Difference between revisions of "Team:Peking/Description"

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            <title>Overview</title>
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  <title>Standard</title>
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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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    <meta name="author" content="Li Jiamian & Wang Yuqing">
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</div>
 
</div>
 
<!--/Navigation -->
 
<!--/Navigation -->
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  <!-- Page Title======================================================================== -->
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    <div id="page-title">
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        <div class="row">
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          <div class="twelve columns centered text-center" style="padding-top: 50px; padding-bottom: 35px;">
 +
              <h1>Notebook</h1>
 +
              <p class="title1" style="text-align:center">In this page, you can view how we worked on our project.</p>
 +
          </div>
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        </div>
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    </div>
 +
    <!-- Page Title End-->
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 +
<!--*************************************************************************************************************-->
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<div id="page-content" class="row page">
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      <div id="primary" class="twelve columns">
 +
 
 +
        <section>
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  <div class="row">
 +
  <div class="col-mid-2">
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  </div>
 +
  <div class="col-md-8">
 +
     
 +
<h3 style="text-align:center"> Dairy</h3>
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 +
<div class="panel-group notebook" id="accordion">
 +
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title" style="color:white;">
 +
                    <a data-toggle="collapse" href="#collapseOne">Week 1 (6/26/2016-7/02/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapseOne" class="panel-collapse collapse in">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <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>
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse2">Week 2 (7/03/2016-7/09/2016)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse2" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <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>
 +
 +
 +
 +
        <div class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse3">Week 3 (7/10/2016-7/16/2016)</a>
 +
                </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>Retrivability:</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>Retrivability:</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>
 
          
 
          
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 +
                <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>Retrivability:</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>
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            </div>
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                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse7">Week 7 (8/07/2016-8/13/2016)</a>
 +
                </h4>
 +
            </div>
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 +
                <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>Retrivability:</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>
 
          
 
          
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                 <h4 class="panel-title">
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                     <a data-toggle="collapse" href="#collapse8">Week 8 (8/14/2016-8/20/2016)</a>
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                     <h1>Description</h1>
+
                </h4>
−
                    <p class="title1" style="text-align:center">In this section, we describe the background and our Uranium Reaper project in brief. We advise you to have a quick overall  understanding of our project before viewing result pages. </p>
+
            </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>Retrivability:</b></li>
 +
                            <ul>
 +
                                <li>Constructed the biotin-coated beads.</li>
 +
                        </ul>
 +
                    </ul>
 
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                <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>Retrivability:</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>
 
          
 
          
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" href="#collapse10">Week 10 (8/28/2015-9/03/2015)</a>
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                </h4>
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                             <div class="twelve columns">
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                                <div class="texttitle">Overview</div>
+
            <div id="collapse10" class="panel-collapse collapse">
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                                <p class="lead add-bottom" style="color:#5E5656">Uranium (U), which is a radionuclide and heavy metal elemant, has been released into the environment in increasing amounts, mainly due to activities related to the booming nuclear industry<sup>1</sup>. This has resulted in persistent anthropogenic uranium contamination<sup>2</sup>, which contributes to ecotoxicological problems<sup>3</sup>, environmental degradation<sup>4</sup> and could cause severe negative health effects. Inhalation, ingestion<sup>5</sup> and skin contact are the main routes of uranium exposure<sup>6</sup>, and this, combined with a tendency to accumulate in the body <sup>7</sup>, as well as uranium’s radioactivity and heavy-metal toxicity<sup>8</sup>, often leads to significant adverse health effects on the human body, including DNA damage<sup>9</sup>, reproductive toxicity<sup>10-12</sup> and nephrotoxicity, inter alia<sup>13</sup>. Therefore, wastewater containing uranium must be thoroughly treated in order to prevent the release of uranium contamination into the environment<sup>14</sup>.</p>
+
                <div class="panel-body">
−
                               
+
                    <ul>
−
                                <p class="lead add-bottom" style="color:#5E5656">Several physical, biological and chemical methods have been developed for the removal of uranium from aqueous waste. These include physico-chemical methods such as ion-exchange, reverse osmosis, precipitation, flocculation etc. <sup>18</sup>, or phytoremediation<sup>1,15</sup>, rhizofiltration and other types of bioremediation<sup>16,17</sup>. However, these methods are often expensive, time-consuming and tedious, or of insufficient efficiency for the treatment of large volumes of wastewater with low concentrations of the target contaminants<sup>19</sup>.After interviewing the Hunan Nuclear Geology 311 Brigade, a geological exploration unit with Grade A qualification in Hunan Province of China, it became apparent that the favored method of dealing with excavation sites consists of simply filling the ground with fresh soil and growing appropriate plants on it<sup>20,21</sup>. This minimalist approach is likely also favored due to the high-cost of the alternative methods mentioned above. Hence, efforts are needed to develop suitable alternative technologies to complement or replace the existing methods<sup>19</sup>.</p>
+
                        <li><b>Protein Secretion:</b></li>
−
                               
+
                             <ul>
−
                                <p class="lead add-bottom" style="color:#5E5656">To obviate such shortcomings, the Peking iGEM 2016 team developed a novel remediation method, Uranium Reaper, which could remove uranyl ions (the predominant form of aqueous uranium) <sup>15,22,23</sup>, with high efficiency at an affordable cost, thus offering great convenience. Uranium Reaper utilizes a smart covalent crosslinking hydrogel which is able to self-assemble in aqueous solution. The addition of biotin-coated magnetic particles to the solution enables the clearance of the complex self-assembled uranium-containing hydrogel by a simple magnet. In this way, uranium pollution is alleviated and the uranyl ions could be cleared and enriched for further use.</p>
+
                                <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>
−
                                <p class="lead add-bottom" style="color:#5E5656">Materials modeled on Uranyl Reaper are not limited to uranium remediation, and could obtain endless functions and applications by attaching different modules of interest to the autonomously covalently cross-linking protein hydrogel. For example, by replacing SUP with Cadmium-Binding Protein (CBP) or Lead-Binding Protein (LBP) <sup>24,25</sup>, this bio-functional hydrogel is capable of adsorbing a variety of heavy metals<sup>26</sup> as confirmed by our experiments. By complementing the leaching and elution circuit in mining, this hydrogel would simplify the mining procedures and reduce the amount of contaminated wastewater produced<sup>27,28</sup>. What’s more, by optimizing the number of crosslinking modules, it may be possible to use similar biomaterials for 3D printing. We also aimed to develop a Uranium Reaper Kit, in order to facillitate the use of the material worldwide.</p>
+
                        <li><b>Secretion examination: </b></li>
−
                               
+
                            <ul>
−
                               
+
                            <li>Evaluated the secretion effect for 3ASUP of different signal peptides using western blot. </li>
−
                                <hr>
+
                        </ul>
−
                                  
+
                        <li><b>Uranyl Absorption:</b></li>
−
                                 <div class="texttitle">General views</div>
+
                            <ul>
−
                                  
+
                                <li>We received the ICP-MS results. </li>
−
                                 <p>the overall illustrations</p>
+
                    </ul>
−
                                <hr/>
+
                        <li><b>Retrivability:</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>
−
                                <div class="texttitle">Important sections</div>
+
                    </ul>
−
                                <div class="five columns">
+
                    </ul>
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                                    <img class="home_img" src="https://static.igem.org/mediawiki/2016/e/ed/T--Peking--images_description_waster_water.png" alt=""/>
+
                </div>
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                                </div>
+
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                                <div class="seven columns">
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                                    <div class="home_text">
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                                        <h3>Sampling of waste water</h3>
+
        <div class="panel panel-default">
−
                                        <p> Uranium, a heavy metal element, is weakly radioactive and poses a threat to both the environment and human health. A person can be exposed to uranium by inhaling dust in the air or by ingesting contaminated water and food. Long-term exposure to uranium increases the risk of various diseases and health issues including cancer, kidney problems and immune system damage. </p>
+
            <div class="panel-heading">
−
                                    </div>
+
                <h4 class="panel-title">
−
                                </div>
+
                    <a data-toggle="collapse" href="#collapse11">Week 11 (9/04/2015-9/10/2015)</a>
−
                               
+
                </h4>
−
                                 <div class="seven columns">
+
            </div>
−
                                    <div class="home_text">
+
            <div id="collapse11" class="panel-collapse collapse">
−
                                        <h3>Design</h3>
+
                <div class="panel-body">
−
                                        <p>To alleviate these problems, the Peking iGEM team aims to construct a novel functional biological material, which can absorb uranyl ion with the employment of a specific uranium-binding protein. With some modification, the design can be applied to deal with uranyl ion in polluted water and soil, demonstrating its impressive potential. We believe that the material can effectively solve the increasingly serious uranium pollution in the near future.</p>
+
                    <ul>
−
                                    </div>
+
                        <li><b>Protein Secretion:</b></li>
−
                                </div>
+
                            <ul>
−
                                <div class="five columns">
+
                                <li>Constructed chromoproteins with the signal peptide, PhoA : </li>
−
                                    <img class="home_img" src="https://static.igem.org/mediawiki/2016/c/cc/T--Peking--images_description_design.png" alt=""/>
+
                                <li>PhoA-mRFP-pET28a, PhoA-eforRed-pET28a, PhoA-Yellow-pET28a</li>
−
                                </div>
+
                                <li>Handed all the vectors with desired elements to the Test Group for examining the secretion efficiency. </li>
−
                               
+
                    </ul>
−
                                <div class="five columns">
+
                        <li><b>Secretion examination: </b></li>
−
                                    <img class="home_img" src="https://static.igem.org/mediawiki/2016/e/e6/T--Peking--images_description_bacterial.png" alt=""/>
+
                            <ul>
−
                                </div>
+
                            <li>Evaluated the secretion effect for 3A mSA and 3B of different signal peptides using western blot.</li>
−
                                <div class="seven columns">
+
                        </ul>
−
                                    <div class="home_text">
+
                        <li><b>Protein Purification:</b></li>
−
                                        <h3>Protein Secretion</h3>
+
                            <ul>
−
                                        <p> Uranium, a heavy metal element, is weakly radioactive and poses a threat to both the environment and human health. A person can be exposed to uranium by inhaling dust in the air or by ingesting contaminated water and food. Long-term exposure to uranium increases the risk of various diseases and health issues including cancer, kidney problems and immune system damage. </p>
+
                            <li>Purified the cell lysate as well as medium of OmpA SUP using Ni-NTA chromatography.</li>
−
                                    </div>
+
                        </ul>
−
                                </div>
+
                        <li><b>Retrivability:</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>Retrivability:</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>Retrivability:</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 class="panel panel-default">
 +
            <div class="panel-heading">
 +
                <h4 class="panel-title">
 +
                    <a data-toggle="collapse" href="#collapse15">Week 15 (10/02/2015-10/08/2015)</a>
 +
                </h4>
 +
            </div>
 +
            <div id="collapse15" class="panel-collapse collapse">
 +
                <div class="panel-body">
 +
                    <ul>
 +
                        <li><b>Parts Construction:</b></li>
 +
                            <ul>
 +
 
 +
                        </ul>
 +
                        <li><b>Protein Secretion:</b></li>
 +
                            <ul>
 +
 
 +
                        </ul>
 +
                        <li><b>Protein Purification:</b></li>
 +
                            <ul>
 +
 
 +
                        </ul>
 +
                        <li><b>Secretion examination: </b></li>
 +
                            <ul>
 
                                  
 
                                  
−
                                <div class="seven columns">
+
                        </ul>
−
                                    <div class="home_text">
+
                        <li><b>Uranyl Absorption:</b></li>
−
                                        <h3>Network Formation</h3>
+
                            <ul>
−
                                        <p>To alleviate these problems, the Peking iGEM team aims to construct a novel functional biological material, which can absorb uranyl ion with the employment of a specific uranium-binding protein. With some modification, the design can be applied to deal with uranyl ion in polluted water and soil, demonstrating its impressive potential. We believe that the material can effectively solve the increasingly serious uranium pollution in the near future.</p>
+
 
−
                                    </div>
+
                        </ul>
−
                                </div>
+
                        <li><b>Retrivability:</b></li>
−
                                <div class="five columns">
+
                            <ul>
−
                                    <img class="home_img" src="https://static.igem.org/mediawiki/2016/d/de/T--Peking--images_description_Network_formation.png" alt=""/>
+
                                <li>We changed the module of SUP to other kinds of heavy metal binding proteins, such as LBP ( Lead binding protein) or CBP ( cadmium binding protein). As the same protocol of Uranium Reaper Kit</li>
−
                                </div>
+
                    </ul>
−
                               
+
                    </ul>
−
                               
+
                </div>
−
                               
+
            </div>
−
                               
+
        </div>
−
                                <div class="five columns">
+
       
−
                                    <img class="home_img" src="https://static.igem.org/mediawiki/2016/c/c2/T--Peking--images_description_harvest.png" alt=""/>
+
       
−
                                </div>
+
    </div>
−
                                <div class="seven columns">
+
    </div><!--col-md-8 ended-->
−
                                    <div class="home_text">
+
 
−
                                        <h3>Harvest</h3>
+
 
−
                                        <p> Uranium, a heavy metal element, is weakly radioactive and poses a threat to both the environment and human health. A person can be exposed to uranium by inhaling dust in the air or by ingesting contaminated water and food. Long-term exposure to uranium increases the risk of various diseases and health issues including cancer, kidney problems and immune system damage. </p>
+
 
−
                                    </div>
+
<h3 style="text-align:center"> Protocols</h3>
−
                                </div>
+
<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>
−
                                <div class="seven columns">
+
<p style="text-align:center"><b>Ligation</b></p>
−
                                    <div class="home_text">
+
<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>
−
                                        <h3>Modularization</h3>
+
<p style="text-align:center"><b>Temperature Gradient Experiment</b></p>
−
                                        <p>To alleviate these problems, the Peking iGEM team aims to construct a novel functional biological material, which can absorb uranyl ion with the employment of a specific uranium-binding protein. With some modification, the design can be applied to deal with uranyl ion in polluted water and soil, demonstrating its impressive potential. We believe that the material can effectively solve the increasingly serious uranium pollution in the near future.</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>
−
                                    </div>
+
<p style="text-align:center"><b>PCR</b></p>
−
                                </div>
+
<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>
−
                                <div class="five columns">
+
<p style="text-align:center"><b>Integrating Experiment</b></p>
−
                                    <img class="home_img" src="https://static.igem.org/mediawiki/2016/e/e6/T--Peking--images_modularization.png" alt=""/>
+
</div>
−
                                </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>
−
                                <div class="references">
+
<p style="text-align:center"><b>Transformation</b></p>
−
                                    <h3>References:</h3>
+
<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>
+
<p style="text-align:center"><b>Testing Adsorption Capacity of Protein</b></p>
−
                                    [1] Piyush Malaviya, Asha Singh. Phytoremediation Strategies for Remediation of Uranium-Contaminated Environments: A Review. Critical Reviews in Environmental Science &amp; Technology, volume 42(24), 2575-2647 (2012)
+
<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><p>
+
<p style="text-align:center"><b>Agarose Gel Electrophoresis</b></p>
−
                                    [2] Merkel B. Long term fate of uranium tailings in mountain areas. Uranium in the Environment. Springer Berlin Heidelberg, 47-56 (2006)
+
<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><p>
+
<p style="text-align:center"><b>Concentration Gradient Experiment</b></p>
−
                                    [3] Antunes S C, Figueiredo D R D, Marques S M, et al. Evaluation of water column and sediment toxicity from an abandoned uranium mine using a battery of bioassays. Science of the Total Environment, 374(2-3):252-259 (2007)
+
 
−
                                    </p><p>
+
</div>
−
                                    [4] Boulois H D D, Joner E J, Leyval C, et al. Impact of arbuscular mycorrhizal fungi on uranium accumulation by plants. Journal of Environmental Radioactivity, 99(5):775-84 (2008)
+
<div class="four columns">
−
                                    </p><p>
+
<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>
−
                                    [5] Neves O, Abreu M M, Vicente E M. Uptake of Uranium by Lettuce ( Lactuca sativa L.) in Natural Uranium Contaminated Soils in Order to Assess Chemical Risk for Consumers. Water Air &amp; Soil Pollution, 195(1):73-84 (2008)
+
<p style="text-align:center"><b>Biotin-associated Experiment</b></p>
−
                                    </p><p>
+
 
−
                                    [6] Anke M, Seeber O, Müller R, et al. Uranium transfer in the food chain from soil to plants, animals and man. Chemie der Erde - Geochemistry, 69(1):75-90 (2009)
+
<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>
−
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+
<p style="text-align:center"><b>SDS-Page</b></p>
−
                                    [7] Schnug E, Steckel H, Haneklaus S. Contribution of uranium in drinking waters to the daily uranium intake of humans - A case study from Northern Germany. Landbauforschung Volkenrode, 55(4) (2005)
+
<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><p>
+
<p style="text-align:center"><b>Western Blot</b></p>
−
                                    [8] Kratz S, Schnug E. Rock phosphates and P fertilizers as sources of U contamination in agricultural soils. Uranium in the Environment. 57-67 (2006)
+
<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><p>
+
<p style="text-align:center"><b>pH Gradient Experiment</b></p>
−
                                    [9] Chang P, Kim K W, Yoshida S, et al. Uranium accumulation of crop plants enhanced by citric acid. Environmental Geochemistry &amp; Health, 27(5-6):529-38 (2005)
+
<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><p>
+
<p style="text-align:center"><b>Crosslinking</b></p>
−
                                    [10] Bednar A J, Medina V F, Ulmer-Scholle D S, et al. Effects of organic matter on the distribution of uranium in soil and plant matrices. Chemosphere, 70(2):237-47 (2007)
+
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−
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+
 
−
                                    [11] Arfsten D P, Still K R, Ritchie G D. A review of the effects of uranium and depleted uranium exposure on reproduction and fetal development. Toxicology &amp; Industrial Health,2001, 17(5-10):180-91 (2001)
+
 
−
                                    </p><p>
+
−
                                    [12] Sztajnkrycer M D, Otten E J. Chemical and radiological toxicity of depleted uranium. Military Medicine, 169(3):212-6 (2004)
+
 
−
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+
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−
                                    [13] Craft E S, Abuqare A W, Flaherty M M, et al. DEPLETED AND NATURAL URANIUM: CHEMISTRY AND TOXICOLOGICAL EFFECTS. Journal of Toxicology &amp; Environmental Health Part B, 7(4):297-317 (2004)
+
               
−
                                    </p><p>
+
               
−
                                    [14] Jing B, Yao H, Fan F, et al. Biosorption of uranium by chemically modified Rhodotorula glutinis. Journal of Environmental Radioactivity, 101(11):969-973 (2010)
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                                    [15] Stojanovic´, M. D, Stevanovic´, D. R, Milojkovic´, J. V, et al. Phytotoxic Effect of the Uranium on the Growing Up and Development the Plant of Corn. Water Air &amp; Soil Pollution, 209(1):401-410 (2010)
+
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+
   
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                                    [16] Xie S, Yang J, Chen C, et al. Study on biosorption kinetics and thermodynamics of uranium by Citrobacter freudii. Journal of Environmental Radioactivity, 99(1):126-33 (2008)
+
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+
 
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                                    [17] Newsome L, Morris K, Lloyd J R. The biogeochemistry and bioremediation of uranium and other priority radionuclides. Chemical Geology, 363(1):164-184 (2014)
+
 
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+
 
−
                                    [18] Bhalara P D, Punetha D, Balasubramanian K. A review of potential remediation techniques for uranium(VI) ion retrieval from contaminated aqueous environment. Journal of Environmental Chemical Engineering, 2(3):1621-1634 (2014)
+
     
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+
</body>
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                                    [19] Bhainsa K C, D'Souza S F. Uranium(VI) biosorption by dried roots of Eichhornia crassipes (water hyacinth). Journal of Environmental Science &amp; Health Part A Toxic/hazardous Substances &amp; Environmental Engineering, 36(9):1621-31 (2001)
+
 
−
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+
 
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                                    [20] Neves M O, Figueiredo V R, Abreu M M. Transfer of U, Al and Mn in the water-soil-plant (Solanum tuberosum L.) system near a former uranium mining area (Cunha Baixa, Portugal) and implications to human health. Science of the Total Environment, 416(2):156–163 (2012)
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                                    [21] Stojanović M, Pezo L, Lačnjevac Č, et al. Biometric approach in selecting plants for phytoaccumulation of uranium. International Journal of Phytoremediation, 18(5):255-262 (2015)
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                                    [22] Kalin M, Wheeler W N, Meinrath G. The removal of uranium from mining waste water using algal/microbial biomass. Journal of Environmental Radioactivity, 78(2):151-177 (2005)
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                                    [23] Handley-Sidhu S, Keith-Roach M J, Lloyd J R, et al. A review of the environmental corrosion, fate and bioavailability of munitions grade depleted uranium. Science of the Total Environment, 408(23):5690-700 (2010)
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                                    [24] Song L, Caguiat J, Li Z, et al. Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site. Journal of Bacteriology, 186(6):1861-8 (2004)
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                                    [25] Brown N L, Stoyanov J V, Kidd S P, et al. The MerR family of transcriptional regulators. Fems Microbiology Reviews, 27(2-3):145–163 (2003)
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                                    [26] Mulligan C N, Yong R N, Gibbs B F. Remediation technologies for metal-contaminated soils and groundwater: an evaluation. Engineering Geology, 60(1-4):193-207 (2001)
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                                    [27] Campbell K M, Gallegos T J, Landa E R. Biogeochemical aspects of uranium mineralization, mining, milling, and remediation. Applied Geochemistry, 57:206-235 (2015)
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                                    [28] Quan, C., Collier, D., Bowell, R.J., van Noort, E.. Optimization of the calcrete-uranium process flowsheet: the implications of metallurgical testwork for the Marenica project. In: Taylor, A. (Ed.), Alta 2010 Uranium Conference, Perth, Australia (2010).
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Revision as of 16:34, 16 October 2016

Standard

Notebook

In this page, you can view how we worked on our project.

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:
  • Retrivability:
    • 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:
  • Retrivability:
    • 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%
  • Retrivability:
    • 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.
  • Retrivability:
    • 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.
  • Retrivability:
    • 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.
  • Retrivability:
    • 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.
  • Retrivability:
    • 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.
  • Retrivability:
    • 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.
  • Retrivability:
    • 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).
  • Retrivability:
    • 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.

Week 15 (10/02/2015-10/08/2015)

  • Parts Construction:
  • Protein Secretion:
  • Protein Purification:
  • Secretion examination:
  • Uranyl Absorption:
  • Retrivability:
    • We changed the module of SUP to other kinds of heavy metal binding proteins, such as LBP ( Lead binding protein) or CBP ( cadmium binding protein). As the same protocol of Uranium Reaper Kit

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