Difference between revisions of "Team:Peking/Notebook"

 
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      <meta name="description" content="Wiki of Peking iGEM 2016" />
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    <meta name="description" content="Wiki of Peking iGEM 2016" />
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      <meta name="author" content="Li Jiamian & Wang Yuqing">
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        <div id="logo" style="max-width:170px"><a class="" href="https://2016.igem.org/Team:Peking"></a></div>
 
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        <ul class="nav">
 
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          <li class="menu-1"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking" >Home</a></li>
 
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          <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a>
 
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                    <ul class="dropdown-menu">
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Description" >Decription</a></li>
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li>
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Proof" >Proof of Concept</a></li>
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Demonstrate</a></li>
 
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                    </ul>
 
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                  </li>
 
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          <li class="dropdown menu-3"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Parts</a>
 
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                    <ul class="dropdown-menu">
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Basic_Part" >Basic parts</a></li>
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Composite_Part" >Composite parts</a></li>
 
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                        <li><a href="https://2016.igem.org/Team:Peking/Part_Collection" >Parts collection</a></li>
 
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                    </ul>
 
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                  </li>
 
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          <li class="menu-4"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Human_Practices" >Practices</a></li>
 
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          <li class="menu-5"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Collaborations" >Collaboration</a></li>
 
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          <li class="menu-6"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Model" >Modeling</a></li>
 
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                    <ul class="dropdown-menu">
 
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                          <li><a class="" href="https://2016.igem.org/Team:Peking/Team" >Team</a></li>
 
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                          <li><a class="" href="https://2016.igem.org/Team:Peking/Attributions" >Attribution</a></li>
 
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                          <li><a class="" href="https://2016.igem.org/Team:Peking/Notebook" >Notebook</a></li>
 
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            </ul>
 
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                  </li>
 
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                  <li class="dropdown menu-8"><a class="colapse-menu1" data-toggle="dropdown" href="https://2016.igem.org/Team:Peking/Interlab" >Interlab</a>
 
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      </div>
 
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    </div>
 
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    </div>
 
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              <h1>Attributions<span>.</span></h1>
 
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              <p class="title1" style="text-align:center">The idea of this project was conceived by the team; advisors and instructors gave advices and suggestions on the implementation of this idea.</p>
 
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                    <div id="logo" style="max-width:170px"><a class="" href="https://2016.igem.org/Team:Peking"></a></div>
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                    <div class="nav-collapse collapse">
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                            <li class="menu-1"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking" >Home</a></li>
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                            <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#" > Achievements</a>
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                                <ul class="dropdown-menu">
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                                    <li><a href="https://2016.igem.org/Team:Peking/Results" >Results</a></li>
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                                    <li><a href="https://2016.igem.org/Team:Peking/Basic_Part" >Parts</a></li>
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                                    <li><a href="https://2016.igem.org/Team:Peking/Collaborations" >Collaborations</a></li>
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                                </ul>
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                            </li>
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                            <li class="dropdown menu-3"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a>
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                                <ul class="dropdown-menu">
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                                    <li><a href="https://2016.igem.org/Team:Peking/Description" >Overview</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Crosslinking" >Crosslinking</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption" >Uranyl adsorption</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Clearance" >Clearance</a></li>
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                                    <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Final Performance</a></li>
 +
                                </ul>
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                            </li>
 +
                            <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a>
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                                <ul class="dropdown-menu">
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                                    <li><a href="https://2016.igem.org/Team:Peking/Model/GelPoint" > Model of Gel Point </a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Model/MassDistribution" > Model of Mass Distribution</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/Software" >Software</a></li>
 +
                                </ul>
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                            </li>
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                            <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a>
 +
                                <ul class="dropdown-menu">
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/311" >Field research</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/questionnaire" >Questionnaire</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/consulting" >Consulting</a></li>
 +
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/otherHP" >Education&nbsp;&amp;&nbsp;Other</a></li>
 +
                                </ul>
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                            </li>
 +
                            <li class="menu-6"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Safety" >Safety</a>
 +
                                <li class="dropdown menu-7"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Lab</a>
 +
                                    <ul class="dropdown-menu">
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                                        <li><a class="" href="https://2016.igem.org/Team:Peking/Team" >Team</a></li>
 +
                                        <li><a class="" href="https://2016.igem.org/Team:Peking/Attributions" >Attribution</a></li>
 +
                                        <li><a class="" href="https://2016.igem.org/Team:Peking/Notebook" >Notebook</a></li>
 +
                                    </ul>
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                                </li>
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                                <li class="menu-8"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Interlab" >Interlab</a>
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                                </li>
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                                </div>
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              <h1>Notebook</h1>
 +
              <p class="title1" style="text-align:center"> Thin as it is, the notebook serves as a best gift to look back on our struggle for iGEM.
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</p>
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<div id="page-content" class="row page">
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        <section>
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  <div class="row">
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  <div class="col-mid-2">
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  </div>
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<h3 style="text-align:center"> Dairy</h3>
  
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<div class="panel-group notebook" id="accordion">
  
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        <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title" style="color:white;">
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                    <a data-toggle="collapse" href="#collapseOne">Week 1 (6/26/2016-7/02/2016)</a>
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                </h4>
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            </div>
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                <div class="panel-body">
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                    <ul>
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                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
                                <li><a>3SpyTag (A) </a>assemble</li>
 +
                                <li><a>SUP</a> PCR</li>
 +
                                <li><a>pET28a backbone </a>PCR</li>
 +
                                <li><a>SpyCatcher (B) </a>PCR</li>
 +
                                <li><a>Monomeric Streptavidin (mSA)</a> PCR</li>
 +
                        </ul>
 +
                    </ul>
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                </div>
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            </div>
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        </div>
  
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" href="#collapse2">Week 2 (7/03/2016-7/09/2016)</a>
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                </h4>
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                    <ul>
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                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
                                <li><a>His-3A-SUP-pET28a</a> construction</li>
 +
                                <li><a>His-3A-mSA-pET28a</a> construction</li>
 +
                                <li><a>His-2B-pET28a </a>construction</li>
 +
                        </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Sequenced the plasmid pBES and confirmed that there are no additional BsaI sites</li>
 +
                                <li>Transformed the plasmid pSB1C3 and extracted it for future use</li>
 +
                                <li>Constructed the signal peptide ImdA on pSB1C3 vector</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
  
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" href="#collapse3">Week 3 (7/10/2016-7/16/2016)</a>
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+
                </h4>
 +
            </div>
 +
            <div id="collapse3" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
                                <li><a>mRFP</a> PCR</li>
 +
                                <li><a>effoRED </a>PCR</li>
 +
                                <li><a>YFP </a>PCR</li>
 +
                        </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the signal peptide OmpA, LTIIb, PhoA, YjfA, NprE</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>BB</a>: 15.440 mg/ml</li>
 +
                                <li><a>3A-sup</a>: 9.281 mg/ml</li>
 +
                                <li><a>3B (15*linker)</a>: 105.857 mg/ml</li>
 +
                                <li><a>3A(15*linker)</a>: fail</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li><a>Pre-experiment:</a>We got familiar with the apparatuses in the lab we performed experiments related to uranyl and tried the Arsenazo III method. Finally, we obtained a standard curve of the concentration ranging from 0uM-30uM</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
  
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse4">Week 4 (7/17/2016-7/23/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse4" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>                 
 +
                                <li><a>His-4A-SUP-pET28a </a>constructione</li>
 +
                                <li><a>His-6A-SUP-pET28a </a>construction</li>
 +
                                <li><a>His-3A-mRFP-pET28a </a>construction</li>
 +
                            </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the signal peptide LipA, SacB</li>
 +
                                <li>Constructed the kil protein</li>
 +
                                <li>Tried to construct the OmpA-SUP-pET28a expression plasmid but failed</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>3A(15*linker)</a>: 50.393 mg/ml</li>
 +
                                <li><a>3A-sup</a>: 10.25 mg/ml</li>
 +
                                <li><a>3A-msa</a>: 10.971 mg/ml</li>
 +
                                <li><a>His-sup</a>: 6.353 mg/ml</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>We broke the E.coli with ultrasonication, and extracted 3A-mSA protein using a affinity column. Mixed 3A-mSA and 3B in TBS buffer. Then we did a SDS-PAGE electrophoresis and analysis the results ( Fig.1 ).We could find out that 3A-mSA had the ability to crosslink with 3B.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse5">Week 5 (7/24/2016-7/30/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse5" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
                                <li><a>His-3A-effoRED-pET28a </a>construction</li>
 +
                                <li><a>His-3A-YFP-pET28a </a>construction</li>
 +
                                <li><a>3A-SUP-pSB1C3</a> construction</li>
 +
                                <li><a>3A-mSA-pSB1C3</a> construction</li>
 +
                        </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the expression plasmids OmpA-SUP-pET28a and LTIIb-SUP-pET28a and transformed them into BL21(DE3) strain</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>4A-sup</a>: 6.182 mg/ml</li>
 +
                                <li><a>6A-sup</a>: 22.320 mg/ml</li>
 +
                                <li><a>3A-red</a>: 93.560 mg/ml</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>We tried to measure the reaction capacity of the mSA part of the fusion protein with Biotin-Atto 488 which could show autofluorescence. But results were not useful, because there might be some noncovalent interactions between 3 kDa cutoff centrifuge filters and the molecular.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse6">Week 6 (7/31/2016-8/06/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse6" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>                     
 +
                                <li><a>His-4A-mRFP-pET28a</a> construction</li>
 +
                                <li><a>His-4A-effoRED-pET28a</a> construction</li>
 +
                                <li><a>His-4A-YFP-pET28a</a> construction</li>
 +
                                <li><a>His-6A-mRFP-pET28a</a> construction</li>
 +
                                <li><a>His-6A-effoRED-pET28a</a> construction</li>
 +
                                <li><a>His-6A-YFP-pET28a</a> construction</li>
 +
                                <li><a>His-4A-mSA-pET28a</a> construction</li>
 +
                                <li><a>His-6A-mSA-pET28a</a> construction</li>
 +
                        </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed additional three signal peptides Bpr, Epr and PelB</li>
 +
                                <li>Tried to construct the expression vector in B.subtilis but all failed</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>3A-yellow</a>: fail</li>
 +
                                <li><a>3A-pink</a>: fail</li>
 +
                                <li><a>3A-sup</a>: 14.374 mg/ml</li>
 +
                                <li><a>3A-msa</a>: 14.262 mg/ml</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li>We improved our experimental methods and supplemented equipment we needed. In these two days, we reduced our reaction volume from 3mL to 400uL and increased our efficiency. Also, standard curve of concentration ranging from 0uM-30uM was tested.</li>
 +
                                <li>The adsorption capacity of 6A-SUP was tested, even though the data were not parallel, we confirmed that 6A-SUP can absorb uranyl. The highest adsorption rate was 81.56%</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>Last week we got nothing useful. So we changed the protocol about how to use Biotin-Atto 488. We prepared several little affinity columns, immobilized 3A-mSA containing 6x His-Tag, and perfused the columns with solution of Biotin-Atto 488. After measuring the fluorescence intensity, we got a qualitative result that 3A-mSA can react with biotin.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse7">Week 7 (8/07/2016-8/13/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse7" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
                                <li><a>3A-SUP-pSB1C3</a> construction</li>
 +
                                <li><a>3A-mSA-pSB1C3</a> construction</li>
 +
                        </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the expression plasmids of SUP, mSA with different signal peptides: PhoA-SUP-pET28a, PelB-SUP-pET28a, OmpA-mSA-pET28a, LTIIb-mSA-pET28a, PhoA-mSA-pET28a and PelB-mSA-pET28a</li>
 +
                                <li>Constructed the expression plasmids of kil: J23110-J61110-kil-pSB4C5</li>
 +
                                <li>Constructed the expression plasmids of mRFP (E1010), yellow (K592010), eforRed (K592012): OmpA-mRFP-pET28a, LTIIb-mRFP-pET28a, PhoA-mRFP-pET28a, PelB-mRFP-pET28a, OmpA-Yellow-pET28a, LTIIb-Yellow-pET28a, PhoA-Yellow-pET28a, PelB-Yellow-pET28a, OmpA-eforRed-pET28a, LTIIb-eforRed-pET28a, PhoA-eforRed-pET28a and PelB-eforRed-pET28a</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>4A-sup</a>: 15.133 mg/ml</li>
 +
                                <li><a>His-sup</a>: 30.663 mg/ml</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li>The adsorption capacity of 3A-SUP and 3A-SUP+3B was tested. We confirmed that 3A-SUP can absorb uranyl, so can 3A-SUP+3B. In today’s experiments, the data was more parallel. We used solution containing uranyl only as control and its uranyl concentration after filtration as the actual concentration of uranyl.</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>To absorb the proteins in the environment, we wanted to use biotin-coated beads. So we bought the reagents and magnetic beads with amino group.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse8">Week 8 (8/14/2016-8/20/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse8" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the expression plasmids of SUP and Spycatcher of different signal peptides for B.subtilis: ImdA-SUP-pBES, NprE-SUP-pBES, SacB-SUP-pBES, YjfA-SUP-pBES, LipA-SUP-pBES, ImdA-Spycatcher-pBES, NprE-Spycatcher-pBES, SacB-Spycatcher-pBES, YjfA-Spycatcher-pBES, LipA-Spycatcher-pBES, Bpr-Spycatcher-pBES</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>3A-msa in the form of inclusion body</a>: 2.273 mg/ml</li>
 +
                                <li><a>3A</a>: 29.768 mg/ml</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li>The adsorption capacity of His-SUP was tested, we confirmed that His-SUP can absorb uranyl and the result was close to the data performed by the author of the paper we referred.</li>
 +
                                <li>We tested the adsorption capacity of 3/4/6A-SUP and 3/4/6A-SUP+3B in one day to exclude unnecessary effects. Then we analyzed the data and found the results were compromising. The capacity were fair and even though the proteins formed colloid, the adsorption capacity barely decreased.</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the biotin-coated beads.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse9">Week 9 (8/21/2015-8/27/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse9" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the expression plasmids of Spycatcher, mSA and Red with different signal peptides : PhoA-Spycatcher-pET28a, PelB-Spycatcher-pET28a, LTIIb-Spycatcher-pET28a, ImdA-mSA-pBES, NprE-mSA-pBES, SacB-mSA-pBES, YjfA-mSA-pBES, LipA-mSA-pBES, ImdA-Red-pBES, NprE-Red-pBES, SacB-Red-pBES, YjfA-Red-pBES, LipA-Red-pBES and SUP-pBES without any signal peptide as a control plasmid</li>
 +
                    </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li><a>3A-sup</a>: 20.349 mg/ml</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li>Water from Weiming lake was collected and simulated sea water was prepared.</li>
 +
                                <li>We tested the adsorption capacity of 3A-SUP+3B in different conditions, including TBS buffer, Weiming lake and simulated sea water. </li>
 +
                                <li>we changed the protein-uranyl ratio from 1:1 to 10:1 to determine whether the adsorption capacity increased. </li>
 +
                                <li>we decreased the uranyl concentration to 5uM. </li>
 +
                                <li>we decreased the uranyl concentration to 13nM and increased the protein-uranyl ratio to 6000:1.</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>Prepared the solution containing 3A protein or 3A-mSA protein, added the beads we made last week, shocked the reaction system adequately for 1h, precipitated the beads with magnetic shelf, and measured the concentration of proteins in the liquid supernatant.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse10">Week 10 (8/28/2015-9/03/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse10" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li> Constructed the expression plasmids of inducible kil with different signal peptides E. coli and SUP with P43 promotor for B.subtilis: T7-Lac promotor-OmpA-SUP, T7-Lac promotor-PhoA-SUP; P43 promotor-ImdA- SUP -PBES; P43 promotor-NprE- SUP -PBES, P43 promotor-SacB- SUP -PBES, P43 promotor-LipA- SUP -PBES, P43 promotor-YjfA- SUP -PBES </li>
 +
                    </ul>
 +
                        <li><b>Secretion examination: </b></li>
 +
                            <ul>
 +
                            <li>Evaluated the secretion effect for 3ASUP of different signal peptides using western blot. </li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li>We received the ICP-MS results. </li>
 +
                    </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>Prepared to attend CCiC, also known as Central China iGEM Consortium. We had a great week in this meeting, in Sun Yat-Sen University, Guang Zhou, China.</li>
 +
                    </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse11">Week 11 (9/04/2015-9/10/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse11" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed chromoproteins with the signal peptide, PhoA : </li>
 +
                                <li>PhoA-mRFP-pET28a, PhoA-eforRed-pET28a, PhoA-Yellow-pET28a</li>
 +
                                <li>Handed all the vectors with desired elements to the Test Group for examining the secretion efficiency. </li>
 +
                    </ul>
 +
                        <li><b>Secretion examination: </b></li>
 +
                            <ul>
 +
                            <li>Evaluated the secretion effect for 3A mSA and 3B of different signal peptides using western blot.</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                            <li>Purified the cell lysate as well as medium of OmpA SUP using Ni-NTA chromatography.</li>
 +
                        </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>Measured the adsorption capacity of protein network with biotin coated beads. (Crosslinked 3A-mSA and 3B for 1 hour, and then added the beads into the reaction system. The remaining proportion of protein in the environment was measured.</li>
 +
                    </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse12">Week 12 (9/11/2015-9/17/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse12" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
                                <li>Constructed all the Secretion Parts on pSB1C3 vector and the expression plasmids LBP-pET28a, CBP-pET28a and MBP-pET28a</li>
 +
                    </ul>
 +
                        <li><b>Secretion examination: </b></li>
 +
                            <ul>
 +
                                <li>Quantified the secretion effect of 3B of different signal peptides using anti-Histag ELISA.</li>
 +
                        </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse13">Week 13 (9/18/2015-9/24/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse13" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
                                <li>3A-LBP, 3A-MBP and 3A-CBP.</li>
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
                                <li>Purified 3A-SUP, 3A-LBP, 3A-CBP and 3A-MBP. Unfortunately, we didn’t get 3A-MBP due to the damage of chromatography column.</li>
 +
                        </ul>
 +
                        <li><b>Secretion examination: </b></li>
 +
                            <ul>
 +
                                <li>Quantified the secretion effect of 3B and 3ASUP of different signal peptides using anti-Histag ELISA.</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li> We repeated experiments on adsorption in different water conditions(boiled and without CO2).</li>
 +
                                <li> We tested 3A-SUP+3B adsorption capacity in TBS buffer with different Ph ranging from 6-9.</li>
 +
                    </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>Prepared the Integrating experiment for the next week.</li>
 +
                    </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse14">Week 14 (9/25/2015-10/01/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse14" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                      <li><b>Secretion examination: </b></li>
 +
                            <ul>
 +
                                <li>Quantified the secretion effect of 3AmSA and 3ASUP of different signal peptides using anti-Histag ELISA.</li>
 +
                                <li>During this week we also tesedt the influence of IPTG on the secreted concentration. A concentration gradient of IPTG was applied to evaluate the secretion effect of OmpA3B at a time gradient from 1 h to 7 h incubated at 37 ℃.</li>
 +
                        </ul>
 +
                        <li><b>Uranyl Absorption:</b></li>
 +
                            <ul>
 +
                                <li> We repeated experiments on 3A-SUP+3B adsorption capacity in different water conditions(boiled and without CO2).</li>
 +
                    </ul>
 +
                        <li><b>Clearance:</b></li>
 +
                            <ul>
 +
                                <li>We prepared the kit to adsorb uranyl from the environment. We used the biotin coated beads to harvest the protein network which had accommodated uranyl of simulate contaminative sea water and fresh water.</li>
 +
                    </ul>
 +
                    </ul>
 +
                </div>
 +
            </div>
 +
        </div>
 +
       
 +
     
 +
       
 +
       
 +
    </div>
 +
    </div><!--col-md-8 ended-->
  
−
  </script>
 
  
  
 +
<h3 style="text-align:center"> Protocols</h3>
 +
<div class="four columns">
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:Gel_Extraction"><img src="https://static.igem.org/mediawiki/2016/0/03/T--Peking--image_protocol_Gel_Extraction.png"></a>
 +
<p style="text-align:center"><b>Gel Extraction</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:ligation"><img src="https://static.igem.org/mediawiki/2016/2/2f/T--Peking--image_protocol_ligation.png"></a>
 +
<p style="text-align:center"><b>Ligation</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:temperature_gradient_experiment"><img src="https://static.igem.org/mediawiki/2016/c/c1/T--Peking--image_protocol_temperature_gradient_experiment.png"></a>
 +
<p style="text-align:center"><b>Temperature Gradient Experiment</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:PCR"><img src="https://static.igem.org/mediawiki/2016/0/09/T--Peking--image_protocol_PCR.png"></a>
 +
<p style="text-align:center"><b>PCR</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:integrating_experiments"><img src="https://static.igem.org/mediawiki/2016/d/d4/T--Peking--image_protocol_integrating_experiment.png"></a>
 +
<p style="text-align:center"><b>Integrating Experiment</b></p>
 +
</div>
  
 +
<div class="four columns">
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:purification_of_recombinant_proteins"><img src="https://static.igem.org/mediawiki/2016/4/48/T--Peking--image_protocol_purification_of_recombinant_proteins.png"></a>
 +
<p style="text-align:center"><b>Purification of Recombinant Proteins</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:transformation"><img src="https://static.igem.org/mediawiki/2016/5/52/T--Peking--image_protocol_transformation.png"></a>
 +
<p style="text-align:center"><b>Transformation</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:testing_adsorption_capacity_of_protein"><img src="https://static.igem.org/mediawiki/2016/8/85/T--Peking--image_protocol_testing_adsorption_capacity_of_protein.png"></a>
 +
<p style="text-align:center"><b>Testing Adsorption Capacity of Protein</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:AGE"><img src="https://static.igem.org/mediawiki/2016/5/51/T--Peking--image_protocol_AGE.png"></a>
 +
<p style="text-align:center"><b>Agarose Gel Electrophoresis</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:concentration_gradient_experiment"><img src="https://static.igem.org/mediawiki/2016/e/ed/T--Peking--image_protocol_concentration_gradient_experiment.png"></a>
 +
<p style="text-align:center"><b>Concentration Gradient Experiment</b></p>
  
 +
</div>
 +
<div class="four columns">
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:biotin-associated_Experiment"><img src="https://static.igem.org/mediawiki/2016/b/b4/T--Peking--image_protocol_biotin-associated_Experiment.png"></a>
 +
<p style="text-align:center"><b>Biotin-associated Experiment</b></p>
  
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:SDS-Pages"><img src="https://static.igem.org/mediawiki/2016/d/d4/T--Peking--image_protocol_SDS_Pages.png"></a>
 +
<p style="text-align:center"><b>SDS-Page</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:Western_Blot"><img src="https://static.igem.org/mediawiki/2016/1/10/T--Peking--image_protocol_Western_Blot.png"></a>
 +
<p style="text-align:center"><b>Western Blot</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:pH_Gradient_Experiment"><img src="https://static.igem.org/mediawiki/2016/9/97/T--Peking--image_protocol_pH_Gradient_Experiment.png"></a>
 +
<p style="text-align:center"><b>pH Gradient Experiment</b></p>
 +
<a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:crosslinking"><img src="https://static.igem.org/mediawiki/2016/7/76/T--Peking--image_protocol_crosslinking.png"></a>
 +
<p style="text-align:center"><b>Crosslinking</b></p>
 +
</div>
  
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    <h4>Attributions</h4>
+
−
      <ul>
+
−
          <li><a href="#members">Members'</a></li>
+
−
          <li><a href="#acknowledgement">Acknowledgement</a></li>
+
−
          <li><a href="#sponsors">Sponsors</a></li>
+
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      </ul>
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  </ul>
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                  <a id="members"></a>     
 
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                  <h3 class="classic-title";"><span>Member’s Attributions</span></h3>                     
 
−
                  <p>All the data presented on this wiki was measured, collected, and analyzed by the team members. All the plasmids directly involved in the data presented on this wiki were constructed by the team members. The idea conceiving, content planning, execution, and result analysis of human practice were all done by the team members. The following is the detailed attribution:
 
−
  </p>
 
−
                  <p>
 
−
                  <strong>LI Cheng</strong> is the team leader of Peking iGEM 2016. He participated in conceiving the project, took charge of troubleshooting, and managed the experiments, guaranteeing the normal operation of the lab.<br/>
 
−
                  <strong>YANG Xiaoyu</strong>  was committed to ameliorate the paired dCas9 (PC) reporter system, namely, to enhance the sensitivity and robustness. Early in the project, he was also responsible for information gathering, experiment design, and lab management<br/>
 
−
 
−
                  <strong>BAI Ke</strong> was in charge of Human Practice and also worked in the wet lab to construct and prepare plasmids for gRNA in vitro transcription.<br/>
 
−
                  <strong>DONG Yiming</strong>
 
−
                  was responsible for designing and manufacturing the portable electronic device that convert optical signal into analogue electrical signal.<br/>
 
−
                  <strong>LI Yuexuan</strong> worked as the financial manager of the team. She was also responsible for plasmid construction and submission.<br/>
 
−
                  <strong>YANG Changru</strong> was in charge of building <i>M.tuberculosis</i>-sensing system using Molecule Beacon. He was also engaged in the protein purification of dCas9 fusion proteins.<br/>
 
−
                  <strong>ZHAO Shijun</strong> was in charge of measuring the luminescence intensity of PC reporter system. She analyzed the entire experimental data.<br/>
 
−
                  <strong>LV Nayun</strong> helped to complete the plasmid construction of sgRNA and was in part responsible for human practice.<br/>
 
−
                  <strong>WANG Dingyu</strong> was in charge of the construction of CRISPR gRNA as well as RNA scaffold in the beginning. Later she was working on the purification of dCas9 fusion protein.<br/>
 
−
                  <strong>LI Jiamian</strong> took the task of cloning dCas9-luciferase. She was also responsible for the testing of PC reporter system.<br/>
 
−
                  <strong>WEI Jingyi</strong> participated in plasmid construction, protein purification, and wiki building. She was also the designer of our PowerPoint slides.<br/>
 
−
                  <strong>WANG Yuqing</strong> was in charge of Modeling. His main task was to computationally search for the suitable guide sequences. Also, he was the lab manager for the normal operation of our lab.<br/>
 
−
                  <strong>TENG Huaiyuan</strong>was in charge of Modeling. His main task was to computationally search for the suitable guide sequences. Also, he was the lab manager for the normal operation of our lab.<br/>
 
−
                  </p>
 
−
              </div>
 
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−
              <div> 
 
−
                  <a id="acknowledgement"></a>
 
−
                    <h3 class="classic-title";"><span>Acknowledgement</span></h3>                             
 
−
                    <p>We'd like to thank all those who have helped us over the summer, without whose sincere support The Uranium Reaper Project goals would have not been achieved.
 
−
  </p>
 
−
                 
 
−
                  <h4>General support</h4>
 
−
                  <p><strong>Prof. OUYANG Qi, Prof. LOU Chunbo, Dr. ZHANG Haoqian</strong> and other teachers helped us during our brainstorming and gave us useful suggestions.<br>
 
−
                  <strong>Prof. OUYANG Qi</strong> and<strong> Prof. LIU Chunli</strong> kindly provided the laboratory to us for experiments.
 
−
                  </p>
 
−
 
−
                  <h4>Materials support</h4>
 
−
                  <p><strong>Dr. ZHANG Wenbing</strong> provided us with the plasmid of 3B(Tri-SpyCatcher within 15X linker).<br>
 
−
                      <strong> Prof. ZHOU Lu</strong> provided us with Uranyl-binding Protein, also called SUP.<br>
 
−
                      <strong>Prof. LIU Chunli</strong>allowed us to use his lab to do experiments on uranyl ion. The uranyl nitrate solution and Arsenazo III was provided by Prof. LIU.<br>
 
−
                    <strong>Prof. LOU Chunbo</strong> provided us with the strain B.Subtilis and mSA, heavy metal ions binding proteins: lead-binding protein, mercury-binding protein and cadmium-binding protein.<br>
 
−
                    <strong>Dr. DU Pei</strong> provided the beads to us and gave us experimental guidance.<br>
 
−
                    <strong>BIT-China</strong> gave us the important Interlab kit.
 
−
                  </p>
 
−
 
−
                  <h4>Experiment equipment support</h4>
 
−
                  <p><strong>Prof. OUYANG Qi</strong> lent the Akta Pure to us for all proteins purification.<br>
 
−
                      <strong>Prof. CHANG Zengyi</strong> and <strong>Dr. WANG Qingsong</strong> provided us with Scanner and helped with the setup and scan of gels.<br>
 
−
                    <strong>Prof. LIU Chunli</strong> provided us with Geiger counter to test the radiation level in and around our lab.<br>
 
−
                    <strong>Core Facilities</strong>, School of Life Sciences, Peking University,assisted us with ITC.<br>
 
−
                    <strong>the State Key Laboratory of environmental simulation and pollution control</strong> provided the Aurora M90 mass spectrograph.
 
−
                  </p>
 
−
 
−
                  <h4>Theoretical guidance</h4>
 
−
                  <p> <strong>ZHANG Yihao</strong> and <strong>WANG Yu</strong>,The wiki designers of Peking iGEM 2015,gave us many suggestions about wiki building.<br>
 
−
                      <strong>Dr. YU Daqi</strong> of Prof. OUYANG Qi’s lab helped us a lot in experimental operating and data analysis.<br>
 
−
                      <strong>Prof. ZHANG Wenbing, Prof. LOU Chunbo</strong> and <strong>Dr. YU Daqi</strong> kindly helped our team with modeling of the project.<br>
 
−
                    <strong>Prof. OUYANG Qi, Prof. LOU Chunbo, Dr. ZHANG Haoqian</strong> and <strong>ZHANG Yihao</strong>,the instructors, coached us for the presentation.<br>
 
−
                      <strong>Prof. LAI Luhua</strong> and <strong>Prof. RAO Yi</strong> participated in our presentation rehearsal and gave suggestions for revisions.
 
−
                  </p>
 
−
                   
 
−
                  <h4>Synthetic biology course</h4>
 
−
                  <p>From March to June, an introductory course of synthetic biology was hold by College of Life Sciences, Peking University.During the course, the teachers, also the instructors gave us kind guidance and judged our project design. In late June, we started in the lab to start our project.
 
−
                  </p>
 
−
              </div>
 
−
             
 
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              <div> 
 
−
                  <a id="sponsors"></a>                             
 
−
                <h3 class="classic-title";"><span>Sponsors</span></h3>
 
−
              <div class="row,row1">
 
−
              <ul class="footer-social">
 
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                              <li class="col-md-6" id="PKU-administration" style="margin-bottom:25px;max-width:300px">
 
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                                  <a href="http://dean.pku.edu.cn/pkudean/index.html"><img src="https://static.igem.org/mediawiki/2016/2/2e/T--Peking--images_sponsors_PKU_Administration.png"></a>
 
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                              </li>
 
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                              </li>
 
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Latest revision as of 20:02, 19 October 2016

Standard

Notebook

Thin as it is, the notebook serves as a best gift to look back on our struggle for iGEM.

Dairy

Week 1 (6/26/2016-7/02/2016)

Week 2 (7/03/2016-7/09/2016)

  • Parts Construction:
  • Protein Secretion:
    • Sequenced the plasmid pBES and confirmed that there are no additional BsaI sites
    • Transformed the plasmid pSB1C3 and extracted it for future use
    • Constructed the signal peptide ImdA on pSB1C3 vector

Week 3 (7/10/2016-7/16/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the signal peptide OmpA, LTIIb, PhoA, YjfA, NprE
  • Protein Purification:
  • Uranyl Absorption:
    • Pre-experiment:We got familiar with the apparatuses in the lab we performed experiments related to uranyl and tried the Arsenazo III method. Finally, we obtained a standard curve of the concentration ranging from 0uM-30uM

Week 4 (7/17/2016-7/23/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the signal peptide LipA, SacB
    • Constructed the kil protein
    • Tried to construct the OmpA-SUP-pET28a expression plasmid but failed
  • Protein Purification:
  • Clearance:
    • We broke the E.coli with ultrasonication, and extracted 3A-mSA protein using a affinity column. Mixed 3A-mSA and 3B in TBS buffer. Then we did a SDS-PAGE electrophoresis and analysis the results ( Fig.1 ).We could find out that 3A-mSA had the ability to crosslink with 3B.

Week 5 (7/24/2016-7/30/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the expression plasmids OmpA-SUP-pET28a and LTIIb-SUP-pET28a and transformed them into BL21(DE3) strain
  • Protein Purification:
  • Clearance:
    • We tried to measure the reaction capacity of the mSA part of the fusion protein with Biotin-Atto 488 which could show autofluorescence. But results were not useful, because there might be some noncovalent interactions between 3 kDa cutoff centrifuge filters and the molecular.

Week 6 (7/31/2016-8/06/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed additional three signal peptides Bpr, Epr and PelB
    • Tried to construct the expression vector in B.subtilis but all failed
  • Protein Purification:
  • Uranyl Absorption:
    • We improved our experimental methods and supplemented equipment we needed. In these two days, we reduced our reaction volume from 3mL to 400uL and increased our efficiency. Also, standard curve of concentration ranging from 0uM-30uM was tested.
    • The adsorption capacity of 6A-SUP was tested, even though the data were not parallel, we confirmed that 6A-SUP can absorb uranyl. The highest adsorption rate was 81.56%
  • Clearance:
    • Last week we got nothing useful. So we changed the protocol about how to use Biotin-Atto 488. We prepared several little affinity columns, immobilized 3A-mSA containing 6x His-Tag, and perfused the columns with solution of Biotin-Atto 488. After measuring the fluorescence intensity, we got a qualitative result that 3A-mSA can react with biotin.

Week 7 (8/07/2016-8/13/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the expression plasmids of SUP, mSA with different signal peptides: PhoA-SUP-pET28a, PelB-SUP-pET28a, OmpA-mSA-pET28a, LTIIb-mSA-pET28a, PhoA-mSA-pET28a and PelB-mSA-pET28a
    • Constructed the expression plasmids of kil: J23110-J61110-kil-pSB4C5
    • Constructed the expression plasmids of mRFP (E1010), yellow (K592010), eforRed (K592012): OmpA-mRFP-pET28a, LTIIb-mRFP-pET28a, PhoA-mRFP-pET28a, PelB-mRFP-pET28a, OmpA-Yellow-pET28a, LTIIb-Yellow-pET28a, PhoA-Yellow-pET28a, PelB-Yellow-pET28a, OmpA-eforRed-pET28a, LTIIb-eforRed-pET28a, PhoA-eforRed-pET28a and PelB-eforRed-pET28a
  • Protein Purification:
  • Uranyl Absorption:
    • The adsorption capacity of 3A-SUP and 3A-SUP+3B was tested. We confirmed that 3A-SUP can absorb uranyl, so can 3A-SUP+3B. In today’s experiments, the data was more parallel. We used solution containing uranyl only as control and its uranyl concentration after filtration as the actual concentration of uranyl.
  • Clearance:
    • To absorb the proteins in the environment, we wanted to use biotin-coated beads. So we bought the reagents and magnetic beads with amino group.

Week 8 (8/14/2016-8/20/2016)

  • Protein Secretion:
    • Constructed the expression plasmids of SUP and Spycatcher of different signal peptides for B.subtilis: ImdA-SUP-pBES, NprE-SUP-pBES, SacB-SUP-pBES, YjfA-SUP-pBES, LipA-SUP-pBES, ImdA-Spycatcher-pBES, NprE-Spycatcher-pBES, SacB-Spycatcher-pBES, YjfA-Spycatcher-pBES, LipA-Spycatcher-pBES, Bpr-Spycatcher-pBES
  • Protein Purification:
  • Uranyl Absorption:
    • The adsorption capacity of His-SUP was tested, we confirmed that His-SUP can absorb uranyl and the result was close to the data performed by the author of the paper we referred.
    • We tested the adsorption capacity of 3/4/6A-SUP and 3/4/6A-SUP+3B in one day to exclude unnecessary effects. Then we analyzed the data and found the results were compromising. The capacity were fair and even though the proteins formed colloid, the adsorption capacity barely decreased.
  • Clearance:
    • Constructed the biotin-coated beads.

Week 9 (8/21/2015-8/27/2015)

  • Protein Secretion:
    • Constructed the expression plasmids of Spycatcher, mSA and Red with different signal peptides : PhoA-Spycatcher-pET28a, PelB-Spycatcher-pET28a, LTIIb-Spycatcher-pET28a, ImdA-mSA-pBES, NprE-mSA-pBES, SacB-mSA-pBES, YjfA-mSA-pBES, LipA-mSA-pBES, ImdA-Red-pBES, NprE-Red-pBES, SacB-Red-pBES, YjfA-Red-pBES, LipA-Red-pBES and SUP-pBES without any signal peptide as a control plasmid
  • Protein Purification:
  • Uranyl Absorption:
    • Water from Weiming lake was collected and simulated sea water was prepared.
    • We tested the adsorption capacity of 3A-SUP+3B in different conditions, including TBS buffer, Weiming lake and simulated sea water.
    • we changed the protein-uranyl ratio from 1:1 to 10:1 to determine whether the adsorption capacity increased.
    • we decreased the uranyl concentration to 5uM.
    • we decreased the uranyl concentration to 13nM and increased the protein-uranyl ratio to 6000:1.
  • Clearance:
    • Prepared the solution containing 3A protein or 3A-mSA protein, added the beads we made last week, shocked the reaction system adequately for 1h, precipitated the beads with magnetic shelf, and measured the concentration of proteins in the liquid supernatant.

Week 10 (8/28/2015-9/03/2015)

  • Protein Secretion:
    • Constructed the expression plasmids of inducible kil with different signal peptides E. coli and SUP with P43 promotor for B.subtilis: T7-Lac promotor-OmpA-SUP, T7-Lac promotor-PhoA-SUP; P43 promotor-ImdA- SUP -PBES; P43 promotor-NprE- SUP -PBES, P43 promotor-SacB- SUP -PBES, P43 promotor-LipA- SUP -PBES, P43 promotor-YjfA- SUP -PBES
  • Secretion examination:
    • Evaluated the secretion effect for 3ASUP of different signal peptides using western blot.
  • Uranyl Absorption:
    • We received the ICP-MS results.
  • Clearance:
    • Prepared to attend CCiC, also known as Central China iGEM Consortium. We had a great week in this meeting, in Sun Yat-Sen University, Guang Zhou, China.

Week 11 (9/04/2015-9/10/2015)

  • Protein Secretion:
    • Constructed chromoproteins with the signal peptide, PhoA :
    • PhoA-mRFP-pET28a, PhoA-eforRed-pET28a, PhoA-Yellow-pET28a
    • Handed all the vectors with desired elements to the Test Group for examining the secretion efficiency.
  • Secretion examination:
    • Evaluated the secretion effect for 3A mSA and 3B of different signal peptides using western blot.
  • Protein Purification:
    • Purified the cell lysate as well as medium of OmpA SUP using Ni-NTA chromatography.
  • Clearance:
    • Measured the adsorption capacity of protein network with biotin coated beads. (Crosslinked 3A-mSA and 3B for 1 hour, and then added the beads into the reaction system. The remaining proportion of protein in the environment was measured.

Week 12 (9/11/2015-9/17/2015)

  • Protein Secretion:
    • Constructed all the Secretion Parts on pSB1C3 vector and the expression plasmids LBP-pET28a, CBP-pET28a and MBP-pET28a
  • Secretion examination:
    • Quantified the secretion effect of 3B of different signal peptides using anti-Histag ELISA.

Week 13 (9/18/2015-9/24/2015)

  • Parts Construction:
    • 3A-LBP, 3A-MBP and 3A-CBP.
  • Protein Purification:
    • Purified 3A-SUP, 3A-LBP, 3A-CBP and 3A-MBP. Unfortunately, we didn’t get 3A-MBP due to the damage of chromatography column.
  • Secretion examination:
    • Quantified the secretion effect of 3B and 3ASUP of different signal peptides using anti-Histag ELISA.
  • Uranyl Absorption:
    • We repeated experiments on adsorption in different water conditions(boiled and without CO2).
    • We tested 3A-SUP+3B adsorption capacity in TBS buffer with different Ph ranging from 6-9.
  • Clearance:
    • Prepared the Integrating experiment for the next week.

Week 14 (9/25/2015-10/01/2015)

  • Secretion examination:
    • Quantified the secretion effect of 3AmSA and 3ASUP of different signal peptides using anti-Histag ELISA.
    • During this week we also tesedt the influence of IPTG on the secreted concentration. A concentration gradient of IPTG was applied to evaluate the secretion effect of OmpA3B at a time gradient from 1 h to 7 h incubated at 37 ℃.
  • Uranyl Absorption:
    • We repeated experiments on 3A-SUP+3B adsorption capacity in different water conditions(boiled and without CO2).
  • Clearance:
    • We prepared the kit to adsorb uranyl from the environment. We used the biotin coated beads to harvest the protein network which had accommodated uranyl of simulate contaminative sea water and fresh water.

Protocols

Gel Extraction

Ligation

Temperature Gradient Experiment

PCR

Integrating Experiment

Purification of Recombinant Proteins

Transformation

Testing Adsorption Capacity of Protein

Agarose Gel Electrophoresis

Concentration Gradient Experiment

Biotin-associated Experiment

SDS-Page

Western Blot

pH Gradient Experiment

Crosslinking