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| | {{BNU-CHINA/partials/header}} | | {{BNU-CHINA/partials/header}} |
| − | {{BNU-CHINA/partials/nav | team=active}} | + | {{BNU-CHINA/partials/nav | TEAM=active}} |
| − | {{BNU-CHINA/article/start | Project | Safety}} | + | {{BNU-CHINA/article | Team | We are Sirens |9/95/Bnubgl}} |
| | + | |
| | <html> | | <html> |
| − | <html lang="en"> | + | <!-- |
| | + | <body onload="javascript: document.location.href='https://2015.igem.org/Team:BNU-CHINA/Team#attribution'"> |
| | + | </body> |
| | + | --> |
| | + | <h2 id="attribution">Attribution</h2> |
| | + | <div class="panel-group" id="accordion"> |
| | + | <div class="panel panel-info"> |
| | + | <div class="panel-heading"> |
| | + | <h3 class="panel-title">Project Design and Wet Lab Experiments</h3> |
| | + | </div> |
| | | | |
| − | <body> | + | <div class="panel-body"> |
| | + | Under the supervision of <strong>Prof. Zhu Xudong</strong>, <strong>Prof. Yang Dong</strong>, and <strong>Prof. Xiang Benqiong</strong> etc, nearly all team members have taken part in the project designing process, especially those of wet lab group. <strong>Beichen Gao</strong> , <strong>Yuanyi Dai</strong> , <strong>Yufei Cao</strong> and <strong>Jia Li</strong> are the group leaders who mainly designed the experimental patterns and guided different parts of the project. |
| | + | </div> |
| | + | </div> |
| | | | |
| − | <div class=" content container"> | + | <div class="panel panel-info"> |
| − | <sidebar class="col-md-3 bs-docs-sidebar">
| + | <div class="panel-heading"> |
| − | <ul id="sidebar" class="nav nav-stacked fixed">
| + | <h3 class="panel-title">Modeling</h3> |
| − | <li><a href="#section-1">Attribution</a>
| + | </div> |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#section-2">Collaboration</a>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#section-3">Aknowledgement</a>
| + | |
| − | <ul class="nav nav-stacked">
| + | |
| − | <li><a href="#section-3-1">Thanks</a></li>
| + | |
| − | <li><a href="#section-3-2">Sponsors</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#section-4">Notebook</a>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#section-5">Protocol</a>
| + | |
| − | <ul class="nav nav-stacked">
| + | |
| − | <li><a href="#section-5-1">Cloning</a></li>
| + | |
| − | <li><a href="#section-5-2">Transformation</a></li>
| + | |
| − | <li><a href="#section-5-3">Detection</a></li>
| + | |
| − | <li><a href="#section-5-4">Nematode</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </sidebar>
| + | |
| | | | |
| − | <article class="col-md-9">
| + | <div class="panel-body"> |
| − | <header class="page-header">
| + | <strong>Cheng Li</strong> is the leader of modeling group, who mainly enrolled in designing the device 2.0. <strong>Yuan Yin</strong> , <strong>Qiuyue Yuan</strong> and <strong>Xueting Zhao</strong> also do a great many works in the modeling group, such as information searching, paper writing and equipment appilication. |
| − | <h1>Team <small>Beijing Normal University</small>
| + | </div> |
| − | </h1>
| + | </div> |
| | | | |
| − | </header>
| + | <div class="panel panel-info"> |
| − | <section id="section-1" class="group">
| + | <div class="panel-heading"> |
| | + | <h3 class="panel-title">Wiki Construction</h3> |
| | + | </div> |
| | + | <div class="panel-body"> |
| | + | <strong>Jiajun Zhang</strong> , who has led the web design group to build the wiki, together with group members <strong>Zhiyao Chen</strong> and <strong>Chengfei Peng</strong> complete the main work in database construction and maintenance. |
| | + | </div> |
| | + | </div> |
| | | | |
| − | <h3>1. Attribution</h3>
| + | <div class="panel panel-info"> |
| − | <div class="panel- group" id=" accordion"> | + | <div class="panel-heading"> |
| | + | <h3 class="panel-title">Graphic design</h3> |
| | + | </div> |
| | | | |
| − | <div class=" panel panel- default"> | + | <div class="panel-body"> |
| − | <div class="panel-heading">
| + | <strong>Lu Xu</strong>( group leader), <strong>Lee</strong> and <strong>Yalei Cao</strong> really do a fantastic work in graphic and web design. They all make great efforts in designing our team logo, website, poster, souvenir, T-shirt and so on. |
| − | <h4 class="panel-title"><a data-toggle="collapse" data-parent="#accordion" href="#collapseOne">Project Design and Wet Lab Experiments</a></h4>
| + | </div> |
| − | </div>
| + | </div> |
| − | <div id="collapseOne" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | Under the supervision of Prof. Zhu Xudong, Prof. Yang Dong, and Prof. Xiang Benqiong etc, nearly all team members have taken part in the project designing process, especially those of wet lab group.Beichen Gao , Yuanyi Dai , Yufei Cao and Jia Li are the group leaders who mainly design the experimental patterns and guide the different parts of the experiments.
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div class="panel panel- default"> | + | <div class="panel panel-info"> |
| − | <div class="panel-heading"> | + | <div class="panel-heading"> |
| − | <h4 class="panel-title"><a data-toggle="collapse" data-parent="#accordion" href="#collapseTwo">Modeling</a></h4>
| + | <h3 class="panel-title">Human practice</h3> |
| − | </div>
| + | </div> |
| − | <div id="collapseTwo" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | Cheng Li is the leader of modeling group, who mainly enrolled in designing the device 2.0. Yuan Yin , Qiuyue Yuan and Xueting Zhao also do a great many works in the modeling group, such as information searching, paper writing and equipment appilication.
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div class=" panel panel- default"> | + | <div class="panel-body"> |
| − | <div class="panel-heading">
| + | <strong>Meng Tang</strong> , the leader of human practice group, has organized a series of activities for Policy and Practice, like survey and publicize in some middle schools etc. Some group members as <strong>Yuanyi Dai</strong>, <strong>Jia Li</strong> , <strong>Jiajun Zhang</strong> , <strong>Beichen Gao</strong> , <strong>Zhiyao Chen</strong> , <strong>Jingyu Zhao</strong> , <strong>Xiaofei Feng</strong> also play a significant role in planning and organizing the activities . Other group members also take an active part in some other activities of human practice. |
| − | <h4 class="panel-title"><a data-toggle="collapse" data-parent="#accordion" href="#collapseThree">Wiki Construction</a></h4>
| + | </div> |
| − | </div>
| + | </div> |
| − | <div id="collapseThree" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | Jiajun Zhang , who has led the web design group to build the wiki, together with group members Zhiyao Chen and Chengfei Peng complete the main work in database construction and maintenance.
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading">
| |
| − | <h4 class="panel-title"><a data-toggle="collapse" data-parent="#accordion" href="#collapseFour">Graphic design</a></h4>
| |
| − | </div>
| |
| − | <div id="collapseFour" class="panel-collapse collapse">
| |
| − | <div class="panel-body">
| |
| − | Lu Xu( group leader), Lee and Yalei Cao really do a fantastic work in graphic and web design. They all make great efforts in designing our team logo, website, poster, souvenir, T-shirt and so on.
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| | | | |
| − | <div class="panel panel-default">
| + | <h3>Attributions Details</h3> |
| − | <div class="panel-heading">
| + | <dl class="dl-horizontal"> |
| − | <h4 class="panel-title"><a data-toggle="collapse" data-parent="#accordion" href="#collapseFive">Human practice</a></h4>
| + | <dt style="color: #258D79;">Team Member</dt> |
| − | </div>
| + | <dd style="color: #258D79;font-weight: bold;">Attributions</dd> |
| − | <div id="collapseFive" class="panel-collapse collapse">
| + | |
| − | <div class="panel-body">
| + | |
| − | Meng Tang , the leader of human practice group, has organized a series of activities for Policy and Practice, like survey and publicize in some middle schools etc. Some group members as Yuanyi Dai,Jia Li , Jiajun Zhang , Beichen Gao , Zhiyao Chen , Jingyu Zhao , Xiaofei Feng also play a significant role in planning and organizing the activities . Other group members also take an active part in some other activities of human practice.
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <section id="section-2" class="group">
| + | |
| − | <h3>2. Collaboration</h3>
| + | |
| − | <p>Our modeling can be divided into four parts. Firstly, in order to enable our project to be applied in life, we design a device placed in soil, which can attract and kill nematodes by modified engineering bacteria inside. Secondly, assuming there is a farmland, we take advantage of nematodes’ movement analogue simulation to find the best position where the device should be placed and the best concentration of attracting substance. Thirdly, according to this concentration, we obtain the best size of the device by calculating. At last, we establish a database to enlarge applied range of our method to kill other pests. We would appreciate that new synthetic biological and environment-friendly methods can be shared and improved with the science researchers all over the world.</p>
| + | |
| − | </section>
| + | |
| − | <section id="section-3" class="group">
| + | |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <h3>3. Acknowledgement</h3>
| + | |
| − | <div id="section-3-1" class="subgroup">
| + | |
| − | <h4 align='center'>Thanks:</h4>
| + | |
| − | <br/>
| + | |
| − | <center>
| + | |
| − | <h5>Prof. Zhu Xudong, Prof. Yang Dong and Prof. Xiang Benqiong
| + | |
| − | </h5>
| + | |
| − | <p>Offered us much precious advice on the project.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Hao Xiaoran, Chi Xiaodong, Huo Liang, Ai Ying and Wang Xuan</h5>
| + | |
| − | <p>Offered us advice on our experiments and helped us solve problems.</p>
| + | |
| − | <br/>
| + | |
| − | <h5>The College of Chemistry, Beijing Normal University
| + | |
| − | </h5>
| + | |
| − | <p>Helped us do GC-MS.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Prof. Yang Chonglin of Chinese Academy of Sciences
| + | |
| − | </h5>
| + | |
| − | <p>Offered us C.elegens and taught us how to cultivate them.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Prof. Li Hongmei of Department of Plant Pathology，Nanjing Agricultural University
| + | |
| − | </h5>
| + | |
| − | <p>Helped us to do Species identification of nematodes.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>BIT-China & ZJU-China & FAFU-CHINA
| + | |
| − | </h5>
| + | |
| − | <p>Offered us some helpful advice and we also had a collaboration together.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Agricultural Service Center of Ceyu, Hebei Province
| + | |
| − | </h5>
| + | |
| − | <p>Helped us learn more information about the damage caused by local nematodes and villager’s attitude towards agricultural insecticide.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Shang Zhong National Middle School of Guizhou Province
| + | |
| − | </h5>
| + | |
| − | <p>Supported us in our voluntary education.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>National Chiao Tung University &Peking University
| + | |
| − | </h5>
| + | |
| − | <p>Provided us opportunities to communicate with other teams.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Feng Xiaofei and her family in Hebei Province
| + | |
| − | </h5>
| + | |
| − | <p>Helped us get soybeans samples and did a survey in the locality.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <h5>Prof. Wang Yingdian
| + | |
| − | </h5>
| + | |
| − | <p>Discussed the bright future of our project with us.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | </center>
| + | |
| − | </div>
| + | |
| − | <hr/>
| + | |
| − | <div id="section-3-2" class="subgroup">
| + | |
| − | <h4 align='center'>Sponsors:</h4>
| + | |
| − | <br/>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/e/ec/TEAM-BNU.png"></center>
| + | |
| − | <br/>
| + | |
| − | <p>Beijing Normal University (BNU), is a public research university located in Beijing with strong emphasis on basic disciplines of humanities and sciences. It is one of the oldest and most prestigious universities in China. Our school supported us to our iGEM Journey and provided us adequate resources to complete the competition. Students of different colleges in our school took part in our team, and all these colleges supported us very much.</p>
| + | |
| − | <center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/d/df/TEAM-LIFE.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/3/36/TEAM-PHY.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/3/34/TEAM-CS.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/8/85/TEAM-MATH.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/e/e3/TEAM-CHE.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/d/da/TEAM-LIYUN.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/d/df/TEAM-Geo.png">
| + | |
| − | </center>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <section id="section-4" class="group">
| + | |
| − | <h3>4. Notebook</h3>
| + | |
| − | <div class="box-frame">
| + | |
| − | <div class="box-top">
| + | |
| − | Week 1
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | We found two kinds of toxic proteins that can kill nematodes specially—Bace16 and MpL—through reviewing literatures which confirmed the function of poison nematodes of these two proteins. We found the gene sequence of bace16(AAV3D0845) and the cDNA sequence of MpL(GeneBank accession number HQ449739) from the Genebank, and optimized the coded sequences in E.coli.
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | After we determined the theme of our project, we recuited more undergraduates from related colleges, including college of Mathematics, Physics and Computer Science. After a short meeting, four undergraduates joined our team. Congratulations!
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <div class="box-frame">
| + | |
| − | <div class="box-top">
| + | |
| − | Week 2
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | We tried some simple experimental operation such as the enlarge culture of bacteria, culture preservation and transformation to be familiar with the laboratory apparatus and the solexa.
| + | |
| − | <br/>
| + | |
| − | <br/> We designed parts rMpL and bace16 by adding pBAD promoter (BBa_K206000) and RBS(BBa_B0034) to the upstream region of rMpL gene and bace16 gene, and we committed the GENEWIZ company to synthesize the rMpL and bace16 gene segment.
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">Modeling group members read some related materials and papers, including wikis of former teams especially their modeling parts as well as papers introducing nematodes. </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-content">
| + | <dt>Cao Yalei</dt> |
| − | </div>
| + | <dd>Graphic Design(Design of logo, banner, poster, jersey and souvenir)</dd> |
| | | | |
| − | <div class="box-frame">
| + | <dt>Cao Yufei</dt> |
| − | <div class="box-top">
| + | <dd>Wet lab group leader(parts design for expressing lemonene in <em>E. coli</em> and coresponding experiments)</dd> |
| − | Week 3
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | There occurs some problems of rMpL and bace16 gene synthesis. GENEWIZ gave us the relevant feedback in time and modified the two sequences to synthesize de novo.
| + | |
| − | <br/>
| + | |
| − | <br/>We did the simple experimental operation such as the enlarge culture of bacteria, culture preservation and transformation again to be further familiar with the laboratory apparatus and the solexa. We transformed the plasmid rfp-pSB1C3(BBa_J04450) in DH5α and proceeded amplification to obtain the standard plasmid backbone.
| + | |
| − | <br/>
| + | |
| − | <br/> We prepared some common used reagent such as TAE, antibiotics and LB cultural medium.
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">Modeling group members read some related materials and papers, including wikis of former teams especially their modeling parts as well as papers introducing nematodes.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Chen Zhiyao</dt> |
| − | <div class="box-top">
| + | <dd>Wiki construction(database construction; UI design)<br>Human practice</dd> |
| − | Week 4
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | We extracted the plasmid and did restriction enzyme digestion to test and verify the plasmid backbone PSB1C3, and preserve the correct plasmid for subsequent experimental use.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| | + | <dt>Dai Yuanyi</dt> |
| | + | <dd>Daily activities arrangement and personnel management<br>Wet lab group leader(biobricks construction, Bace16 expression and testification)<br>Human practice</dd> |
| | | | |
| | + | <dt>Feng Xiaofei</dt> |
| | + | <dd>Wet lab experiments(limonene synthesis)<br>Human practice</dd> |
| | | | |
| − | <div class="box-frame">
| + | <dt>Gao Beichen</dt> |
| − | <div class="box-top">
| + | <dd>Team leader<br>Wet lab group leader（biobricks construction, nematocidal protein expression, rMpL's toxicity identification<br>Human practice</dd> |
| − | Week 5
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;"> We designed the sequence of Limonene synthase (LS) that would be transformed to E.coli to express. Our designed sequences was based on the two cDNA sequences of LS expression sequence--Citrus unshiu CitMTSE1 for d-limonene synthase and Mentha spicata 4S-limonene synthase, together with prefix and suffix codes and restriction enzyme cutting site were sent to the company for optimum synthesis.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">We obtained the plasmid of gene rMpL with PUC57 backbone in the form of dry power. We dissolved this dry power and inoculated it on LB solid cultural medium through which we obtained the monoclonal colonies. We inoculated 10 mL centrifuge tubes with the monoclonal colony to produce bacterium solution for preservation and plasmid extraction, and we named the obtained plasmid pUC57-rMpL.
| + | |
| − | <br/>
| + | |
| − | <br/> We digested the plasmid PSB1C3-RFP sent by iGEM and the pUC57-rMpL with restriction enzyme separately and obtained the PSB1C3 backbone and rMpL gene segment which were connected together later on to assemble the plasmid PSB1C3-rMpL.
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">Together with members majoring in physics, mathematics and biology, we drafted an abstract settlement concentrating on how to simulate the concentration of nematodes in chemotaxis. The goal of our modeling project is to give a best attractant of concentration so that we can determine the most economical plan.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Lao Kejing</dt> |
| − | <div class="box-top">
| + | <dd>Wet lab experiments(plasmid purification; SDS-PAGE; nematodes synchronization)<br>Helping Beijing Institute of Technology with extracting membrane proteins, NhaA and NhaB</dd> |
| − | Week 6
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;">We prepared the L-limonene and D-limonene and the synthases expression E.coli (BL21,pGEX-4T-1-sls-gpps) without standardization of them both, which were cryopreserved with glycerol.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We prepared the BW25113 competent cells in terms of the component efficiency kit, and we detected their efficiency of transformation by transforming rfp-PSB1C3 plasmids of different concentration into the cells.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">We transformed pSB1C3-rMpL into BW25113 competent cells to cultivate on the LB plate and inoculated 10 mL centrifuge tubes with the obtained monoclonal colony to produce bacterium solution for preservation and plasmid extraction.
| + | |
| − | <br/>
| + | |
| − | <br/> We did restriction enzyme digestion to check the extracted plasmid.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Lee</dt> |
| − | <div class="box-top">
| + | <dd>Graphic Design( website design, logo of the team and T-shirt)</dd> |
| − | Week 7
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;"> We revived the limonene synthases expression E.coli strain preserved in glycerol.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We assembled bace16-pSB1C3 and transformed it into E.coli DH5α. After the result of digestion exam was correct we extracted the plasmid and did sequencing, and we transformed the plasmid with correct sequence into E.coli BW25113 to establish expression strain.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">We activated the preserved culture and enlarge cultivated it in 250 mL conical flask, inducting expression with different concentration gradient of L-arabinose, we collected the precipitation after centrifuging the bacterium solution. We resuspended the precipitation with buffer A and after ultrasonication we obtained homogenate, then we did high speed centrifugation to get the supernatant. However, we regrettably observed that the effect of expression is not ideal from the SDS-PAGE gel electrophoresis of both the homogenate and the supernatant.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">We realized that our original assumption that we regard nematodes as static models cannot react to some essential properties of nematode moving. So we changed our minds into using dynamic methods to simulate the diffusion of nematodes in chemotaxis. We decided to apply Celluar Automata to solve the problem.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Li Cheng</dt> |
| − | <div class="box-top">
| + | <dd>Modeling leader(design of the device 2.0 ; parts improving)<br>Wiki<br>Graphic design</dd> |
| − | Week 8
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;">We fetched the N2 wild type caenorhabditis elegans favored by professor Yang Huilin of Institute of Genetics and Development Biology, Chinese Academy of Science, and studied the methods of cultivating nematodes as well as synchronization. Additionally, we learned the life history, growth properities and feeding method of nematodes.
| + | |
| − | <br/>
| + | |
| − | <br/>We prepared he nematodes for enlarge cultivation and induced expression of the LS expression E.coli, and we synchronized a batch of nematodes for verification.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We digested rfp-pSB1C3 with two isocaudamas Spe I and Xba I and prepared empty vector plasmid as negative control of whether the desired gene expressed.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;"> We prepared NGM cultural medium and cultivated OP50 E.coli in LB liquid medium.
| + | |
| − | <br/>We cultivated nematodes.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">Since we can get the solution of best concentration of attractant and the diffusion of nematodes, we decided to go towards a further step. What is needed urgently in agriculture product is a low-cost and more importantly environment-friendly device to kill nematodes. Finally our plan was to design and build a debug device.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Li Jia</dt> |
| − | <div class="box-top">
| + | <dd>Wet lab group leader(chemical attractants' function testification, LS and GPPS expression, <em>C.elegans</em> cultivation )<br>Human practice</dd> |
| − | Week 9
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;"> We cultivated nematodes and proceeded synchronization, and we did replication experiment of baiting nematodes with linalool using pure chemical compound (linalool). We optimized the protocol of replication experiment on and on, and the growth condition of nematodes is favorable. However, according to our statistical result we were not able to conclude that linalool can attract nematodes. And we processed nematodes’ chemotaxis replication experiment of limonene as well.
| + | |
| − | <br/>
| + | |
| − | <br/> We also did expression replication experiments by dong SDS-PAGE under the protocol mentioned above (reserve the homogenate as protein sample) which came out an obvious band (89kd).
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We ensured the empty vector plasmid was correct with three groups of restriction digestion.
| + | |
| − | <br/>
| + | |
| − | <br/>We transformed bace16- pSB1C3 assembled by GENEWIZ and ourselves into the expression strain BW25113 to express bace16 as an attempt. We added 500 uL 1M L-Arabinose into 100mL bacterium solution to induce expression at 26C for 5h. We obtained proteins by precipitation with ammonium sulfate and concentration through dialysis which was dissolved with buffer A and then went through ultrasonication to get homogenate, and then high speed centrifugation for the supernatant. We did SDS-PAGE separately for the concentrated proteins, homogenate and supernatant. However, as the protein loading buffer was not commercial, the outcome effect was not satisfied.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Group number
| + | |
| − | </td>
| + | |
| − | <td>Nematodes amount(\(\mu\)L)
| + | |
| − | </td>
| + | |
| − | <td>Sample amount(\(\mu\)L)
| + | |
| − | </td>
| + | |
| − | <td>bace16</td>
| + | |
| − | <td>Heated bace16</td>
| + | |
| − | <td>pSB1C3
| + | |
| − | </td>
| + | |
| − | <td>M9</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>1</td>
| + | |
| − | <td>50</td>
| + | |
| − | <td>20</td>
| + | |
| − | <td>Died immediately，and the cells lysed</td>
| + | |
| − | <td>Died immediately，and the cells lysed</td>
| + | |
| − | <td>Died immediately，and the cells lysed</td>
| + | |
| − | <td>Alive, active</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>2</td>
| + | |
| − | <td>750</td>
| + | |
| − | <td>30</td>
| + | |
| − | <td>Died totally while the morphology was completed.</td>
| + | |
| − | <td>--</td>
| + | |
| − | <td>Died totally while the morphology was completed.
| + | |
| − | </td>
| + | |
| − | <td>Partly died</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>3</td>
| + | |
| − | <td>750</td>
| + | |
| − | <td>10</td>
| + | |
| − | <td>Partly died</td>
| + | |
| − | <td>A few died, inactive
| + | |
| − | </td>
| + | |
| − | <td>A few died, inactive
| + | |
| − | </td>
| + | |
| − | <td>In good condition, active</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">We repeated the induction experiment with different concentration gradient of arabinose as before, while we just collected the precipitation without extracting proteins this time.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">We built a device all by handwork! There was a big change in the process. At first, we wanted to design in CAD software and built it by 3D print. However, the cost of product by 3D print was too high. Finally, we chose traditional technology to produce devices in the future if it was verified that our device had high value of application. Of course, we also cannot afford the cost of custom-built sample. That’s why we had to make it by hand.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Li Yuehuan</dt> |
| − | <div class="box-top-double">
| + | <dd>Wet lab experiments(rMpL verification)</dd> |
| − | Week 10
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;"> We tried another protocol of replication experiment as increasing the amount of sample whereas the effect was still not ideal indicated by the statistical result.
| + | |
| − | <br/>
| + | |
| − | <br/> We obtained gene l-limonene and d-limonene connected with pUC57 backbone in the form of dry power. We dissolved these dry power and transformed them into E.coli DH5α, coating on LB plate. We inoculated the obtained monoclonal colony into 10 mL centrifuge tubes to produce bacterium solution for preservation and plasmid extraction. The correct plasmids examined with restricton enzyme digestion was preserved in -20C, named l-limonene-PUC57 and d-limonene-PUC57.
| + | |
| − | <br/>
| + | |
| − | <br/> We digested the plasmid pSB1C3-RFP from iGEM as well as l-limonene-PUC57 and d-limonene-PUC57 separately with restriction enzymes to get pSB1C3 backbone and the two gene segments, among which the brightness of pSB1C3 backbone band was quite weak. Then we recycled the backbone and gene segments following the agarose gel DNA extraction kit and linked them to produce plasmids l-limonene-PSB1C3 and d-limonene-PSB1C3.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We continued to watch the movement of nematodes under toxic test, where the nematodes of M9 group was generally active, partly died of bace16 and heated bace16 groups, and acted normally in pSB1C3 group.
| + | |
| − | <br/>
| + | |
| − | <br/>Another SDS-PAGE of extracellular proteins extract was run while the objective band was still not observed. So we extracted the bace16-pSB1C3 from the expression strain BW25113 for restriction enzyme digestion and no objective band was extracted still. Meanwhile, it appeared to be some problems of other groups’ digestion too, against which we speculated that the cause may be the pollution of reagent, star activity or something wrong with the expression strain. We transformed into E.coli DH5α the bace16-pSB1C3 to find that the result of plasmid extraction and enzyme digestion was correct, thus we confirmed the problem was from the expression strain.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;"> We re-cultivated the collected bacteria and mixed up the recombined bacteria with the control ones to a certain scale (1:4) to coat on NGM medium, preparing for nematodes cultivation of toxic test.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">Another important module of our project is to build a database which researchers and even farmers can get information of bio-pesticide easily. We started to work on it.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Liu Longping</dt> |
| − | <div class="box-top-double">
| + | <dd>Wet lab experiments(exploraton of how to promote the expression rate of protein rMpL; design of the toxic test)</dd> |
| − | Week 11
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;">We transformed the obtained plasmids l-limonene-PSB1C3、d-limonene-PSB1C3 into E.coli BW25113 and cultivated 10mL centrifuge tube with them to produce bacterium solution for preservation and plasmid extraction. We examined the extracted plasmid with enzyme digestion, no objective band observed as a result.
| + | |
| − | <br/>
| + | |
| − | <br/>We obtained gene l-limonene and d-limonene connected with pUC57 backbone in the form of dry power. We dissolved these dry power and transformed them into E.coli DH5α, coating on LB plate. We inoculated the obtained monoclonal colony into 10 mL centrifuge tubes to produce bacterium solution for plasmid extraction, the enzyme digestion result of which was incorrect that a band around 500~750 bp appeared whereas the backbone band lost. The problem was confirmed to be from the expression strain as mentioned above, so we adopted a new strain of BW25113 and continued our experiment.
| + | |
| − | <br/>
| + | |
| − | <br/>We enlarge cultivated the standard LS expression bacteria (E.coli-BW2513-psb1c3) and expressed the objective produce (in contrast with empty vector). We did SDS-PAGE under the protocol mentioned above (reserve the homogenate as protein sample), no significant difference observed between the bands.
| + | |
| − | <br/>
| + | |
| − | <br/>In addition, the differences between bands of BW25113 and BL21 was obvious when run SDS-PAGE together whereas the objective band of BW25113 cannot be ensured.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We expressed bace16 protein twice and prolonged the expression period to overnight. Also, we changed the concentration of L-Ara and increased the concentration of running gel to 15% when running SDS-PAGE. Still, we failed to observe objective band at all the supernatant of bacterium, homogenate and supernatant of ultrasonic broken cells.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;">We induced expression of rMpL continuously with arabinose in concentration gradient to acquire a stable expression condition. We extracted proteins to do SDS-PAGE, again finding no sign of rMpL expression.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">We reviewed the work we had done together and made a specific plan for following days. First, we had built simulation model and we can use it to build economic model. Second, the device we had made actually was not applicable enough, which required further improvement. Third, we had built a basic frame of the database.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <div class="box-frame">
| + | <dt>Liu Xinyu</dt> |
| − | <div class="box-top-double">
| + | <dd>Wet lab experiments(limonene group and other parts' experiments)<br>Human practice</dd> |
| − | Week 12
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/6/62/BNU-wet.png"></center>
| + | |
| − | <div class="box-content">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/c/ce/BNU-bait.png" style="float:left;"> Sequencing gene l-limonene and d-limonene synthesized by the company to get an unsatisfactory result, we speculated that these plasmids were extracted from the wrong BW25113. So we re-transformed the plasmids from the company into E.coli DH5αand BL21 separately and did all the cultivation, plasmid extraction, enzyme digestion de novo to make sure the examine result correctly. And we sent the plasmids extracted this time for sequencing.
| + | |
| − | <br/>
| + | |
| − | <br/>We re-prepared the homogenate of the revived E.coli-BL21(pGEX-4T-1-sls-gpps) for SDS-PAGE and enlarge cultivated the standard bacteria (BL21-pxb1c3) transformed the second time to express the objective product.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2015/2/25/BNU-bace16.png" style="float:left;">We transformed bace16-pSB1C3 into the newly obtained BW25113 competent cells to express for another time, and examined the result with SDS-PAGE.
| + | |
| − | <br/>
| + | |
| − | <br/><img src="https://static.igem.org/mediawiki/2015/a/a7/BNU-rmpl.png" style="float:left;"> We went on the expression experiment of rMpL as above except that we added parallel controlled groups whereas canceled the control ones expressed under 37C and prolonged the expression period. However, we still failed to observe obvious expression of rMpL, against which we suspected there occurred something wrong with the expression bacteria and planed to change bacteria strain to express de novo.
| + | |
| − | <br/>
| + | |
| − | <br/>We prepared NGM plate and nematodes liquid medium for toxic test of rMpL.
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/9e/BNU-dry.png"></center>
| + | |
| − | <div class="box-content">We put the database on the github.com and it can be edited and added new items by all users.
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| | | | |
| − | </section>
| + | <dt>Ma Hongfei</dt> |
| − | <section id="section-5 " class="group ">
| + | <dd>Wet lab experiments(GC-MS of limonene )<br>Modeling(device 1.0 design and creation)</dd> |
| − | <div id="section－5-1 " class="subgroup ">
| + | |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <h4>5.1 Cloning</h4>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>PCR</h5>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>Reaction System:</h5>
| + | |
| − | <p>H<sub>2</sub>O 38 \(\mu\)L</p>
| + | |
| − | <p>10x Taq buffer 5 \(\mu\)L
| + | |
| − | </p>
| + | |
| − | <p>2.5mM dNTP 4 \(\mu\)L</p>
| + | |
| − | <p>Primer 1 1 \(\mu\)L</p>
| + | |
| − | <p>Primer 2 1 \(\mu\)L
| + | |
| − | </p>
| + | |
| − | <p>Template 0.5 \(\mu\)L
| + | |
| − | </p>
| + | |
| − | <p>Taq 0.5 \(\mu\)L
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <h5>Process:</h5>
| + | |
| − | <p>\(\begin{equation}\left. \begin{array}{lcl} {94°C\ 10min} \\ {94°C\ 30s} \\{58°C\ 30s} \end{array} \right\} Cycle\ 30\end {equation}\)</p>
| + | |
| − | <p>94°C 10min
| + | |
| − | </p>
| + | |
| − | <p>94°C 30s
| + | |
| − | </p>
| + | |
| − | <p>58°C 30s
| + | |
| − | </p>
| + | |
| − | <p>72°C 2min30s
| + | |
| − | </p>
| + | |
| − | <p>72°C 10min
| + | |
| − | </p>
| + | |
| − | <p>10°C ---
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <h5 align='center'>Electrophoresis---Gel Purification
| + | <dt>Peng Chengfei</dt> |
| − | </h5> | + | <dd>Wiki construction(database construction, deployment and operation, Wiki frame construction)</dd> |
| − | <li>
| + | |
| − | <h5>Material:</h5>
| + | |
| − | <p>Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain</p>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <h5>Protocol:</h5>
| + | |
| − | <p>We used gelstain to stain the DNA and imaged it in a Transilluminator.
| + | |
| − | </p>
| + | |
| − | <p>We used the gel extraction kit to get the objective fragment. </p>
| + | |
| − | </li>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <h5 align='center'>Digestion</h5>
| + | <dt>Tang Meng</dt> |
| − | <table class="table table-condensed ">
| + | <dd>Human practice project leader(activities planning and organization)<br>Wet lab experiments</dd> |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>50\(\mu\)L reaction system</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>10x H buffer</td>
| + | |
| − | <td>EcoR I</td>
| + | |
| − | <td>Pat I</td>
| + | |
| − | <td>Plasmid</td>
| + | |
| − | <td>H<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>5 \(\mu\)L</td>
| + | |
| − | <td>1.5 \(\mu\)L</td>
| + | |
| − | <td>1.5 \(\mu\)L</td>
| + | |
| − | <td>15 \(\mu\)L</td>
| + | |
| − | <td>27 \(\mu\)l
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>10\(\mu\)L reaction system</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>10x H buffer</td>
| + | |
| − | <td>EcoR I</td>
| + | |
| − | <td>Pat I</td>
| + | |
| − | <td>Plasmid</td>
| + | |
| − | <td>H<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>1 \(\mu\)L
| + | |
| − | <td>0.3 \(\mu\)L</td>
| + | |
| − | <td>0.3 \(\mu\)L</td>
| + | |
| − | <td>3 \(\mu\)L</td>
| + | |
| − | <td>5.4 \(\mu\)L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <br/>
| + | |
| | | | |
| − | <h5 align='center'>Ligation</h5>
| + | <dt>Wang Ran</dt> |
| − | <table class="table table-condensed ">
| + | <dd>Translation<br>Human practice</dd> |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Ligation reaction system</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>Cph8</td>
| + | |
| − | <td>PSB1C3</td>
| + | |
| − | <td>T4 buffer</td>
| + | |
| − | <td>T4 ligase</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>14 \(\mu\)L
| + | |
| − | <td>3 \(\mu\)L</td>
| + | |
| − | <td>2 \(\mu\)L</td>
| + | |
| − | <td>1 \(\mu\)L</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| | | | |
| − | </ul>
| + | <dt>Wang Yingbing</dt> |
| − | </div>
| + | <dd>Wet lab experiments(Bace16 group work)</dd> |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <div id="section-5-2 " class="subgroup ">
| + | |
| − | <h4>5.2 Transformation</h4>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>Material:
| + | |
| − | </h5></li>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>LB liquid medium</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>Tryptone</td>
| + | |
| − | <td>Yeast extract powder</td>
| + | |
| − | <td>NaCl</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>10 g/L
| + | |
| − | <td>5 g/L</td>
| + | |
| − | <td>10 \(\mu\)L</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <br/>
| + | |
| − | <li>
| + | |
| − | <h5>Protocol:</h5>
| + | |
| − | <p>preparation of the competent cells</p>
| + | |
| − | <p>20\(\mu\)L ligation product + 50\(\mu\)L cells
| + | |
| − | </p>
| + | |
| − | <p>Heatshock of E.coli BW25113(42°C,90s)
| + | |
| − | </p>
| + | |
| − | <p>Put on ice(2min)
| + | |
| − | </p>
| + | |
| − | <p>Add 800\(\mu\)L LB media and incubate for 1.5h(37°C, 150rpm)
| + | |
| − | </p>
| + | |
| − | <p>Centrifuge at 4000rpm for 1min and remove 900\(\mu\)L supernatant
| + | |
| − | </p>
| + | |
| − | <p>Resuspend the pellets using the left supernatant
| + | |
| − | </p>
| + | |
| − | <p>Spread plates(with ampicillin)
| + | |
| − | </p>
| + | |
| − | <p>Incubate for 12~16h(37°C)
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <div id="section-5-3 " class="subgroup ">
| + | |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <h4>5.3 Detetion</h4>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>SDS-PAGE
| + | |
| − | </h5>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>Materials:</h5>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Gel</td>
| + | |
| − | <td>Tris-HCl</td>
| + | |
| − | <td>Acr/Bis 30%
| + | |
| − | </td>
| + | |
| − | <td>SDS 10%
| + | |
| − | </td>
| + | |
| − | <td>ddH<sub>2</sub>O</td>
| + | |
| − | <td>TEMED
| + | |
| − | </td>
| + | |
| − | <td>AP 10%
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Stacking Gel(4%) </td>
| + | |
| − | <td>pH=6.8 500\(\mu\)L
| + | |
| − | </td>
| + | |
| − | <td>500 \(\mu\)L</td>
| + | |
| − | <td>25 \(\mu\)L</td>
| + | |
| − | <td>1350 \(\mu\)L</td>
| + | |
| − | <td>2.5 \(\mu\)L</td>
| + | |
| − | <td>12.5 \(\mu\)L</td>
| + | |
| | | | |
| − | </tr>
| + | <dt>Wu Zekai</dt> |
| − | <tr>
| + | <dd>Wet lab experiments(photoinduced system design; Bace 16 functional verification)</dd> |
| − | <td>Running Gel(12%)
| + | |
| − | <td>pH=8.8 1250 \(\mu\)L</td>
| + | |
| − | <td>2000 \(\mu\)L</td>
| + | |
| − | <td>50 \(\mu\)L</td>
| + | |
| − | <td>1675 \(\mu\)L</td>
| + | |
| − | <td>2.5 \(\mu\)L</td>
| + | |
| − | <td>25 \(\mu\)L</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Running Gel(18%)
| + | |
| − | <td>pH=8.8 1250 \(\mu\)L</td>
| + | |
| − | <td>3000 \(\mu\)L</td>
| + | |
| − | <td>50 \(\mu\)L</td>
| + | |
| − | <td>675 \(\mu\)L</td>
| + | |
| − | <td>2.5 \(\mu\)L</td>
| + | |
| − | <td>25 \(\mu\)L</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Running Buffer</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>Tris-HCl</td>
| + | |
| − | <td>Glycine</td>
| + | |
| − | <td>(w/v) SDS</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>25 mmol/L
| + | |
| − | <td>0.192 mol/L</td>
| + | |
| − | <td>0.1%</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <h5>Protocol:</h5>
| + | |
| − | <p>The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.</p>
| + | |
| − | <p>After ensuring that the equipment is waterproof, the 12 % (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H<sub>2</sub>O on top of the running gel to seal the gel. </p>
| + | |
| − | <p>After polymerization, the remaining H<sub>2</sub>O is removed and the 12 % stacking gel is filled on top. Insert a comb to create sample pockets.</p>
| + | |
| − | <p>After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via the SDS gel.
| + | |
| − | </p>
| + | |
| − | <p>After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>Detection of Baiting Nematodes</h5>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>Materials:
| + | |
| − | </h5>
| + | |
| − | <p>Pure chemical compound: linalool and limonene.
| + | |
| − | </p>
| + | |
| − | <p>Solvent: DMSO
| + | |
| − | </p>
| + | |
| − | <p>Dissolve the compounds with DMSO to set a series of concentration gradient of attractant.
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>Protocol:
| + | |
| − | </h5>
| + | |
| − | <p>Synchronize the nematodes.
| + | |
| − | </p>
| + | |
| − | <p>Divide the NGM solid medium (d = 6 cm) equally into two parts (drawing on the surface of the culture dishes).
| + | |
| − | </p>
| + | |
| − | <p>Put 50 \(\mu\)L compound of different concentration and DMSO as contrast respectively on the two parts. Make 3 repeats of each concentration.
| + | |
| − | </p>
| + | |
| − | <p>Flush nematode from the plate with M9 and inoculate 20 \(\mu\)L (about 30 nematodes) into the center of the medium.
| + | |
| − | </p>
| + | |
| − | <p>Incubate at 20°C for 1hr, and then place them at 4°C refrigerator for 1hr.
| + | |
| − | </p>
| + | |
| − | <p>Count the nematodes of each part under the stereoscope.
| + | |
| − | </p>
| + | |
| − | <p>Compare the results of different compound concentration and make a conclusion.
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>Detection of Killing Nematodes
| + | |
| − | </h5> | + | |
| | | | |
| − | <ul>
| + | <dt>Xiao Shusheng</dt> |
| − | <p>Inoculate 5mL LB liquid mediums added Chl with experimental group E.coli BW25113 (transformed with rMpl or bace16 genes) and blank control group separately. Incubate at 37°C, 190 r.m.p for 3 hr.
| + | <dd>Literature reading<br>Human practice</dd> |
| − | </p>
| + | |
| − | <p>Mix up 800\(\mu\)L control group and 200ul experimental liquid medium Inoculate 1mL mixture mentioned above to NGM plate. Incubate upside down at 37°C overnight.
| + | |
| − | </p>
| + | |
| − | <p>Flush nematodes from the plate with M9, centrifugate (1500 r.p.m, 3min), abandon the supernatant and resuspend the precipitation with 3mL M9.
| + | |
| − | </p>
| + | |
| − | <p>Add 500\(\mu\)L resuspended solution to the NGM plate inoculated with mixed E.coli as mentioned above as experimental group and NGM plate with blank E.coli as control group. 37°C incubate overnight.
| + | |
| − | </p>
| + | |
| − | <p>Observe and compare the activation and size of nematodes of each group and draw a conclusion.
| + | |
| − | </p>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <br/>
| + | |
| − | <hr/>
| + | |
| − | <div id="section-5-4 " class="subgroup ">
| + | |
| − | <h4>5.4 Nematode</h4>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>Materials:
| + | |
| − | </h5></li>
| + | |
| − | <li>
| + | |
| − | <p>NGM medium:</p>
| + | |
| − | </li>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Minimum Medium</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>NaCl</td>
| + | |
| − | <td>Bacto peptone（BD 4059002)</td>
| + | |
| − | <td>Agar</td>
| + | |
| − | <td>ddH<sub>2</sub>O</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>3 g
| + | |
| − | <td>2.5 g/L</td>
| + | |
| − | <td>17 \(\mu\)L</td>
| + | |
| − | <td>975 mL</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <p>Prepare 1mol/L CaCl<sub>2</sub>, 1mol/L MgSO<sub>4</sub> and phosphoric acid buffer meanwhile.
| + | |
| − | </p>
| + | |
| − | <table class="table table-condensed ">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Phosphoric Acid Buffer</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>KH<sub>2</sub>PO<sub>4</sub></td>
| + | |
| − | <td>KHPO<sub>4</sub></td>
| + | |
| − | <td>ddH<sub>2</sub>O</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>108.3 g
| + | |
| − | <td>35.6 g/L</td>
| + | |
| − | <td>1 L</td>
| + | |
| | | | |
| − | </tr>
| + | <dt>Xu Lu</dt> |
| − | </tbody>
| + | <dd>Graphic design leader（mainly on poster and PPT design）</dd> |
| − | </table>
| + | |
| − | <p>Autoclave the above mentioned reagents with 120°C for 30 min and then water bath them to 65°C.</p>
| + | |
| − | <p>Dissolve the cholesterol with ethanol to 5 mg/mL.</p>
| + | |
| − | <p>Add 1ml cholesterol, CaCl<sub>2</sub>, MgSO<sub>4</sub> and 25 mL phosphoric acid buffer into the minimum medium in order (all at 65°C, shake up).</p>
| + | |
| − | <p>Make plate with the NGM medium, storing at 4°C.</p>
| + | |
| − | <br/>
| + | |
| − | <li>
| + | |
| − | <h5>OP50 E.coli deficit type (food of nematodes):</h5></li>
| + | |
| − | <p>Streak inoculate OP50 on LB solid medium, Incubate at 37°C for 6 hr or overnight. Inoculate LB liquid medium with OP50 single colonies, and incubate (37°C, 220 r.p.m) overnight.
| + | |
| − | </p>
| + | |
| − | </ul>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>Cultivate
| + | |
| − | </h5>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <h5>The incubation condition of caenorhabditis elegans N2 wild type:
| + | |
| − | </h5>
| + | |
| − | <p>Generally incubated at 20°C. Grow slowly at 16°C and grow fast at 25°C while egg laying amount declines.</p>
| + | |
| − | </li>
| + | |
| − | <br/>
| + | |
| − | <li>
| + | |
| − | <h5>Protocol:
| + | |
| − | </h5></li>
| + | |
| − | <p>Inoculate NGM medium with 150~200 \(\mu\)L OP50, incubate at 37°C for 12hr. Cut down a square of NGM(about 1cm x 1cm) with nematodes.
| + | |
| − | </p>
| + | |
| − | <p>Put the square on the NGM medium with OP50 and let the surface with nematodes adown to contact the medium.
| + | |
| − | </p>
| + | |
| − | <p>Seal and incubate upside down at 20°C.
| + | |
| − | </p>
| + | |
| − | <p>Watch the growth condition of nematodes under the stereoscope everyday and re-inoculate every 4 to 5 days to avoid the nematodes growing too densely.
| + | |
| − | </p>
| + | |
| − | <p>For inoculating abundant nematodes rapidly, or changing plate to provide more food and better condition for them, we can flush them with M9 and centrifuge them with 1500 r.m.p for 1 min, and incubate the precipitate on a new plate. </p>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>Synchronization</h5>
| + | |
| − | <li>
| + | |
| − | <h5>Materials:
| + | |
| − | </h5>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </li>
| + | |
| | | | |
| − | <table class="table table-condensed ">
| + | <dt>Yin Yuan</dt> |
| − | <tbody>
| + | <dd>Modeling(proposing the solving ideas; searching the data)</dd> |
| − | <tr>
| + | |
| − | <td>M9 NS</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>Na<sub>2</sub>HPO<sub>4</sub></td>
| + | |
| − | <td>KH<sub>2</sub>PO<sub>4</sub></td>
| + | |
| − | <td>NaCl</td>
| + | |
| − | <td>1M MgSO<sub>4</sub></td>
| + | |
| − | <td>ddH<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>6 g
| + | |
| − | <td>3 g</td>
| + | |
| − | <td>5 g</td>
| + | |
| − | <td>1 mL</td>
| + | |
| − | <td>1 L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| | | | |
| − | <table class="table table-condensed ">
| + | <dt>Yuan Qiuyue</dt> |
| − | <tbody>
| + | <dd>Modeling(nematode movement simulation;model of diffusion of the attraction)<br>Human practice</dd> |
| − | <tr>
| + | |
| − | <td>Bleach Buffer</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reagent</td>
| + | |
| − | <td>NaOCl</td>
| + | |
| − | <td>5M NaOH</td>
| + | |
| − | <td>ddH<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Dosage</td>
| + | |
| − | <td>50 \(\mu\)L
| + | |
| − | <td>100 \(\mu\)L</td>
| + | |
| − | <td>850 \(\mu\)L</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <br/>
| + | |
| − | <li>
| + | |
| − | <h5>Protocols:</h5></li>
| + | |
| − | <p>We have tried several methods and two of them succeed as mentioned below, we recommend the first one by comparison.
| + | |
| − | </p>
| + | |
| − | <p>1. Flush and incubate
| + | |
| − | </p>
| + | |
| − | <p>Flush the nematodes with M9 NS
| + | |
| − | </p>
| + | |
| − | <p>Centrifugation (3000 r.m.p, 1min)
| + | |
| − | </p>
| + | |
| − | <p>Abandon the supernatant, add 1 mL bleach buffer to the precipitation
| + | |
| − | </p>
| + | |
| − | <p>Centrifugation (3000 r.m.p, 1min), abandon the supernatant
| + | |
| − | </p>
| + | |
| − | <p>Transfer the precipitation (nematode eggs) to a NGM plate with OP50, then the eggs will incubate synchronously.
| + | |
| − | </p>
| + | |
| − | <p>2. Directly pick
| + | |
| − | </p>
| + | |
| − | <p>Transfer 30 nematodes that are under egg laying period to a new NGM plate with OP50. Generally, the egg laying nematodes can lay about 8 eggs per hour. Remove all nematodes after 2hr.
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | Incubate the plate at 20°C for 3d and therefor can get about 300 to 400 nematodes.
| + | |
| − | </p>
| + | |
| − | <p><strong>Note: all of the procedure outlined above must be conducted under sterile condition.
| + | |
| − | </strong></p>
| + | |
| − | <br/>
| + | |
| − | <h5 align='center'>Isolate nematodes from soil
| + | |
| − | </h5>
| + | |
| − | <li>
| + | |
| − | <h5>modified Baermann funnel method:</h5></li>
| + | |
| − | <p> 1. Place a glass funnel (d = 10~15 cm) on the funnel stand linked with about 10cm rubber tube which is furnished with a flatjaw pinchcock.
| + | |
| − | </p>
| + | |
| − | <p>2. Cut the soil sample into matchstick shape. Package 10 g(wet weight) with gauze and put it into the funnel, and then add water to immersion the sample.
| + | |
| − | </p>
| + | |
| − | <p>3. After 4~24 hr, nematodes leave the plant tissue and swim in the water and ultimately precipitate at the bottom due to their gravity and hydrotaxis.
| + | |
| − | </p>
| + | |
| − | <p>4. Open the flatjaw pinchcock, adopt about 5~15 mL liquid sample from the bottom which obtains most active nematodes of the soil sample. Check under the stereoscope and count nematodes, or centrifugate(1500 r) first and check the precipitation if the nematodes are too few.
| + | |
| − | </p>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| − | </article>
| + | |
| − | </div>
| + | |
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