Difference between revisions of "Team:Peking/Interlab"

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            <h1>Interlab<span>.</span></h1>
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              <h1>Attributions<span>.</span></h1>
            <p class="title1" style="text-align:center">The Peking iGEM 2016 team is participating in the third year of the iGEM Interlab study along with about 100 teams.We focused on quantify expression of GFP in common, comparable or absolute units. </p>
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              <p class="title1" style="text-align:center">The idea of this project was conceived by the team; advisors and instructors gave advices and suggestions on the implementation of this idea.</p>
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    <h4>Attributions</h4>
    <div id="primary" class="twelve columns">
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      <ul>
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          <li><a href="#members">Members'</a></li>
      <section>
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          <li><a href="#acknowledgement">Acknowledgement</a></li>
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           <li><a href="#sponsors">Sponsors</a></li>
             
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  <h4>Methods</h4>
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    <ul>
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        <li><a href="#background">Background</a></li>
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        <li><a href="#design">Design</a></li>
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        <li><a href="#materials">Materials</a></li>
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        <li><a href="#protocol">Protocol</a></li>
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    </ul>
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  <h4>Results</h4>
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  <ul>
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        <li><a href="#sequencing">Sequencing</a></li>
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        <li><a href="#data">Data</a></li>   
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                <a id="background"></a>                           
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                <h3 class="classic-title";"><span>Background</span></h3>
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                <p>“All of the 2016 iGEM teams are invited and encouraged to participate in the Third International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.
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</p>
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                <a id="design"></a>                               
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                <h3 class="classic-title";"><span>Design</span></h3>
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                <p>Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). While this is an indirect measurement, it provides a useful insight into expression levels and has the significant advantage that it can be monitored continuously without disrupting cells.
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</p>
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                <p>Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.</p>
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                <p>We aim to do this using the supplied FITC as a standard reference material. You will measure the fluorescence of your instrument using a dilution series of this reference material to construct a standard curve. We have previously performed this standard curve on our own instrument alongside a standard curve for purified GFP. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.</p>
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                <p>However, we aim to control for instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.</p> 
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                <a id="materials"></a>                              
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                <h3 class="classic-title";"><span>Materials and methods</span></h3>
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              <div> 
                <h5>Used plasmids</h5>
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                  <a id="members"></a>    
                <p style="margin:0 0 0 0">•  Plasmid DNA (100 pg/uL in 10uL of Buffer EB)</p>
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                  <h3 class="classic-title";"><span>Member’s Attributions</span></h3>                      
                <div style="padding-left:20px">
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                  <p>All the data presented on this wiki was measured, collected, and analyzed by the team members. All the plasmids directly involved in the data presented on this wiki were constructed by the team members. The idea conceiving, content planning, execution, and result analysis of human practice were all done by the team members. The following is the detailed attribution:
                <p>
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  </p>
                o Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3<br>
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                  <p>
                o Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3<br>
+
                  <strong>LI Cheng</strong> is the team leader of Peking iGEM 2016. He participated in conceiving the project, took charge of troubleshooting, and managed the experiments, guaranteeing the normal operation of the lab.<br/>
                o Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3<br>
+
                  <strong>YANG Xiaoyu</strong> was committed to ameliorate the paired dCas9 (PC) reporter system, namely, to enhance the sensitivity and robustness. Early in the project, he was also responsible for information gathering, experiment design, and lab management<br/>
                o Positive Control Device: I20270 in pSB1C3 Also located in Kit Plate 3, well 8P<br>
+
                o Negative Control Device: R0040 in pSB1C3 Also located in Kit Plate 2, well 6F<br>
+
                </p>
+
                </div>
+
           
+
  
                <h5>Used strain</h5>
+
                  <strong>BAI Ke</strong> was in charge of Human Practice and also worked in the wet lab to construct and prepare plasmids for gRNA in vitro transcription.<br/>
                <p><i>Escherichia coli</i> TOP10</p>
+
                  <strong>DONG Yiming</strong>
               
+
                  was responsible for designing and manufacturing the portable electronic device that convert optical signal into analogue electrical signal.<br/>
                <h5>Used material</h5>
+
                  <strong>LI Yuexuan</strong> worked as the financial manager of the team. She was also responsible for plasmid construction and submission.<br/>
                <p>
+
                  <strong>YANG Changru</strong> was in charge of building <i>M.tuberculosis</i>-sensing system using Molecule Beacon. He was also engaged in the protein purification of dCas9 fusion proteins.<br/>
                • FITC Standard: one tube with dried down FITC for creating a FITC standard<br>
+
                  <strong>ZHAO Shijun</strong> was in charge of measuring the luminescence intensity of PC reporter system. She analyzed the entire experimental data.<br/>
                • LUDOX: one tube with 30% colloidal silica suspended in 1mL of water<br>
+
                  <strong>LV Nayun</strong> helped to complete the plasmid construction of sgRNA and was in part responsible for human practice.<br/>
                • 1xPBS (phosphate buffered saline)<br>
+
                  <strong>WANG Dingyu</strong> was in charge of the construction of CRISPR gRNA as well as RNA scaffold in the beginning. Later she was working on the purification of dCas9 fusion protein.<br/>
                • Terrific broth (at half strength: 0.5x TB) or can use LB (Luria Bertani) media as an alternative<br>
+
                  <strong>LI Jiamian</strong> took the task of cloning dCas9-luciferase. She was also responsible for the testing of PC reporter system.<br/>
                • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)<br>
+
                  <strong>WEI Jingyi</strong> participated in plasmid construction, protein purification, and wiki building. She was also the designer of our PowerPoint slides.<br/>
                • 50 ml Falcon tube (or equivalent) or 250 ml shake flask for cell growth<br>
+
                  <strong>WANG Yuqing</strong> was in charge of Modeling. His main task was to computationally search for the suitable guide sequences. Also, he was the lab manager for the normal operation of our lab.<br/>
                • 1.5 ml eppendorf tubes for sample storage<br>
+
                  <strong>TENG Huaiyuan</strong>was in charge of Modeling. His main task was to computationally search for the suitable guide sequences. Also, he was the lab manager for the normal operation of our lab.<br/>
                • Ice bucket with ice<br>
+
                  </p>
                • Pipettes<br>
+
              </div>
                • 96 well plate <br>
+
     
</p>
+
              <div>
               
+
                  <a id="acknowledgement"></a>  
                <h5>Used machines</h5>
+
                    <h3 class="classic-title";"><span>Acknowledgement</span></h3>                             
                <p>
+
                    <p>We'd like to thank all those who have helped us over the summer, without whose sincere support The Uranium Reaper Project goals would have not been achieved.
                • Thermo VARIOSKAN FLASH<br>
+
  </p>
                • MAPADA UV-3100PC SPECTROPHOTOMETER<br>
+
                 
                • YKKY(FM40)<br>
+
                  <h4>General support</h4>
                • AISITE electro-heating standing-temperature cultivator<br>
+
                  <p><strong>Prof. OUYANG Qi, Prof. LOU Chunbo, Dr. ZHANG Haoqian</strong> and other teachers helped us during our brainstorming and gave us useful suggestions.<br>
                • HONOUR INCURATOR SHAKER<br>
+
                  <strong>Prof. OUYANG Qi</strong> and<strong> Prof. LIU Chunli</strong> kindly provided the laboratory to us for experiments.
               
+
                  </p>
</p>
+
  
                <h5>Used software</h5>
+
                  <h4>Materials support</h4>
                <p>
+
                  <p><strong>Dr. ZHANG Wenbing</strong> provided us with the plasmid of 3B(Tri-SpyCatcher within 15X linker).<br>
                • Microsoft Excel<br>
+
                      <strong> Prof. ZHOU Lu</strong> provided us with Uranyl-binding Protein, also called SUP.<br>
               
+
                      <strong>Prof. LIU Chunli</strong>allowed us to use his lab to do experiments on uranyl ion. The uranyl nitrate solution and Arsenazo III was provided by Prof. LIU.<br>
</p>
+
                    <strong>Prof. LOU Chunbo</strong> provided us with the strain B.Subtilis and mSA, heavy metal ions binding proteins: lead-binding protein, mercury-binding protein and cadmium-binding protein.<br>
 +
                    <strong>Dr. DU Pei</strong> provided the beads to us and gave us experimental guidance.<br>
 +
                    <strong>BIT-China</strong> gave us the important Interlab kit.
 +
                  </p>
  
                <h5>Used methods</h5>
+
                  <h4>Experiment equipment support</h4>
                <p style="margin:0 0 0 0">•  Calibration</p>
+
                  <p><strong>Prof. OUYANG Qi</strong> lent the Akta Pure to us for all proteins purification.<br>
                <div style="padding-left:45px">
+
                      <strong>Prof. CHANG Zengyi</strong> and <strong>Dr. WANG Qingsong</strong> provided us with Scanner and helped with the setup and scan of gels.<br>
                <p>
+
                    <strong>Prof. LIU Chunli</strong> provided us with Geiger counter to test the radiation level in and around our lab.<br>
                o OD600 Reference point<br>
+
                    <strong>Core Facilities</strong>, School of Life Sciences, Peking University,assisted us with ITC.<br>
                o FITC fluorescence standard curve<br>
+
                    <strong>the State Key Laboratory of environmental simulation and pollution control</strong> provided the Aurora M90 mass spectrograph.
                o Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3<br>
+
                  </p>
                </p>
+
                </div>
+
                <p style="margin:0 0 0 0">•  Cell measurement</p>
+
                <div style="padding-left:45px">
+
                <p>
+
                o Transformation<br>
+
                o Measurements <br>
+
                </p>
+
                </div>
+
  
 +
                  <h4>Theoretical guidance</h4>
 +
                  <p> <strong>ZHANG Yihao</strong> and <strong>WANG Yu</strong>,The wiki designers of Peking iGEM 2015,gave us many suggestions about wiki building.<br>
 +
                      <strong>Dr. YU Daqi</strong> of Prof. OUYANG Qi’s lab helped us a lot in experimental operating and data analysis.<br>
 +
                      <strong>Prof. ZHANG Wenbing, Prof. LOU Chunbo</strong> and <strong>Dr. YU Daqi</strong> kindly helped our team with modeling of the project.<br>
 +
                    <strong>Prof. OUYANG Qi, Prof. LOU Chunbo, Dr. ZHANG Haoqian</strong> and <strong>ZHANG Yihao</strong>,the instructors, coached us for the presentation.<br>
 +
                      <strong>Prof. LAI Luhua</strong> and <strong>Prof. RAO Yi</strong> participated in our presentation rehearsal and gave suggestions for revisions.
 +
                  </p>
 +
                   
 +
                  <h4>Synthetic biology course</h4>
 +
                  <p>From March to June, an introductory course of synthetic biology was hold by College of Life Sciences, Peking University.During the course, the teachers, also the instructors gave us kind guidance and judged our project design. In late June, we started in the lab to start our project.
 +
                  </p>
 +
              </div>
 +
             
  
</div>
+
              <div>  
 
+
                  <a id="sponsors"></a>                              
 
+
                 <h3 class="classic-title";"><span>Sponsors</span></h3>  
                  <a id="protocol"></a>
+
              <div class="row,row1">
                 <h3 class="classic-title";"><span>Protocols</span></h3>
+
              <ul class="footer-social">
                  <div style="padding-left:45px">
+
                              <li class="col-md-6" id="PKU-administration" style="margin-bottom:25px;max-width:300px">
                <p><a href="#">>>Calibration</a><br>
+
                                  <a href="http://dean.pku.edu.cn/pkudean/index.html"><img src="https://static.igem.org/mediawiki/2016/2/2e/T--Peking--images_sponsors_PKU_Administration.png"></a>
                <a href="#">>>Cell measurement</a></p>
+
                              </li>
 +
                              <li class="col-md-6" id="PKU-SLS" style="margin-bottom:25px;max-width:300px">
 +
                                  <a href="http://www.bio.pku.edu.cn/"><img src="https://static.igem.org/mediawiki/2016/7/74/T--Peking--images_sponsors_PKU_SLS.png"></a>
 +
                              </li>
 +
                              <li class="col-md-6" id="IMCAS" style="margin-bottom:25px;max-width:300px">
 +
                                  <a href="http://english.im.cas.cn/"><img src="https://static.igem.org/mediawiki/2016/7/77/T--Peking--images_sponsors_PKU_IMCAS.png"></a>
 +
                              </li>                           
 +
                              <li class="col-md-6" id="PKU-CQB" style="margin-bottom:25px;max-width:300px">
 +
                                  <a href="http://cqb.pku.edu.cn/en/"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Peking--images_sponsors_PKU_CQB.png"></a>
 +
                              </li>
 +
                              <!--<li class="col-md-6" id="BluePha" style="margin-bottom:25px; max-width:300px">
 +
                                  <a href="http://www.bluepha.com/"><img src="images/PKU/Bluepha-logo.png"></a>
 +
                              </li>-->
 +
                              <li class="col-md-6" id="PKU-CCME" style="margin-bottom:25px;max-width:300px">
 +
                                  <a href="http://www.chem.pku.edu.cn/index.php?styleid=2"><img src="https://static.igem.org/mediawiki/2016/b/ba/T--Peking--images_sponsors_PKU_CCME.png"></a>
 +
                              </li>
 +
                          </ul>
 
                 </div>
 
                 </div>
 +
        </div>
 +
                </div> <!-- row End -->
 +
               
 
                  
 
                  
 
+
               </section> <!-- section end -->
                <h3 class="classic-title";"><span>Description</span></h3>
+
          </div> <!-- primary end -->
                <p>Plasmids containing promoters and GFP were taken from The 2016 DNA Distribution Kit and all devices were transformed into E.coli. Fluorescence of colonies was checked up under UV light. </p>
+
      </div> <!-- page-content End-->
                <p>5 ml of liquid LB-M medium with chloramphenicol were inoculated with two chosen colonies of each device. Liquid cultures were incubated for 16~18 hours in HONOUR INCURATOR SHAKER placed in incubator. OD of these cultures was measured by MAPADA UV-3100PC SPECTROPHOTOMETER and diluted to 0.02. Fluorescence of biological and also technical replicates was measured using Thermo VARIOSKAN FLASH following our protocol.
+
    </div> <!-- Content End-->
                </p>
+
 
+
                <h3 class="classic-title";"><span>Results</span></h3>
+
                  <a  id="sequencing"></a>
+
                <h5>Sequencing</h5>
+
                <p> • Device 1: J23101+I13504<br>
+
                    • Device 2: J23106+I13504<br>
+
                    • Device 3: J23117+I13504<br>
+
                    • Positive Control Device: I20270 in pSB1C3<br>
+
                    • Negative Control Device: R0040 in pSB1C3 </p>
+
<p>
+
>BBa_J23101 Part-only sequence (35 bp)<br>
+
    <span style="padding-left:45px">Tttacagctagctcagtcctaggtattatgctagc</span></p><p>
+
>BBa_J23106 Part-only sequence (35 bp)<br>
+
    <span style="padding-left:45px">Tttacggctagctcagtcctaggtatagtgctagc</span></p><p>
+
>BBa_J23117 Part-only sequence (35 bp)<br>
+
    <span style="padding-left:45px">Ttgacagctagctcagtcctagggattgtgctagc</span></p><p>
+
>BBa_I13504 Part-only sequence (875 bp)<br>
+
    <span style="padding-left:45px">Aaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata</span></p><p>
+
>BBa_I20270 Part-only sequence (919 bp)<br>
+
    <span style="padding-left:45px">Ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata</span></p><p>
+
>BBa_R0040 Part-only sequence (54 bp)<br>
+
    <span style="padding-left:45px">tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac</span>
+
</p></p>
+
                 
+
                  <a  id="data"></a>
+
                  <h5>Data</h5>
+
                  <p><b>2.1 OD600 Reference Point</b></p>
+
                  <p><b>2.2 FITC Standard Curve</b></p>
+
                    <p><b>2.3 Normalisation</b></p>
+
                    <p><b>2.4 Cell Measurement</b></p>
+
               
+
 
+
                  <h5>Discussion</h5>
+
                  <p>It is noticeable that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3.</p>
+
 
+
                  <h3 class="classic-title";"><span>Appendix</span></h3>
+
                  <p>Individuals responsible for conducting InterLab study
+
• Dong Yiming measured the devices.
+
• Li Cheng processed the data.
+
                  </p>
+
       
+
 
+
 
+
</div>
+
              </div> <!-- row End -->
+
             
+
                
+
            </section> <!-- section end -->
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+
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Revision as of 15:03, 29 September 2016

  
  
  
  
      
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Attributions.

The idea of this project was conceived by the team; advisors and instructors gave advices and suggestions on the implementation of this idea.

Member’s Attributions

All the data presented on this wiki was measured, collected, and analyzed by the team members. All the plasmids directly involved in the data presented on this wiki were constructed by the team members. The idea conceiving, content planning, execution, and result analysis of human practice were all done by the team members. The following is the detailed attribution:

LI Cheng is the team leader of Peking iGEM 2016. He participated in conceiving the project, took charge of troubleshooting, and managed the experiments, guaranteeing the normal operation of the lab.
YANG Xiaoyu was committed to ameliorate the paired dCas9 (PC) reporter system, namely, to enhance the sensitivity and robustness. Early in the project, he was also responsible for information gathering, experiment design, and lab management
BAI Ke was in charge of Human Practice and also worked in the wet lab to construct and prepare plasmids for gRNA in vitro transcription.
DONG Yiming was responsible for designing and manufacturing the portable electronic device that convert optical signal into analogue electrical signal.
LI Yuexuan worked as the financial manager of the team. She was also responsible for plasmid construction and submission.
YANG Changru was in charge of building M.tuberculosis-sensing system using Molecule Beacon. He was also engaged in the protein purification of dCas9 fusion proteins.
ZHAO Shijun was in charge of measuring the luminescence intensity of PC reporter system. She analyzed the entire experimental data.
LV Nayun helped to complete the plasmid construction of sgRNA and was in part responsible for human practice.
WANG Dingyu was in charge of the construction of CRISPR gRNA as well as RNA scaffold in the beginning. Later she was working on the purification of dCas9 fusion protein.
LI Jiamian took the task of cloning dCas9-luciferase. She was also responsible for the testing of PC reporter system.
WEI Jingyi participated in plasmid construction, protein purification, and wiki building. She was also the designer of our PowerPoint slides.
WANG Yuqing was in charge of Modeling. His main task was to computationally search for the suitable guide sequences. Also, he was the lab manager for the normal operation of our lab.
TENG Huaiyuanwas in charge of Modeling. His main task was to computationally search for the suitable guide sequences. Also, he was the lab manager for the normal operation of our lab.

Acknowledgement

We'd like to thank all those who have helped us over the summer, without whose sincere support The Uranium Reaper Project goals would have not been achieved.

General support

Prof. OUYANG Qi, Prof. LOU Chunbo, Dr. ZHANG Haoqian and other teachers helped us during our brainstorming and gave us useful suggestions.
Prof. OUYANG Qi and Prof. LIU Chunli kindly provided the laboratory to us for experiments.

Materials support

Dr. ZHANG Wenbing provided us with the plasmid of 3B(Tri-SpyCatcher within 15X linker).
Prof. ZHOU Lu provided us with Uranyl-binding Protein, also called SUP.
Prof. LIU Chunliallowed us to use his lab to do experiments on uranyl ion. The uranyl nitrate solution and Arsenazo III was provided by Prof. LIU.
Prof. LOU Chunbo provided us with the strain B.Subtilis and mSA, heavy metal ions binding proteins: lead-binding protein, mercury-binding protein and cadmium-binding protein.
Dr. DU Pei provided the beads to us and gave us experimental guidance.
BIT-China gave us the important Interlab kit.

Experiment equipment support

Prof. OUYANG Qi lent the Akta Pure to us for all proteins purification.
Prof. CHANG Zengyi and Dr. WANG Qingsong provided us with Scanner and helped with the setup and scan of gels.
Prof. LIU Chunli provided us with Geiger counter to test the radiation level in and around our lab.
Core Facilities, School of Life Sciences, Peking University,assisted us with ITC.
the State Key Laboratory of environmental simulation and pollution control provided the Aurora M90 mass spectrograph.

Theoretical guidance

ZHANG Yihao and WANG Yu,The wiki designers of Peking iGEM 2015,gave us many suggestions about wiki building.
Dr. YU Daqi of Prof. OUYANG Qi’s lab helped us a lot in experimental operating and data analysis.
Prof. ZHANG Wenbing, Prof. LOU Chunbo and Dr. YU Daqi kindly helped our team with modeling of the project.
Prof. OUYANG Qi, Prof. LOU Chunbo, Dr. ZHANG Haoqian and ZHANG Yihao,the instructors, coached us for the presentation.
Prof. LAI Luhua and Prof. RAO Yi participated in our presentation rehearsal and gave suggestions for revisions.

Synthetic biology course

From March to June, an introductory course of synthetic biology was hold by College of Life Sciences, Peking University.During the course, the teachers, also the instructors gave us kind guidance and judged our project design. In late June, we started in the lab to start our project.

Sponsors