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

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{{ZJU-China}}
 
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<html>
 
  
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<html lang="zh-CN">
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/*文中自定义*/
 +
</style>
 +
 +
  <body id="page-top" data-spy="scroll" data-target=".navbar-fixed-top">
 +
  <div class="page-loader"></div>
 +
      <!-- Docs master nav -->
 +
                  <!-- <h1><a class="navbar-brand" href="index.html">MuMei Lab</a></h1> -->
 +
 +
    <div class="container">
 +
      <!-- header -->
 +
      <div class="header-w3layouts">
 +
          <!-- Navigation -->
 +
          <nav class="navbar navbar-default navbar-fixed-top">
 +
              <div class="navbar-header page-scroll">
 +
                  <button type="button" class="navbar-toggle" data-toggle="collapse" data-target=".navbar-ex1-collapse">
 +
                  </button>
 +
 +
                  <a href="https://2017.igem.org/Team:ZJU-China">
 +
                      <img style="margin-top:11px" class="navbar-brand"  src="https://static.igem.org/mediawiki/2017/d/d5/ZJUChina_logo.png">
 +
                  </a>
 +
 +
                  <!-- <h1><a class="navbar-brand" href="index.html">My Design</a></h1> -->
 +
 +
              </div>
 +
              <!-- Collect the nav links, forms, and other content for toggling -->
 +
              <div class="collapse navbar-collapse navbar-ex1-collapse">
 +
                  <ul class="nav navbar-nav navbar-right cl-effect-15">
 +
                      <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
 +
                      <li class="hidden"><a class="page-scroll" href="#page-top"></a> </li>
 +
 +
                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Overview<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Overview">Project Description</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Demonstrate">Demonstrate</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Achievements">Achievements</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Applied_Design">Applied Design</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Improve">Improve Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/InterLab">InterLab</a></li>
 +
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Project<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st">Chemical Transduction</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/mt">Medium Wave Transduction</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/tp">Trichoderma Proof</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/voc">VOC sensors</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/Downstream">Downstream</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/conclusion">Conclusion</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Notebook">Notebook</a></li>
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Model<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <!--<li><a href="https://2017.igem.org/Team:ZJU-China/Model">Summery</a></li>-->
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Model">VOC analysis</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Model/Coculture">Coculture</a></li>
 +
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Parts<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Composite_Part">Composite Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Part_Collection">Part Collection</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Basic_Part">Basic Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Parts">All Parts</a></li>
 +
 +
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li><a href="https://2017.igem.org/Team:ZJU-China/Hardware">Hardware</a></li>
 +
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Safety<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Safety">Environment</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Safety/Lab">Laboratory</a></li>
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">HP<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Human_Practices">Summary</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/HP/Silver">Silver</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/HP/Gold_Integrated">Gold Integrated</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Engagement">Engagement</a></li>
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Team<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Collaborations">Collaboration</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Team">Teammates</a></li>
 +
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<h1>Improve</h1>
 
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Regisrty. Please include a link to your improved part on this page.</p>
 
  
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<h3>Gold Medal Criterion #2</h3>
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<div style="width: 100%" class="container zjuContent">
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<p><b>Standard Tracks:</b> Improve the function of an existing BioBrick Part. The original part must NOT be from your 2017 part number range. If you change the original part sequence, you must submit a new part. In addition, both the new and original part pages must reference each other. This working part must be different from the part documented in bronze #4 and silver #1.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
+
  <h1 id="improve" class="ArticleHead GreenAH">Improve Part</h1>
 +
      <p class="PP">It is well known that T7 promoter is a kind of common promoter used for expression of heterogenous protein in some E.coli strains such as BL21(DE3). Though the wild-type T7 promoter is of high efficiency, in order to meet some specific demands, we need a series of modified T7 promoters with different strength of expression. Hence, we tried to transform the Wild-type T7 promoter to get a collection of modified T7 promoters.</p>
 +
      <p class="PP">T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+ 1) and extends from -17 to +6. As reported in some papers, the sequence specificty of T7 promoter is so strong that some mutation may make T7 promoter fail to work. Thus, with the help of some previous research, we carefully chose the site which would be mutated by PCR. These sites mainly distribute in the site from -20 to -12. The sequences of these modified promoters are shown in the Table below.</p>
 +
    <h2 id="sms" class="H2Head">Sequences of Modified Promoters</h2>
 +
          <table class="table">
 +
              <tr><th class="yellowTable">Part number</th><th class="yellowTable">Sequence(-20~+6)</th></tr>
 +
              <tr><th class="grayTable">BBa_K525998(wild type)</th><th>gagtaatacgactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207024</th><th><a class="redgene">t</a>agtaatacgactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207025</th><th>aa<a class="redgene">g</a>taatacgactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207026</th><th>ga<a class="redgene">a</a>taatacgactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207027</th><th>ga<a class="redgene">t</a>taatacgactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207028</th><th>ga<a class="redgene">t</a>taata<a class="redgene">a</a>gactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207029</th><th>ga<a class="redgene">t</a>taata<a class="redgene">t</a>gactcactatagggaaa</th></tr>
 +
              <tr><th class="grayTable">BBa_K2207030</th><th><a class="redgene">t</a>a<a class="redgene">t</a>taatacgactcactatagggaaa</th></tr>
 +
          </table>
 +
 
 +
      <h2 id="test" class="H2Head">Test</h2>
 +
          <p class="PP">To test the function of mutant promoters, we chose the <a class="cite" href="http://parts.igem.org/Part:BBa_E1010">mRFPas</a> our reporter. By assessing the relative fluorescence units(RFU) and OD600, we can conclude the relative strength of all promoters. When the E.coli BL21(DE3) is cultured at the stage of logarithmic phase, we added IPTG to induce the expression of mRFP in strains BL21(DE3) for 4 hours. And the result is shown as Figure below.</p>
 +
          <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/2/23/ZJU_China_imporve_1.png"></div>
 +
            <p class="capture">Relative Strength of wildtype T7 promoter and mutant promoters</p>
 +
          <p class="PP">As we can see from the figure, except the part K2207024, all mutant promoters showed increased strength compared with wild type T7 promoter. Therefore, our part collection enables users to control the expression of protein using T7 promoter, especially offering more opportunity for increasing the efficiency of protein expression.</p>
  
  
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        <li><a href="#sms">Sequences of Modified Promoters</a></li>
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Revision as of 11:30, 29 October 2017

Improve Part

It is well known that T7 promoter is a kind of common promoter used for expression of heterogenous protein in some E.coli strains such as BL21(DE3). Though the wild-type T7 promoter is of high efficiency, in order to meet some specific demands, we need a series of modified T7 promoters with different strength of expression. Hence, we tried to transform the Wild-type T7 promoter to get a collection of modified T7 promoters.

T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+ 1) and extends from -17 to +6. As reported in some papers, the sequence specificty of T7 promoter is so strong that some mutation may make T7 promoter fail to work. Thus, with the help of some previous research, we carefully chose the site which would be mutated by PCR. These sites mainly distribute in the site from -20 to -12. The sequences of these modified promoters are shown in the Table below.

Sequences of Modified Promoters

Part numberSequence(-20~+6)
BBa_K525998(wild type)gagtaatacgactcactatagggaaa
BBa_K2207024tagtaatacgactcactatagggaaa
BBa_K2207025aagtaatacgactcactatagggaaa
BBa_K2207026gaataatacgactcactatagggaaa
BBa_K2207027gattaatacgactcactatagggaaa
BBa_K2207028gattaataagactcactatagggaaa
BBa_K2207029gattaatatgactcactatagggaaa
BBa_K2207030tattaatacgactcactatagggaaa

Test

To test the function of mutant promoters, we chose the mRFPas our reporter. By assessing the relative fluorescence units(RFU) and OD600, we can conclude the relative strength of all promoters. When the E.coli BL21(DE3) is cultured at the stage of logarithmic phase, we added IPTG to induce the expression of mRFP in strains BL21(DE3) for 4 hours. And the result is shown as Figure below.

Relative Strength of wildtype T7 promoter and mutant promoters

As we can see from the figure, except the part K2207024, all mutant promoters showed increased strength compared with wild type T7 promoter. Therefore, our part collection enables users to control the expression of protein using T7 promoter, especially offering more opportunity for increasing the efficiency of protein expression.


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