Difference between revisions of "Team:UESTC-China/Improve"

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<link rel="stylesheet" type="text/css" href="https://2018.igem.org/Template:UESTC-China/CSS/zzcs?action=raw&amp;ctype=text/css">
 
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.hehe{
 
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nav.navbar.bootsnav{
 
nav.navbar.bootsnav{
 
border: none;
 
border: none;
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.navbar-nav{
 
.navbar-nav{
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font-weight: 500;
 
font-weight: 500;
 
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nav.navbar.bootsnav ul.nav > li > a{ margin: 0; }
 
nav.navbar.bootsnav ul.nav > li > a{ margin: 0; }
 
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             border-radius: 9px;
 
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             margin-bottom: 15px;
 
             margin-bottom: 15px;
 
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         }
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</style>
 
</style>
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<script type="text/javascript" src="https://2018.igem.org/Template:UESTC-China/Javascript/jquery?action=raw&amp;ctype=text/javascript"></script>
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<script type="text/javascript">
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$(document).ready(function(e) {
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t = $('.fixed').offset().top;
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mh = $('.main').height();
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fh = $('.fixed').height();
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$(window).scroll(function(e){
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</head>
 
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<body>
 
<body>
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<img src="https://static.igem.org/mediawiki/2018/7/7d/T--UESTC-China--up.png" width="100%">
 
<img src="https://static.igem.org/mediawiki/2018/7/7d/T--UESTC-China--up.png" width="100%">
 
</div>
 
</div>
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<div class="collapse navbar-collapse" id="navbar-menu" style="border-bottom-style:solid; border-bottom-width:1px" style="z-index:9999">
 
<div class="collapse navbar-collapse" id="navbar-menu" style="border-bottom-style:solid; border-bottom-width:1px" style="z-index:9999">
 
<ul class="nav navbar-nav" data-in="fadeInDown" data-out="fadeOutUp">
 
<ul class="nav navbar-nav" data-in="fadeInDown" data-out="fadeOutUp">
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                                                         <li><a href="#">HOME</a></li>
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                                                         <li><a href="https://2018.igem.org/Team:UESTC-China">HOME</a></li>
−
<li><a href="#">ACHIEVEMENT</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/achievement">ACHIEVEMENT</a></li>
 
<li class="dropdown">
 
<li class="dropdown">
 
<a href="#" class="dropdown-toggle" data-toggle="dropdown">PROJECT</a>
 
<a href="#" class="dropdown-toggle" data-toggle="dropdown">PROJECT</a>
 
<ul class="dropdown-menu animated fadeOutUp">
 
<ul class="dropdown-menu animated fadeOutUp">
−
<li><a href="#">Introduction</a></li>
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<li><a href="https://2018.igem.org/Team:UESTC-China/project_introduction">Introduction</a></li>
−
<li><a href="#">Design</a></li>
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<li><a href="https://2018.igem.org/Team:UESTC-China/project_design">Design</a></li>
−
<li><a href="#">Demonstrate</a></li>
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<li><a href="https://2018.igem.org/Team:UESTC-China/Demonstrate">Demonstrate</a></li>
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<li><a href="#">Part</a></li>
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                                                                        <li><a href="https://2018.igem.org/Team:UESTC-China/Improve">Improve</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
−
<li><a href="#">MODELING</a></li>
+
                                                        <li class="dropdown">
−
<li><a href="#">ATTRIBUTIONS</a></li>
+
<a href="#" class="dropdown-toggle" data-toggle="dropdown">PART</a>
 +
<ul class="dropdown-menu animated fadeOutUp">
 +
<li><a href="https://2018.igem.org/Team:UESTC-China/Part">Part</a></li>
 +
<li><a href="https://2018.igem.org/Team:UESTC-China/Improve">Improve</a></li>
 +
                                                                </ul> 
 +
        </li>
 +
<li><a href="https://2018.igem.org/Team:UESTC-China/Model">MODELING</a></li>
 +
<li><a href="https://2018.igem.org/Team:UESTC-China/Attributions">ATTRIBUTIONS</a></li>
 
<li class="dropdown">
 
<li class="dropdown">
 
<a href="#" class="dropdown-toggle" data-toggle="dropdown">H&nbsp;&nbsp;P</a>
 
<a href="#" class="dropdown-toggle" data-toggle="dropdown">H&nbsp;&nbsp;P</a>
 
<ul class="dropdown-menu animated fadeOutUp">
 
<ul class="dropdown-menu animated fadeOutUp">
−
<li><a href="#">Our Story</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/hp_ourstory">Our Story</a></li>
−
<li><a href="#">Supporting Research</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/Human_Practice">Supporting Research</a></li>
−
<li><a href="#">Engagement</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/Public_Engagement">Engagement</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
<li class="dropdown">
 
<li class="dropdown">
−
<a href="#" class="dropdown-toggle" data-toggle="dropdown">TEAM</a>
+
<a href="https://2018.igem.org/Team:UESTC-China/team" class="dropdown-toggle" data-toggle="dropdown">TEAM</a>
 
<ul class="dropdown-menu animated fadeOutUp">
 
<ul class="dropdown-menu animated fadeOutUp">
−
<li><a href="#">Team Introduction</a></li>
+
                                                                        <li><a href="https://2018.igem.org/Team:UESTC-China/team">Team</a></li>
−
<li><a href="#">Collaborations</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/team_teamintroduction">Team Introduction</a></li>
 +
<li><a href="https://2018.igem.org/Team:UESTC-China/Collaborations">Collaborations</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
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<a href="#" class="dropdown-toggle" data-toggle="dropdown">NOTEBOOK</a>
 
<a href="#" class="dropdown-toggle" data-toggle="dropdown">NOTEBOOK</a>
 
<ul class="dropdown-menu animated fadeOutUp">
 
<ul class="dropdown-menu animated fadeOutUp">
−
<li><a href="#">Day Note</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/notebook_daynote">Day Note</a></li>
−
<li><a href="#">protocol</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/notebook_protocol">Protocol</a></li>
−
<li><a href="#">safety</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/notebook_safety">Safety</a></li>
−
<li><a href="#">..........</a></li>
+
<li><a href="https://2018.igem.org/Team:UESTC-China/InterLab">Interlab</a></li>
−
<li><a href="#">........</a></li>
+
 
</ul>
 
</ul>
 
</li>
 
</li>
 +
                                                   
 
</ul>
 
</ul>
 
                                              
 
                                              
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</div>
 
</div>
 
                     <div class="logo" style="height:45px; width:83.3px; position: absolute;right: 20px;z-index: 9999;top: 10px;">
 
                     <div class="logo" style="height:45px; width:83.3px; position: absolute;right: 20px;z-index: 9999;top: 10px;">
−
                         <a href="#">
+
                         <a href="https://2018.igem.org/Team:UESTC-China">
 
    <img src="https://static.igem.org/mediawiki/2018/b/b2/T--UESTC-China--logoyuan.gif" width="100" height="100" >
 
    <img src="https://static.igem.org/mediawiki/2018/b/b2/T--UESTC-China--logoyuan.gif" width="100" height="100" >
 
</a>
 
</a>
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</div>
 
</div>
 
</div>
 
</div>
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<div class="team" style="margin-top:-30px; position:relative; width:100%; z-index:1; background-color:#fff">
 
<div class="team" style="margin-top:-30px; position:relative; width:100%; z-index:1; background-color:#fff">
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     <div class="neirong" style="position:relative; z-index:1 ;padding:50px 8%; background-color:#fff">
 
     <div class="neirong" style="position:relative; z-index:1 ;padding:50px 8%; background-color:#fff">
−
        <div class="row member">
+
<div class="row member">
−
           
+
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            <div class="col-md-3 col-sm-4">
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              <div class="fixed">
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              <menu id="tocc" class="hide-on-med-and-down" style="padding: 0.1px; left: 80px; z-index:1; font-family:'Candara',sans-serif;">
+
−
                <h2><strong>Attribution</strong></h3>
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−
                <ul>
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−
                    <li><a href="#yi" style="color: black;">Attribution</a></li>
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−
                    <li><a href="#2" style="color: black;">Advisor</a></li>
+
−
                    <li><a href="#3" style="color: black;">PI</a></li>
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−
                    <li><a href="#4" style="color: black;">Acknowledgement</a></li>
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                </ul>
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−
              </menu>
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<div class="col-md-3 col-sm-4">
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                    <div class="fixed" style="position:fixed; top:250px; ">
−
            </div>
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                        <div class="fl_l"><h3>IMPROVE PART</h3></div>
−
            <div class="main col-md-9 col-sm-8 col-xs-12">
+
<menu id="tocc" class="hide-on-med-and-down" style="padding: 0.1px; left: 0px; z-index:1; font-family:'Candara',sans-serif;">
−
<div class="bigtitle" id="yi">
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−
    Attribution
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<ul class="fl_l" style="margin-top:200px; opacity:0;">
−
   
+
<li><a href="#">Overview</a></li>
−
</div>
+
<li><a href="#">Vector design for the validation of expression system</a></li>
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Team Leader</div>
+
<li><a href="#">Validate the extracellular expression of PETase by SDS-PAGE</a></li>
−
<div class="zhengwen">Jianzhe Yang and Changyu Li are our team leaders. As team leaders, they were responsible for the coordination of the team, and supervised experimental details to promote the experimental process. At the meantime, Jianzhe Yang contacted with biological companies to buy related reagents. The role Changyu Li played in the experiment was mainly to finish the construction and verification of vectors.</div>
+
<li><a href="#">Detect the activity of PETase</a></li>
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Part Construction & Interlab</div>
+
<li><a href="#">References</a></li>
−
<div class="zhengwen">Changyu Li, Huishuang Tan, Shizhi Ding, Yinsong Xu, Yansong Wang and Yetao Zou all contributed to part construction. All parts that need to submit were constructed by them.  Additionally, Shizhi Ding and Yinsong Xu were both responsible for interlab and making great effort in it.</div>
+
</ul>
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Experiments in Straw Degradation</div>
+
</menu>
−
<div class="zhengwen">Shizhi Ding and Yinsong Xu were mainly responsible for experiments in straw degradation. They construct the plasmid of straw degradation and conduct it into different E. coli strains. For the final product in this step, they finish the detection of ferulic acid by gas chromatography as well as xylose and glucose qualitatively by TLCA</div>
+
                    </div>
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Experiments in Butanol Production</div>
+
</div>
−
<div class="zhengwen">Changyu Li and Huishuang Tan were mainly responsible for experiments in butanol production. Additionally, they measured the titer of butanol by gas chromatography. They also studied the improvement of the resistance of E. coli to butanol by GroESL.</div>
+
<div class="main col-md-9 col-sm-8 col-xs-12">
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Cellulose Enzyme Assay</div>
+
−
<div class="zhengwen">Yetao Zou and Liang Zhao were mainly responsible for enzyme assay. They detected the activity and the extracellular expression of cellulose. </div>
+
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Experiments in Hydrogen Production</div>
+
−
<div class="zhengwen">Jiayi Yin and Yansong Wang were mainly responsible for experiments in hydrogen production. They construct the plasmid of straw degradation and conduct it into different E. coli strains. In addition, they finish the detection of Hydrogen by gas chromatography and another team member, Qi Wang set up a hydrogen collection device.</div>
+
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Human Practices</div>
+
−
<div class="zhengwen">Yuxian Zhang, Longjing Pan, Jianzhe Yang were mainly responsible for human practices and Jianzhe Yang, Changyu Li, Yinsong Xu, Xiang Zhou and Qi Wang also make contribution to it. They had made the following contributions ：</div>
+
−
<div class="xstitle"><strong>Support project：</strong></div>
+
−
<div class="zhengwen">Longjing Pan and Yuxian Zhang carried out a variety of questionnaires to survey the current situation of treating straw and citizen’s attitude toward clean energy. To make our project more practical, they interact with expert in Biogas Institute of Ministry of Agriculture and Rural Affairs to learn more information about agricultural waste and bioenergy. They also interviewed synthetic biology expert, Dr. Junbiao Dai to optimize our pathway.</div>
+
−
<div class="xstitle"><strong>Public Engagement：</strong></div>
+
−
<div class="zhengwen">Yuxian Zhang and Changyu Li organized a large event of Education of Public Engagement called “Gene go”, interacting with more than 300 families in Sichuan Science and Technology Museum. They are also making effort in high school students, inspiring their interest in synthetic biology. There are other team members designed some product such as “Gene Card” online programed by Xiang Zhou and Qi Wang, Crazy Lab designed by Yinsong Xu and Plasmid Rubik made by Longjing Pan. Collaboration and meet-up is in the charge of Yuxian Zhang and Jianzhe Yang to combine universities to solve the problems that was hardly solved by themselves.</div>
+
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Modeling</div>
+
−
<div class="zhengwen">Zijian Wu was mainly responsible for modeling. In order to maximize the production of butanol and hydrogen, it is necessary to optimize the fermentation of butanol and hydrogen. He first screened out the significant influence factors of the fermentation reaction. Next, he used response surface methodology to establish a functional relationship between the product and the significant influence factor. He used Box-Behnken to design test points to get the data he needed. He finally found the optimum conditions for the fermentation reaction.</div>
+
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Art Design</div>
+
−
<div class="zhengwen">Xieyi Liu and Jiayi Yin were mainly responsible for art design. As art designers, they focused on cooperation and contacted with Human Practice, Wiki and experiment. Their work included our team logo, team uniform, the design of the page and illustrations of Wiki as well as the posters and PPTs required for meetings such as Southwest Alliance and CCiC. They also joined in the design of our educational product, such as “Crazy Lab” etc.</div>
+
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Wiki</div>
+
−
<div class="zhengwen">Qichen Pan was mainly responsible for the establishment of our Wiki page. During the whole project, he warmly cooperated with art designers and designed a set of Wiki style. He was in charge of coding and debugging. Meanwhile, he collected project content from other members and delivered it to the website and ensured complete display of Wiki.</div>
+
−
<p id="2" class="konghang" style="text-indent:30.0pt;line-height:200%;"><span style="font-family:'Candara',sans-serif; font-size:15.0pt; color:black; ">&nbsp;</span></p>
+
  
−
<div class="bigtitle">
+
<ul class="fl_r">
−
    Advisor
+
<li>
−
</div>
+
        <div class="bigtitle">
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Binglin Liu</div>
+
            Overview
−
<div class="zhengwen">He taught us how to design construction and primer for vector, and checked the sequence of primers whether it’s right or not. He also guided us to finish the construction of vector efficiently by teaching us how to make Gibson assembly.</div>
+
        </div>
−
<p id="3" class="konghang" style="text-indent:30.0pt;line-height:200%;"><span style="font-family:'Candara',sans-serif; font-size:15.0pt; color:black; ">&nbsp;</span></p>
+
        <div class="zhengwen">
 +
            This year, we have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. The combination of promoter, RBS and signal peptide makes it convenient to use as a BioBrick and gives it the function of extracellular expression.
 +
        </div>
  
−
<div class="bigtitle">
+
    </li>
−
     PI
+
     <li>
−
</div>
+
        <div class="bigtitle">
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Yong Zhang</div>
+
            Vector design for the validation of expression system
−
<div class="zhengwen">He was one of our instructors. He guided us to design the whole project and helped us to check construction strategy whether it’s feasible. Especially, he gave us very professional guidance on molecular cloning. He also helped us to comb the pathway and analyzed the result. What’s more, he gave some useful suggestions on wiki and presentation.</div>
+
        </div>
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Lixia Tang</div>
+
        <div class="zhengwen">
−
<div class="zhengwen">She was one of our instructors. She gave us some guidance on modeling. She also gave us a hand on experiment of protein expression and detection of enzyme activity. In addition, she helped us to make the data analysis. Moreover, she came up with a series of valuable advices about the design of wiki and ppt.</div>
+
            For the verification of function, we decided to use the PETase as our reporter protein. Two plasmids were constructed.
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Xuelian Zheng</div>
+
        </div>
−
<div class="zhengwen">She was one of our instructors. She gave us a lot of suggestions in details during the process of design and experiments. She helped our team leader to coordinate the team works and had communication with each member friendly to appease our tension. She also raised some idea about the style of our wiki, poster, banner and so on.</div>
+
        图
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Juan Feng</div>
+
        <div class="zhengwen">
−
<div class="zhengwen">She was one of our instructors. She was concerned with our interlab and gave us a lot of useful suggestions on it. Meanwhile, she gave us many theoretical guidance on modeling and taught us way of using various software to found model. She also gave us precious advices on TLC design.</div>
+
            Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful.
−
<p id="4" class="konghang" style="text-indent:30.0pt;line-height:200%;"><span style="font-family:'Candara',sans-serif; font-size:15.0pt; color:black; ">&nbsp;</span></p>
+
        </div>
 +
        图
 +
    </li>
 +
    <li>
 +
        <div class="bigtitle">
 +
            Validate the extracellular expression of PETase by SDS-PAGE
 +
        </div>
 +
        <div class="zhengwen">
 +
            To validate the extracellular expression of PETase, the supernatant fractions of LB culture were separated by centrifugation (8000rpm,4℃). Then we used the absorbing column to do the protein concentration. Added 30mL supernatant into absorbing column, and got 1mL even less concentrated supernatant through refrigerated centrifugation(4℃, 4000g，30min).After SDS-PAGE analysis, we found that the supernatant of piGEM2016-001 which carrying pelB-5D showed a target band while no such a band was detected in piGEM2016-002 （FigXX).The result demonstrated that our improved part can increase the extracellular expression of the protein.
 +
        </div>
 +
        图
  
−
<div class="bigtitle">
+
    </li>
−
     Acknowledgement
+
    <li>
 +
        <div class="bigtitle">
 +
            Detect the activity of PETase
 +
        </div>
 +
        <div class="zhengwen">
 +
            The activity of PETase was determined after expression of PETase in E.coli BL21 (DE3) for 24h.pNP (p-nitrophenol) assay is commonly used method for quantitative detection of lipase activity. The assay is based on the production of pNP which has a maximum absorption at 405 nm. For measuring the activity of PETase, we chose pNPB (p-nitrophenol butyrate) as the substrate which can be hydrolyzed to pNP by PETase. One unit (U) of PETase activity was defined as the amount of PETase that could release 1 mmol pNP from pNPB per minute (Kim et al., 2015). For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured (Figure 8).
 +
        </div>
 +
        图
 +
     </li>
 +
    <li>
 +
        <div class="bigtitle">
 +
            Reference
 +
        </div>
 +
    </li>
 +
</ul>
 +
</div>
 +
</div>
 +
<div style="clear: both;"></div>
 
</div>
 
</div>
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;UESTC-China iGEM 2018 team gratefully acknowledges the following institutes:</div>
 
−
<div class="zhengwen">
 
−
<ul>
 
−
    <li>School of Life Sciences, University of Electronic Science and Technology of China.</li>
 
−
    <li>Office of Educational Administration, University of Electronic Science and Technology of China.</li>
 
−
    <li>Office of Students' Affairs, University of Electronic Science and Technology of China.</li>
 
−
    <li>Plant Genome Engineering Lab, University of Electronic Science and Technology of China.</li>
 
−
    <li>Protein Engineering Lab, University of Electronic Science and Technology of China.</li>
 
−
    <li>College Of Life Sciences, Sichuan University.</li>
 
−
</ul>
 
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</div>
 
−
<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Experiment equipment support</div>
 
−
<div class="zhengwen">
 
−
<ul>
 
−
    <li>Thanks to School of Medicine (University of Electronic Science and Technology of China) for giving us support on Multifunctional enzyme marker.</li>
 
−
    <li>Thanks to School of Biotechnology (Jiangnan University) for presenting us the strain of Escherichia coli B0016-050</li>
 
−
</ul>
 
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</div>
 
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<div class="smtitle"><i class="fa fa-chevron-right"></i>&nbsp;Human practices support</div>
 
−
<div class="zhengwen">
 
−
    <ul>
 
−
        <li>Thanks to Biogas Institute of Ministry of Agriculture and Rural Affairs for giving us some suggestions in our project</li>
 
−
    </ul>
 
 
</div>
 
</div>
  
  
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Revision as of 15:11, 14 October 2018

team

IMPROVE PART

  • Overview
    This year, we have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. The combination of promoter, RBS and signal peptide makes it convenient to use as a BioBrick and gives it the function of extracellular expression.
  • Vector design for the validation of expression system
    For the verification of function, we decided to use the PETase as our reporter protein. Two plasmids were constructed.
    图
    Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful.
    图
  • Validate the extracellular expression of PETase by SDS-PAGE
    To validate the extracellular expression of PETase, the supernatant fractions of LB culture were separated by centrifugation (8000rpm,4℃). Then we used the absorbing column to do the protein concentration. Added 30mL supernatant into absorbing column, and got 1mL even less concentrated supernatant through refrigerated centrifugation(4℃, 4000g，30min).After SDS-PAGE analysis, we found that the supernatant of piGEM2016-001 which carrying pelB-5D showed a target band while no such a band was detected in piGEM2016-002 （FigXX).The result demonstrated that our improved part can increase the extracellular expression of the protein.
    图
  • Detect the activity of PETase
    The activity of PETase was determined after expression of PETase in E.coli BL21 (DE3) for 24h.pNP (p-nitrophenol) assay is commonly used method for quantitative detection of lipase activity. The assay is based on the production of pNP which has a maximum absorption at 405 nm. For measuring the activity of PETase, we chose pNPB (p-nitrophenol butyrate) as the substrate which can be hydrolyzed to pNP by PETase. One unit (U) of PETase activity was defined as the amount of PETase that could release 1 mmol pNP from pNPB per minute (Kim et al., 2015). For quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured (Figure 8).
    图
  • Reference
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