
<th>Unknown</th>
 +
<th>17.2</th>
 +
<th>33/192</th>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<div style="margin-top:30px; margin-bottom:0">
 +
                <p><b>Awareness of tuberculosis</b></p>
 +
                <p>Most of the 194 tourists were well aware of the core issues of tuberculosis. In detail, the awareness rate of severity, the infection sources and the transmission routes of tuberculosis were 77.8%, 71.1% and 70.1%, respectively. And the awareness rate of the clinical symptoms of tuberculosis was as high as 88.7%. However, the national tuberculosis control policy awareness is much worse, only 40 people answer the related question correctly, accounting for 20.6% of the survey population. And the respondents who can answer all of the six core questions accounted for only 6.9%. As for the knowledge about specific diagnostic methods, treatment methods of the tuberculosis, the results were barely satisfactory, the awareness rate of which were just 34.5% and 7.2%, respectively.</p>
 +
</div>
 +
<div style="margin-top:30px;marigin-bottom:0">
 +
  <p class="text-center"><small><b>Table.2  The Acknowledgment of Tuberculosis</b></small></p>
 +
                <table class="table" style="color:#666;font-size:14px">
 +
<thead>
 +
<tr>
 +
<th>Crucial questions</th>
 +
<th>The percentage of correctly answered(%)</th>
 +
<th>Ratio </th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<th>1. Do you think the Tuberculosis is a disease greatly threatened human health?</th>
 +
<th>77.8</th>
 +
<th>151/194</th>
 +
</tr>
 +
<tr>
 +
<th>2. Which is main source of infection?</th>
 +
<th>71.1</th>
 +
<th>138/192</th>
 +
</tr>
 +
<tr>
 +
<th>3. Which is main transmission route of TB?</th>
 +
<th>70.1</th>
 +
<th>136/194</th>
 +
</tr>
 +
<tr>
 +
<th>4. If someone get cough and expectoration more than two weeks with Hemoptysis and blood sputum. What should he or she do?</th>
 +
<th>88.7</th>
 +
<th>172/194</th>
 +
</tr>
 +
<tr>
 +
<th>5. Whether the tuberculosis can be cured?</th>
 +
<th>90.7</th>
 +
<th>176/194</th>
 +
</tr>
 +
<tr>
 +
<th>6. Are the drugs and treatment services for the infectious tuberculosis patients free?</th>
 +
<th>Male</th>
 +
<th>20.6</th>
 +
<th>40/194</th>
 +
</tr>
 +
<tr>
 +
<th> </th>
 +
<th> </th>
 +
<th> </th>
 +
</tr>
 +
<tr>
 +
<th>The other question</th>
 +
<th>The percentage of correctly answered(%)</th>
 +
<th>Ratio</th>
 +
</tr>
 +
<tr>
 +
<th>7. TB can be confirmed by which of the following symptom?</th>
 +
<th>34.5</th>
 +
<th>67/194</th>
 +
</tr>
 +
<tr>
 +
<th>8. Which drugs are for the tuberculosis treatment?</th>
 +
<th>7.2</th>
 +
<th>14/194</th>
 +
</tr>
 +
<tr>
 +
<th>9. What’s your idea about the positive result of PPD test?</th>
 +
<th>36.1</th>
 +
<th>70/194</th>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<div style="margin-top:30px;marigin-bottom:0">
 +
                <p><b>Awareness of synthetic biology</b></p>
 +
                <p>Among the 194 tourists, only 12 of them has a deep understanding about synthetic biology, while 33.3% of visitors have never heard of synthetic biology. Among the people who had heard of synthetic biology, most of them had not heard of the specific professional terms in the field of synthetic biology, such as "bio-element", "molecular machine", "bio-plastics". The awareness rates of these terms were 26.4%, 21.2%, 31.1%, respectively. It deserves to be mentioned that, 68.4% of the respondents said that they had heard the term "genome", but unfortunately, only 3.6% of people had heard of iGEM. And the main ways the respondents use to access to the knowledge of synthetic biology was through the network (58.1%), the television (52.8%) and classroom lectures (30.4%).</p>
 +
</div>
 +
<div style="margin-top:30px;marigin-bottom:0">
 +
  <p class="text-center"><small><b>Table.3  The Acknowledgment of Synthetic Biology</b></small></p>
 +
<table class="table" style="color:#666;font-size:14px">
 +
<thead>
 +
<tr>
 +
<th>Questions </th>
 +
<th>Options</th>
 +
<th>Percentage</th>
 +
<th>Ratio</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<th rowspan="3">1. Have you ever heard of Synthetic Biology?</th>
 +
<th>Yes. And have some knowledge of it</th>
 +
<th>6.2</th>
 +
<th>12/192</th>
 +
</tr>
 +
<tr>
 +
<th>Yes. But have no idea about it</th>
 +
<th>59.4</th>
 +
<th>114/192</th>
 +
</tr>
 +
<tr>
 +
<th>No</th>
 +
<th>33.3</th>
 +
<th>64/192</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="6">2. Which of the following nouns you have heard before?</th>
 +
<th>Bio-element</th>
 +
<th>26.4</th>
 +
<th>51/193</th>
 +
</tr>
 +
<tr>
 +
<th>Molecule machine</th>
 +
<th>21.2</th>
 +
<th>41/193</th>
 +
</tr>
 +
<tr>
 +
<th>Gene Sequence</th>
 +
<th>68.4</th>
 +
<th>132/193</th>
 +
</tr>
 +
<tr>
 +
<th>iGEM</th>
 +
<th>3.6</th>
 +
<th>7/193</th>
 +
</tr>
 +
<tr>
 +
<th>Bioplastics</th>
 +
<th>31.1</th>
 +
<th>133/193</th>
 +
</tr>
 +
<tr>
 +
<th>None of them</th>
 +
<th>21.2</th>
 +
<th>41/193</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="6">3. Where you heard the above nouns?</th>
 +
<th>Network </th>
 +
<th>58.1</th>
 +
<th>93/160</th>
 +
</tr>
 +
<tr>
 +
<th>TV and radio</th>
 +
<th>52.8</th>
 +
<th>85/161</th>
 +
</tr>
 +
</tr>
 +
<tr>
 +
<th>Newspapers</th>
 +
<th>19.3</th>
 +
<th>31/161</th>
 +
</tr>
 +
<tr>
 +
<th>Neighbors</th>
 +
<th>3.1</th>
 +
<th>5/161</th>
 +
</tr>
 +
<tr>
 +
<th>Classes and lectures</th>
 +
<th>30.4</th>
 +
<th>49/161</th>
 +
</tr>
 +
<tr>
 +
<th>The others</th>
 +
<th>10.5</th>
 +
<th>17/162</th>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<div style="margin-top:30px;marigin-bottom:0">
 +
                <p><b>Attitudes toward synthetic biology</b></p>
 +
                <p> Among the tourists who had heard of synthetic biology, the attitudes toward synthetic biology of the majority were neutral. To be specific, 70.5% of the population held the idea that synthetic biology had both advantages and disadvantages. About 76% of the population were optimistic about the development of the diagnostic methods combining the methods in synthetic biology. And 86% of the investigated people were willing to use the synthetic biology methods if possible, the main reasons were rapidness (46.4% of population choose the answer) and the individuation (51.8% of population believe this point) of the new detection technology, in addition, 37.5% of the population believes that the method is more reliable. To the contrary, the tourists who were not willing to use that detection technology mainly worry about the potential side effects (39.3% of population believe this point), and 40.7% of the population believe that traditional detection methods haven’t been well developed. (Table four)</p>
 +
               </div>  
 +
<div style="margin-top:30px;marigin-bottom:0">
 +
<p class="text-center"><small><b>Table.4 The Atittude Towards Synthetic Biology</b></small></p>
 +
<table class="table" style="color:#666;font-size:14px">
 +
<thead>
 +
              <tr>
 +
<th>Questions </th>  
 +
<th>Options</th>
 +
<th>Percentage</th>
 +
<th>Ratio</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<th rowspan="4">1. What’s your attitude to the Synthetic
 +
 
 +
Biology?</th>
 +
<th>Have no advantages</th>
 +
<th>0.8</th>
 +
<th>1/129</th>
 +
</tr>
 +
<tr>
 +
<th>Have no disadvantages</th>
 +
<th>3.9</th>
 +
<th>5/129</th>
 +
</tr>
 +
<tr>
 +
<th>Have both advantages and disadvantages</th>
 +
<th>70.5</th>
 +
<th>91/129</th>
 +
<tr>
 +
<th>Have no idea</th>
 +
<th>24.0</th>
 +
<th>31/129</th>
 +
</tr>
 +
</tr>
 +
<tr>
 +
<th rowspan="3">2. Do you think it possible to invent a
 +
 
 +
new method of TB diagnosis make use of the knowledge of  
 +
 
 +
Synthetic Biology?</th>
 +
<th>Sure</th>
 +
<th>76.0</th>
 +
<th>98/129</th>
 +
</tr>
 +
<tr>
 +
<th>No way</th>
 +
<th>0.8</th>
 +
<th>1/129</th>
 +
</tr>
 +
<tr>
 +
<th>Have no idea</th>
 +
<th>20.9</th>
 +
<th>27/129</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="2">3. If such a method has been invented,  
 +
 
 +
would you like to try it?</th>
 +
<th>Yes</th>
 +
<th>86.0</th>
 +
<th>111/129</th>
 +
</tr>
 +
<tr>
 +
<th>No</th>
 +
<th>13.2</th>
 +
<th>17/129</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="5">4. Why you would like to have a try?</th>
 +
<th>Cheap</th>
 +
<th>8.0</th>
 +
<th>9/129</th>
 +
</tr>
 +
<tr>
 +
<th>Fast and convenient</th>
 +
<th>46.4</th>
 +
<th>52/112</th>
 +
</tr>
 +
<tr>
 +
<th>Personalized medicine service</th>
 +
<th>51.8</th>
 +
<th>58/112</th>
 +
</tr>
 +
</tr>
 +
<tr>
 +
<th>Efficacy is better</th>
 +
<th>37.5</th>
 +
<th>42/112</th>
 +
</tr>
 +
<tr>
 +
<th>Other reason</th>
 +
<th>0.0</th>
 +
<th>0/112</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="4">5.Why you don’t do that?</th>
 +
<th>The potential side effect</th>
 +
<th>39.3</th>
 +
<th>12/28</th>
 +
</tr>
 +
<tr>
 +
<th>It will pollute the environment</th>
 +
<th>7.4</th>
 +
<th>2/27</th>
 +
</tr>
 +
<tr>
 +
<th>It will be expensive</th>
 +
<th>18.5</th>
 +
<th>5/27</th>
 +
</tr>
 +
<tr>
 +
<th>The technique is not well developed</th>
 +
<th>40.7</th>  
 +
<th>11/27</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="5">6. How much would you like to spend on
 +
 
 +
such a method for your health?</th>
 +
<th>Less than 10 yuan</th>
 +
<th>1.6</th>
 +
<th>2/123</th>
 +
</tr>
 +
<tr>
 +
<th>10 yuan-100 yuan </th>
 +
<th>6.5</th>
 +
<th>8/123</th>
 +
</tr>
 +
<tr>
 +
<th>100 yuan-500 yuan</th>
 +
<th>22.8</th>
 +
<th>28/123</th>
 +
</tr>
 +
<tr>
 +
<th>500 yuan-1000 yuan</th>
 +
<th>25.2</th>
 +
<th>31/123</th>
 +
</tr>
 +
<tr>
 +
<th>1000 yuan-5000 yuan</th>
 +
<th>36.6</th>
 +
<th>45/123</th>
 +
</tr>
 +
<tr>
 +
<th>More than 5000 yuan</th>
 +
<th>6.5</th>
 +
<th>8/123</th>
 +
</tbody>
 +
</table>
 +
</div>
 +
<div style="margin-top:30px;marigin-bottom:0">
 +
 
 +
                <p><big><b>Conclusions</b></big><br/></p>
 +
                <p>Through this investigation, we recognize that Chinese people have a certain understanding of the basic knowledge of tuberculosis, but it is not comprehensive and systematic, especially details about the disease. The majority of the population of the survey has a neutral attitude towards synthetic biology, and most of them are optimistic about the applications and progress of synthetic biology, though only a small portion of people knows this field very well. <br/>Therefore, on the one hand, it is necessary to strengthen the popularization of the knowledge of tuberculosis in public. The process of propaganda to the national for health education of tuberculosis should not be a slogan, and it should be in-depth and systematic. On the other hand, researchers in the field of synthetic biology should actively work for the science popularization of synthetic biology at the same time they are working for the scientific research. They are responsible for improving the people’s understanding of the synthetic biology, and attracting more people to join the development trend of synthetic biology.
 +
</p>
 +
              </div>
 +
 
 +
                <h3 class="classic-title"  style="margin-top:50px"><span>Education for children</span></h3>
  
              <h3 class="classic-title"  style="margin-top:50px"><span>Design</span></h3>
 
 
               <div style="margin-top:30px; margin-bottom:0">
 
               <div style="margin-top:30px; margin-bottom:0">
              <p>We combined dCas9 and split luciferase to obtain a paired dCas9 (PC) reporter system that can not only extract the sequence information of target DNA, but also quantitatively visualize its abundance through bioluminescence emission. As shown in the schematic diagram (Figure 2), when N-luciferase-dCas9:sgRNA complex and C-luciferase-dCas9:sgRNA complex simultaneously bind to adjacent sites on a target DNA, the two fragments of luciferase are brought into proximity and the subsequent complementation brings measurable bioluminescence signal.</p>
+
                <p>To attract children’s more attention toward synthetic biology and to popularize some basic biological concepts among them, we invited near 20 pupils to visit our laboratory. In the morning of August 17th, these children and some of their parents came to Peking University and attended a lecture given by ZHANG Yihao, the team leader. </p>
              <img src="https://static.igem.org/mediawiki/2015/5/5d/Peking-CRISPR-Figure2.png">
+
                <img src="https://static.igem.org/mediawiki/2015/0/02/Peking-Practices-Engagement-Education_for_children1.jpg">
              <p class="text-center"><small><b>Figure 2. Schematic of the paired dCas9 (PC) reporter system. dCas9 was fused with fragments of split luciferase (N-luc and C-luc), respectively, to form two kinds of split luciferase-dCas9:sgRNA complexes. In the presence of target DNA, the complexes, respectively, bind to two adjacent recognition sites, bringing N-luc and C-luc into proximity to produce bioluminescence signal.</b></small></p>
+
                <p>By plain words, he made it easy for these children to understand the basic concepts of biological field and the core ideas of synthetic biology. What is the basic substance of life? What caused creatures on the earth so diversified and fascinating? Moreover, can scientists build life with fancy functions? They got the answers to all these questions above.</p>
              </div>
+
               
              <h3 class="classic-title"  style="margin-top:50px"><span>Results</span></h3>
+
                <p>In the afternoon, they visited the lab of Peking iGEM. Having known how the biological researchers work in the labs, they all indicated that they hope to learn more about synthetic biology. </p>
 +
                <img src="https://static.igem.org/mediawiki/2015/f/fa/Peking-Practices-Engagement-Education_for_children3.jpg">
 +
              </div>
 +
              <h3 class="classic-title"  style="margin-top:50px"><span>Education for High School Students</span></h3>
 +
 
 
               <div style="margin-top:30px; margin-bottom:0">
 
               <div style="margin-top:30px; margin-bottom:0">
              <p><br><big><b>Validating the functional reconstitution of split luciferase </b></big><br/></p>
+
                <img src="https://static.igem.org/mediawiki/2015/b/b6/Peking-Practices-Engagement-Education_for_high_school_students1.jpg">
              <p>We began by examining whether our split luciferase could be really functionally reconstituted by protein fragment-assisted complementation (PFAC) [7,8]. For the complementation-assisting partners, we chose rapamycin-binding domain (FRB) of human mTOR that binds with high affinity to FK-506-binding protein 12 (FKBP); rapamycin is able to induce the dimerization to form a FRB-rapamycin-FKBP complex (Figure 3a).  A pair of widely-used split firefly (Photinus pyralis) luciferase fragments (N-luc 416/C-luc 398) was then selected to construct N-luc-FRB and FKBP-C-luc fusion proteins; they are, therefore, subjected to rapamycin induced complementation [9]. Our results of PFAC assay on our split luciferase fragments confirmed that the luciferase activity are able to be successfully reconstituted in a rapamycin-dependent manner (Figure 3b).</p>
+
                <p>On August 23th, our team member YE Yixuan went to Zhenghe No.1 Middle School of Fujian Province, giving a lecture to the high school students. She mainly introduced the overview of synthetic biology and the project of Peking this year. </p>
              <img src="https://static.igem.org/mediawiki/2015/7/74/Peking-CRISPR-Figure3.png">
+
                <img src="https://static.igem.org/mediawiki/2015/2/26/Peking-Practices-Engagement-Education_for_high_school_students2.jpg">
              <p class="text-center"><small><b>Figure 3. Rapamycin-induced N-luc-FRB/FKBP-C-luc complementation. (a) The working mechanism of rapamycin induced dimerization. The interacting protein partners (FRB and FKBP) get closer and dimerize soon after rapamycin is added (40nM) [10], thus to reconstitute the enzymatic activity of luciferase. (b) The experimental data. Error bars denote s.d.; n=3. </b></small></p>
+
                <p>More than 60 students who were interested in science have attended the lecture. They were confronted with the choice of their major fields in the university, so the lecture really helped them. Ye combined the basic concepts of synthetic biology with what they had learned in school. After the lecture, a heated discussion about biology and future life began. What’s more, the project of Peking iGEM team this year was presented, for the purpose of arousing their interest in this field. </p>
              <p><br><big><b>The location of His-tag</b></big><br/></p>
+
              </div>
              <p>Despite the fact that His-tag is widely used for protein purification, it is well-known that His-tag might have some considerable impact on protein structure. Hence, we first tested the activity of C-terminally and N-terminally His-tagged split luciferase-dCas9 fusion proteins to see whether the location of His-tag matters. We found that when the His-tag is attached to N-luc or C-luc, the activity of fusion protein would dramatically decrease (See working mechanism of PC reporter in Figure 2); this is probably due to the interruption of luciferase reconstitution by His-tag (Figure 4). When the His-tag is attached to dCas9, the enzymatic activity was reconstituted significantly. Therefore, we chose split luciferase-dCas9 fusion proteins with His-tagged dCas9 for the following study.</p>
+
              <h3 class="classic-title"  style="margin-top:50px"><span>Education for Adults</span></h3>
              <img src="https://static.igem.org/mediawiki/2015/f/f3/Peking-CRISPR-Figure4.png">
+
              <p class="text-center"><small><b>Figure 4. The impact of His-tag location on the reconstitution of luciferase activity. When attached to dCas9, His-tag has minimal impact on the complementation of N-luc and C-luc.</b></small></p>
+
                <p><br><big><b>Construction and optimization of PC reporter system</b></big><br/></p>
+
                <p>Considering that dCas9 is a huge protein and is structurally complex, we need to figure out the configuration of PC reporter system. We first fused split luciferase to either N or C terminus of dCas9 (Figure 5a) to see its influence. Then, provided that initial binding of dCas9 depends on the protospacer adjacent motif (PAM, a short 3’ motif adjacent to target sequence) [11], four sets of sgRNA orientation settings were also tested (Figure 5b). One set placed two PAM sequences distal from the spacer sequence between the sgRNA pair, with the 5' end of the sgRNA adjacent to the spacer (PAM-out) while another set put both PAM sequences adjacent to the spacer (PAM-in). PAM-direct 1 and PAM-direct 2 combine both sgRNA orientations with one PAM sequence adjacent to and another distal from the spacer (Figure 5b).</p>
+
  
                <p>In total, 16 pairs of split luciferase-dCas9:sgRNA complexes were constructed and tested (Figure 5c). Most of these pairs worked very well.  Among them we selected N-luc-dCas9/C-luc-dCas9, the most robust protein construct with minimal intergroup difference, and PAM-out, the sgRNA orientation exhibiting the strongest luminescence, for the following study. </p>
+
              <div style="margin-top:30px; margin-bottom:0">
                <img src="https://static.igem.org/mediawiki/2015/e/ea/Peking-CRISPR-Figure5.png">
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                 <p>Handing out propagandistic handbooks is another approach of scientific education for public. In this summer, we have wrote a comprehensive literature review about tuberculosis and synthetic biology and made a propagandist handbook easy for learning.</p>
                 <p class="text-center"><small><b>Figure 5. Construction and optimization of PC reporter system. (a) 4 different split luciferase-dCas9 fusion strategies.  (b) 4 different sgRNA orientation settings. In orientation PAM-out, the pair of PAM sequences are distal from the spacer sequence, with the 5' end of the sgRNA adjacent to the spacer; in orientation PAM-in, the pair of PAM sequences are adjacent to the spacer sequence, with the 3' end of the sgRNA in proximity to the spacer; in orientation PAM-direct 1 and PAM-direct 2, one PAM sequence is adjacent to and another distal from the spacer. (c) The experimental results of PC reporter systems using 16 different (4 different constructs X 4 different sgRNA orientation settings) configurations.</b></small></p>
+
                 <img src="https://static.igem.org/mediawiki/2015/2/2b/Peking-Practices-Engagement-Education_for_adults1.jpg">
                <p>As the reconstitution of enzymatic activity was constrained by the distance between N-luc and C-luc [11], we varied the spacer length from 5 bp to 107 bp (Figure 6) to find how it affects the performance of our PC reporter system. Results showed that the PC reporter system works with a preference for spacer length of about 21 bp and 33 bp, presenting a period of 10-11 bp; this is consistent with the length of DNA double helix in one period (10.4 bp) [12].</p>
+
                 <p>Every time when we finished our questionnaire surveys, we gave our informants handbooks to spread the knowledge of TB control and synthetic biology.</p>
                 <img src="https://static.igem.org/mediawiki/2015/a/a3/Peking-CRISPR-Figure6.png">
+
                 <img src="https://static.igem.org/mediawiki/2015/3/3d/Peking-Practices-Engagement-Education_for_adults2.jpg">
                 <p class="text-center"><small><b>Figure 6. The effect of spacer length variation on the performance of PC reporter system. Spacer is defined as the sequence between the sgRNA pairs. The spacer length varies from 5 bp to 107 bp. Note that the effect of spacer length exhibits a period of 10-11bp, which is consistent with the structure of DNA double helix. </b></small></p>
+
                <p>In the handbook, we introduce the basic information of tuberculothesis, such as route of transmission, diagnosis methods and its treatment, along with the national policies of free diagnosis and treatment on tuberculosis. After that, we give a brief description of the history of synthetic biology, combined with its application. In such a small handbook, many vivid but scientific pictures are shown. We did our best to make the words easy to be understood but rigorous.</p>
                <p>Taking all the above together, the final design of our PC reporter system was determined: both fragments of split luciferase fused to N-terminus of dCas9, PAM-out sgRNA orientation, and spacer length of 21 bp. Next we set out to validate whether PC reporter can read out the sequence-specific information using crude PCR product instead of well-purified DNA samples. </p>
+
                <img src="https://static.igem.org/mediawiki/2015/1/1d/Peking-Practices-Engagement-Education_for_adults3.jpg">
                <p><br><big><b>Applying PC reporter to crude NAD product</b></big><br/></p>
+
              </div>
                <p>We first carried out specificity test using PC reporter system to distinguish the target plasmid (BBa_K909009) from different kinds of non-specific controls (Figure 7a,b,c). The results showed that the luminescence intensity using the positive target is significantly higher than that of non-specific controls (Figure 7d).</p>
+
                 <img src="https://static.igem.org/mediawiki/2015/a/a4/Peking-CRISPR-Figure7.png">
+
                <p class="text-center"><small><b>Figure 7. Specificity test of PC reporter system. (a) Positive target with two sites recognized simultaneously by a pair of split luciferase-dCas9:sgRNA complexes. (b) Control with only one site recognized by "half pair". (c) Negative control with no site to be recognized. (d) Experimental data. Error bars denote s.d.; n=3. </b></small></p>
+
                <p>Also, we need to test the sensitivity of PC reporter system. As shown in Figure 8, PC reporter can still work at the concentration of ~0.1nM target for both plasmid (Figure 8a) and crude PCR product (Figure 8b). </p>
+
                <img src="https://static.igem.org/mediawiki/2015/5/51/Peking-CRISPR-Figure8.png">
+
                <p class="text-center"><small><b>Figure 8. Exploring the sensitivity of PC reporter using plasmid and PCR product as the target. (a) Bioluminescence intensity at different concentrations of plasmid as the target. (b) Bioluminescence intensity at different concentrations of crude PCR product as the target. Both showed that PC reporter was able to detect target DNA at a low concentration (0.1 nM).</b></small></p>
+
                <p>However, we don’t know whether 0.1nM is the bottom limit of detection. We speculated that with multiple PC reporters recognizing more sites on one target, enhanced bioluminescence signal could be observed. In that way we could get a relatively high signal at a lower concentration of target DNA. The results confirmed our speculation (Figure 9).</p>
+
                <img src="https://static.igem.org/mediawiki/2015/a/ad/Peking-CRISPR-Figure9.png">
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                <p class="text-center"><small><b>Figure 9. Bioluminescence intensity with multiple PC reporters. One, three, and five PC reporters were, respectively, subjected to the measurement using PCR product of 0.01nM.</b></small></p>
+
                <p>After validating the specificity and sensitivity of PC reporter system. We were eager to apply it to detect the real MTB genome. Fortunately, we got the absolutely safe MTB genomic DNA from Prof. LIU Cuihua (Chinese Academy of Sciences). </p>
+
                <p>First, we used the most popular commercial TB NAD toolkit (TB-LAMP) to amplify MTB genome and found out that there was a large yield of non-specific product after amplification (Figure 10a,b), which could be mistakenly considered as a positive result if using the conventional diagnosis method. However, our PC reporter system can easily distinguish the true from the false (Figure 10c).</p>
+
                <img src="https://static.igem.org/mediawiki/2015/5/55/Peking-CRISPR-Figure10.png">
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                <p class="text-center"><small><b>Figure 10. (a) Gel electrophoresis of amplification product of LAMP using MTB genome or control genome (E.coli). (b) Amount of amplified nucleic acid measured using the method provided in TB-LAMP toolkit. (c) Bioluminescence intensity generated by PC reporter, which is able to distinguish the true PCR product from the false.</b></small></p>
+
                <p>Then we set out to test whether our PC reporter could be applied to the clinical cases where patients might be in an early stage of TB thus the copies of MTB in their pathological samples could not be that many. The key factor is to figure out the minimum copy numbers of MTB genome in crude sample that can be detected by PC reporter when combined with PCR. We performed a series of dilution on the MTB genome before PCR amplification and found that, when the PCR products are subjected to the measurement, our PC reporter can easily reach the detection limit of only 1 genome copy per tube, which is sensitive enough for real TB diagnosis (Figure 11).</p>
+
                <img src="https://static.igem.org/mediawiki/2015/b/be/Peking-CRISPR-Figure11.png">
+
                <p class="text-center"><small><b>Figure 11. The effect of genome copies/mL for NAD amplification on the bioluminescence intensity of PC reporter.</b></small></p>
+
                <p><br><big><b>From single to array</b></big><br/></p>
+
                <p>We have demonstrated that our Paired dCas9 (PC) reporter system works well in bench and could be applied to the detection of MTB genomic DNA. Particularly, when combined with PCR, our PC reporter is as sensitive as to confidently detect only 1 genome copy per tube.</p>
+
                <p>However, in the clinical practice, the situations are much more complicated considering the fact that strain mutation, sample difference and other environmental factors would cause misdiagnosis for single test. So we got an idea of designing a diagnosis array to extract more sequence information for on our PC Reporter to test multiple sites on the target genome at one time, thus  to guarantee reliability of diagnosis.</p>
+
                <p>The core and also the first step of array design is to enumerate all the dCas9 binding site (PAM site) through the entire genome of MTB and to identify MTB-specific PAM sites as markers via SSPD. After computational screening, we totally obtained 2791 MTB-specific markers. 72 sgRNA pairs was designed according to 72 markers out of 9 fragments on MTB genome (Figure 12). Then we utilized Oligo Generator to design oligos for Golden Gate Cloning in a high-throughput manner, built sgRNA Generators for cell-free transcription, and finally obtained 70 sgRNA pairs (2 pairs failed during cloning).</p>
+
                <img src="https://static.igem.org/mediawiki/2015/a/a6/Peking-CRISPR-Figure12.png">
+
                  <p class="text-center"><small><b>Figure 12. 72 target sites (MTB-specific markers) out of 9 fragments on MTB genome, screened out using SSPD.</b></small></p>
+
                  <p>Then we performed array-based high-throughput measurement using PC reporter system (See Methods). Two separated assays were carried out using MTB and a control bacteria (E. coli), respectively. By data analysis, the results were presented as heat maps below (Figure 13). We can easily discriminate the results of MTB from that of control strain. Note that among 70 successfully constructed sgRNAs, 59 exhibit a significant performance for the detection of MTB; 11, however, fail to be positive. This is consistent with our concern that some factors might disrupt the performance of individual single test of PC reporter.  Reassuringly, the array-based PC reporter detection solved this problem (Figure 13).</p>
+
                  <img src="https://static.igem.org/mediawiki/2015/4/4e/Peking-CRISPR-Figure13.png">
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                  <p class="text-center"><small><b>Figure 13. Results of high-throughput assay for MTB and control strain. F denotes fragments obtained from MTB genome (a) or control strain (b); P denotes markers from each fragment.</b></small></p>
+
                </div>
+
  
                <h3 class="classic-title"  style="margin-top:50px"><span>Methods</span></h3>
 
                  <p><br><big><b>Clone construction of split luciferase-dCas9 fusion protein </b></big><br/></p>
 
                  <p>We cloned our split luciferase-dCas9 fusion protein in the pET-21a by Golden Gate assembly (Figure 14a,b).</p>
 
                  <img src="https://static.igem.org/mediawiki/2015/f/fa/Peking-CRISPR-Figure14.png">
 
                  <p class="text-center"><small><b>Figure 14. Plasmids of split luciferase-dCas9 fusion protein in pET-21a. (a) Plasmid of Nluc-dCas9 fusion architecture with T7 promoter, Cluc-dCas9 coding sequence and T7 terminator. (b) Plasmid of Cluc-dCas9 fusion architecture T7 promoter, Nluc-dCas9 coding sequence and T7 terminator.</b></small></p>
 
                  <p><br><big><b>Selection of test target sequence</b></big><br/></p>
 
                  <p>Before taking the sequence of Mycobacterium tuberculosis (MTB) genome as target of our PC reporter system, we first performed our experiments within a test target gene, which must not only conform to the substrate requirements of dCas9, but also harmless and safe enough to its bacteria carrier after chemical transformation. After screening over iGEM Registry of Standard Biological Parts, we finally took Part: BBa_K909009 (cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana (from iGEM12_ETH_Zurich) as the test target(Figure 15).</p>
 
                  <img src="https://static.igem.org/mediawiki/2015/0/06/Peking-CRISPR-Figure15.png">
 
                  <p class="text-center"><small><b>Figure 15. Schematic of Part: BBa_K909009.</b></small></p>
 
                  <p><br><big><b>sgRNA generation</b></big><br/></p>
 
                  <p>To obtain sgRNA, we used SSPD to screen out all guide sequences from the entire genome and Oligo Genrator to generate oligos for Golden Gate Cloning, then inserted all guide sequences into sgRNA Generator backbone (Figure 16). Through PCR amplification and cell-free transcription using HiScirbe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs), the sgRNA was produced and purified at last.We specially thank ZHAO Xuejin (Research Assistant, Institute of Microbiology, Chinese Academy of Sciences) for providing us with the sgRNA Generator!</p>
 
                  <img src="https://static.igem.org/mediawiki/2015/f/f9/Peking-CRISPR-Figure16.png">
 
                  <p class="text-center"><small><b>Figure 16. Schematic of sgRNA Generator backbone, containing T7 promotor, lacZa’, crRNA(tracrRNA) and T7 terminator.</b></small></p>
 
                  <p><br><big><b>Testing PC reporter system</b></big><br/></p>
 
                  <p>We invented a new protocol for testing our PC reporter system (Figure 17).</p>
 
                  <img src="https://static.igem.org/mediawiki/2015/4/41/Peking-CRISPR-Figure17.png">
 
                  <p class="text-center"><small><b>Figure 17. Flow chart for testing PC reporter system. Since one dCas9 fusion protein would form a complex with a pair of sgRNAs simultaneously, which will decrease the complementation of Nluc and Cluc, we incubated two split luciferase-dCas9:sgRNA complexes separately at 25°C for 10 min. After adding target DNA into the mixture of two complexes and incubated at 37°C for 30 min, they simultaneously bind to two adjacent half-sites on target DNA, bringing Nluc and Cluc into close proximity, thus leading to recovery of its enzymatic activity. Then transfer the mixture to 96-well plates before adding Luciferase Assay Reagent (Promega) and promoptly measure the luminescence in Microplate Reader (Thermo Scientific).</b></small></p>
 
                  <p><br><big><b>High-throughput array detection assay</b></big><br/></p>
 
                  <p>Based on the results we acquired, single-site target detection presents relatively high specificity and sensitivity. However, taking strain mutations, sample differences and other environmental factors in clinical practices into consideration, we set an array (Figure 18) to detect multiple target sites (markers) of MTB to make our PC reporter quantitatively reliable.</p>
 
                  <img src="https://static.igem.org/mediawiki/2015/6/61/Peking-CRISPR-Figure18.png">
 
                  <p class="text-center"><small><b>Figure 18. The 96-well plate layout in array detection assay. 9 fragments (F1-F9) screened out by SSPD from MTB genome were set in each line, and 8 MTB markers (M1-M8) in each fragment were respectively set in each sample pool. In total 8*9=72 markers were detected in one single assay. For each fragment, P9 meant a negative control with a mismatch sgRNA pair added. Two separated assays were carried out using MTB and a control strain (E. coli) as experimental subjects respectively</b></small></p>
 
                  </div>
 
  
                  <h3 class="classic-title"  style="margin-top:50px"><span>References</span></h3>
 
                  <p>1.WHO. Global Tuberculosis Report 2014[M]. World Health Organization, 2014.</p>
 
                  <p>2.Rodolphe Barrangou, Christophe Fremaux, Hélène Deveau, et al. CRISPR provides acquired resistance against viruses. Science, 2007, 315: 1709-1712.</p>
 
                  <p>3.John P Guilinger, David B Thompson, David R Liu. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature biotechnology, 2014, 32, 6.</p>
 
                  <p>4.Lei S. Qi, Matthew H. Larson, Luke A. Gilbert et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152: 1173-1183.</p>
 
                  <p>5.Sujan S Shekhawat, Indraneel Ghosh. Split-protein systems: beyond binary protein-protein interactions. Current Opinion in Chemical Biology, 2011, 15: 789–797.</p>
 
                  <p>6.Taha Azad, Amin Tashakor, Saman Hosseinkani. Split-luciferase complementary assay: applications, recent developments, and future perspectives. Anal Bioanal Chem, 2014, 406: 5541-5560.</p>
 
                  <p>7. Rossi F, Charlton C, Blau. H. Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc. Natl. Acad. Sci. 1994, USA 94: 8405–8410.</p>
 
                  <p>8. Remy I, Galarneau A, Michnick S W. Detection and visualization of protein interactions with protein fragment complementation assays. Methods Mol. Biol. 2002, 185: 447–459.</p>
 
                  <p>9. Kathryn E. Luker, Matthew C. P. Smith, et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS, 2004, 101: 12288-12293.</p>
 
                  <p>10. Ramasamy Paulmurugan and Sanjiv S. Gambhir. Combinatorial Library Screening for Developing an Improved Split-Firefly Luciferase Fragment-Assisted Complementation System for Studying Protein-Protein Interactions. Anal. Chem. 2007, 79: 2346-2353.</p>
 
                  <p>11. Jinek M, Chylinski K, Fonfara I et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096): 816-821.</p>
 
                  <p>12.Ramasamy Paulmurugan, Sanjiv S. Gambhir. An intramolecular folding sensor for imaging estrogen receptor-ligand interactions. The National Academy of Sciences of the USA, 2006, 103: 15883-15888.</p>
 
                  <p>13. Bruce Alberts et al. Molecular Biology of the Cell. Fifth edition.</p>
 
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Revision as of 08:26, 18 September 2015

Project

Study how our work affects the world, and how the world affects our work.

Questionnaire

Questionnaire Report

Investigation Report on the Cognition of Tuberculosis and Synthetic Biology


Abstract

Objective
To investigate the current awareness rate of tuberculosis and synthetic biology knowledge among Chinese people so as to provide the evidence of tuberculosis health education and synthetic biology education in Chinese people.

Methods
194 tourists around the Weiming Lake of Peking University from August 16th to August 18th were selected by random sampling. The anonymous questionnaire was of our own design.

Results
The awareness rates of the sources, routes of transmission, clinical symptoms, severity and treatment effect of tuberculosis are high, which accounted for 70%-90% among 194 tourists. However, the awareness rates of the diagnosis methods, the specific treatment methods and the national prevention and control policy of tuberculosis are less than 40%. As for the part of cognitive attitude of synthetic biology, only 6.2% of the tourists have a deeper understanding of synthetic biology, while 33.3% of the respondents have never heard of synthetic biology. The awareness rates of the selected terms about the field of synthetic biology are generally lower than 35%, of which 58.1% and 52.8% of the tourists have access to relevant knowledge through the Internet and television, respectively. 76% of the tourists agree that applying synthetic biology methods to clinical diagnosis is feasible, and 86% of the visitors are willing to use the relevant technology products if possible, the main reasons of which are rapidness, individuation and the reliable results of the new detection technology. To the contrary, the tourists who are not willing to use that detection technology mainly worry about the potential side effects, and they believe that traditional detection methods are mature enough.

Conclusion
Chinese people have certain knowledge of tuberculosis, but not systematic or comprehensive; they have a low awareness rate of synthetic biology, which means its popularization needs to be strengthened.

Keywords  Tuberculosis; Synthetic Biology; Cognition


Objects and Methods


Objects
194 tourists around the Weiming Lake of Peking University from August 16th to August 18th

Methods
Respondents were selected by random sampling in our investigation. The anonymous questionnaire was of our own design. A total of 200 questionnaires were distributed, and 194 questionnaires were collected, the effective recovery rate was 97% (Table 1).

Statistical analysis:
Using EpiData3.1 to import the questionnaire data based on the methods of data entry, thus to establish a database; then SPSS20.0 was used to statistical analysis.

Results

General demographic situation

Gender and age structure

A total of 194 questionnaires were collected in this investigation, of which the proportion of men was 48.7%; for female, it was 50.8%. Most of the population was among the 15-50 years old age group, accounting for 82.3%. Therefore, the composition of gender and age of the population in the survey was different from the national population in some extent (Table 1).

Region

Among the 194 respondents, 139 were from urban, accounting for 72.4%, and 53 were from rural, accounting for 27.6%.

Education level and economic level.

In the survey population, the educational background of the majority was undergraduate degree or above, accounting for 61.5%. The economic levels of most respondents are not bad because 71.4% of whose family income were more than 2000 yuan.

Table.1 The General Demographic Situation

Aspects Options Percentage Ratio
1.Age Below 15 13.5 26/192
15-30 35.9 69/192
30-50 46.4 89/192
50-70 4.2 8/192
Above 70 0.0 0/192
2.Gender Male 50.8 98/193
Female 48.7 94/193
3.Census register Rural 27.6 53/192
Urban 72.4 139/192
4.Degree of education Untutored 0.5 1/192
Grade school 6.3 12/192
Junior high school 13.0 25/192
High school 18.8 36/192
Undergraduate or graduate 61.5 18/192
5. Month-earning of the family Below 500 RMB 1.0 2/192
500-1999 RMB 10.4 20/192
2000-4999 RMB 40.1 77/192
Above 5000 RMB 31.3 60/192
Unknown 17.2 33/192

Awareness of tuberculosis

Most of the 194 tourists were well aware of the core issues of tuberculosis. In detail, the awareness rate of severity, the infection sources and the transmission routes of tuberculosis were 77.8%, 71.1% and 70.1%, respectively. And the awareness rate of the clinical symptoms of tuberculosis was as high as 88.7%. However, the national tuberculosis control policy awareness is much worse, only 40 people answer the related question correctly, accounting for 20.6% of the survey population. And the respondents who can answer all of the six core questions accounted for only 6.9%. As for the knowledge about specific diagnostic methods, treatment methods of the tuberculosis, the results were barely satisfactory, the awareness rate of which were just 34.5% and 7.2%, respectively.

Table.2 The Acknowledgment of Tuberculosis

Crucial questions The percentage of correctly answered(%) Ratio
1. Do you think the Tuberculosis is a disease greatly threatened human health? 77.8 151/194
2. Which is main source of infection? 71.1 138/192
3. Which is main transmission route of TB? 70.1 136/194
4. If someone get cough and expectoration more than two weeks with Hemoptysis and blood sputum. What should he or she do? 88.7 172/194
5. Whether the tuberculosis can be cured? 90.7 176/194
6. Are the drugs and treatment services for the infectious tuberculosis patients free? Male 20.6 40/194
The other question The percentage of correctly answered(%) Ratio
7. TB can be confirmed by which of the following symptom? 34.5 67/194
8. Which drugs are for the tuberculosis treatment? 7.2 14/194
9. What’s your idea about the positive result of PPD test? 36.1 70/194

Awareness of synthetic biology

Among the 194 tourists, only 12 of them has a deep understanding about synthetic biology, while 33.3% of visitors have never heard of synthetic biology. Among the people who had heard of synthetic biology, most of them had not heard of the specific professional terms in the field of synthetic biology, such as "bio-element", "molecular machine", "bio-plastics". The awareness rates of these terms were 26.4%, 21.2%, 31.1%, respectively. It deserves to be mentioned that, 68.4% of the respondents said that they had heard the term "genome", but unfortunately, only 3.6% of people had heard of iGEM. And the main ways the respondents use to access to the knowledge of synthetic biology was through the network (58.1%), the television (52.8%) and classroom lectures (30.4%).

Table.3 The Acknowledgment of Synthetic Biology

Questions Options Percentage Ratio
1. Have you ever heard of Synthetic Biology? Yes. And have some knowledge of it 6.2 12/192
Yes. But have no idea about it 59.4 114/192
No 33.3 64/192
2. Which of the following nouns you have heard before? Bio-element 26.4 51/193
Molecule machine 21.2 41/193
Gene Sequence 68.4 132/193
iGEM 3.6 7/193
Bioplastics 31.1 133/193
None of them 21.2 41/193
3. Where you heard the above nouns? Network 58.1 93/160
TV and radio 52.8 85/161
Newspapers 19.3 31/161
Neighbors 3.1 5/161
Classes and lectures 30.4 49/161
The others 10.5 17/162

Attitudes toward synthetic biology

Among the tourists who had heard of synthetic biology, the attitudes toward synthetic biology of the majority were neutral. To be specific, 70.5% of the population held the idea that synthetic biology had both advantages and disadvantages. About 76% of the population were optimistic about the development of the diagnostic methods combining the methods in synthetic biology. And 86% of the investigated people were willing to use the synthetic biology methods if possible, the main reasons were rapidness (46.4% of population choose the answer) and the individuation (51.8% of population believe this point) of the new detection technology, in addition, 37.5% of the population believes that the method is more reliable. To the contrary, the tourists who were not willing to use that detection technology mainly worry about the potential side effects (39.3% of population believe this point), and 40.7% of the population believe that traditional detection methods haven’t been well developed. (Table four)

Table.4 The Atittude Towards Synthetic Biology

Questions Options Percentage Ratio
1. What’s your attitude to the Synthetic Biology? Have no advantages 0.8 1/129
Have no disadvantages 3.9 5/129
Have both advantages and disadvantages 70.5 91/129
Have no idea 24.0 31/129
2. Do you think it possible to invent a new method of TB diagnosis make use of the knowledge of Synthetic Biology? Sure 76.0 98/129
No way 0.8 1/129
Have no idea 20.9 27/129
3. If such a method has been invented, would you like to try it? Yes 86.0 111/129
No 13.2 17/129
4. Why you would like to have a try? Cheap 8.0 9/129
Fast and convenient 46.4 52/112
Personalized medicine service 51.8 58/112
Efficacy is better 37.5 42/112
Other reason 0.0 0/112
5.Why you don’t do that? The potential side effect 39.3 12/28
It will pollute the environment 7.4 2/27
It will be expensive 18.5 5/27
The technique is not well developed 40.7 11/27
6. How much would you like to spend on such a method for your health? Less than 10 yuan 1.6 2/123
10 yuan-100 yuan 6.5 8/123
100 yuan-500 yuan 22.8 28/123
500 yuan-1000 yuan 25.2 31/123
1000 yuan-5000 yuan 36.6 45/123
More than 5000 yuan 6.5 8/123

Conclusions

Through this investigation, we recognize that Chinese people have a certain understanding of the basic knowledge of tuberculosis, but it is not comprehensive and systematic, especially details about the disease. The majority of the population of the survey has a neutral attitude towards synthetic biology, and most of them are optimistic about the applications and progress of synthetic biology, though only a small portion of people knows this field very well.
Therefore, on the one hand, it is necessary to strengthen the popularization of the knowledge of tuberculosis in public. The process of propaganda to the national for health education of tuberculosis should not be a slogan, and it should be in-depth and systematic. On the other hand, researchers in the field of synthetic biology should actively work for the science popularization of synthetic biology at the same time they are working for the scientific research. They are responsible for improving the people’s understanding of the synthetic biology, and attracting more people to join the development trend of synthetic biology.

Education for children

To attract children’s more attention toward synthetic biology and to popularize some basic biological concepts among them, we invited near 20 pupils to visit our laboratory. In the morning of August 17th, these children and some of their parents came to Peking University and attended a lecture given by ZHANG Yihao, the team leader.

By plain words, he made it easy for these children to understand the basic concepts of biological field and the core ideas of synthetic biology. What is the basic substance of life? What caused creatures on the earth so diversified and fascinating? Moreover, can scientists build life with fancy functions? They got the answers to all these questions above.

In the afternoon, they visited the lab of Peking iGEM. Having known how the biological researchers work in the labs, they all indicated that they hope to learn more about synthetic biology.

Education for High School Students

On August 23th, our team member YE Yixuan went to Zhenghe No.1 Middle School of Fujian Province, giving a lecture to the high school students. She mainly introduced the overview of synthetic biology and the project of Peking this year.

More than 60 students who were interested in science have attended the lecture. They were confronted with the choice of their major fields in the university, so the lecture really helped them. Ye combined the basic concepts of synthetic biology with what they had learned in school. After the lecture, a heated discussion about biology and future life began. What’s more, the project of Peking iGEM team this year was presented, for the purpose of arousing their interest in this field.

Education for Adults

Handing out propagandistic handbooks is another approach of scientific education for public. In this summer, we have wrote a comprehensive literature review about tuberculosis and synthetic biology and made a propagandist handbook easy for learning.

Every time when we finished our questionnaire surveys, we gave our informants handbooks to spread the knowledge of TB control and synthetic biology.

In the handbook, we introduce the basic information of tuberculothesis, such as route of transmission, diagnosis methods and its treatment, along with the national policies of free diagnosis and treatment on tuberculosis. After that, we give a brief description of the history of synthetic biology, combined with its application. In such a small handbook, many vivid but scientific pictures are shown. We did our best to make the words easy to be understood but rigorous.