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| <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a> | | <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a> |
| <ul class="dropdown-menu"> | | <ul class="dropdown-menu"> |
− | <li><a href="https://2016.igem.org/Team:Peking/Description" >Decription</a></li> | + | <li><a href="https://2016.igem.org/Team:Peking/Description" >Description</a></li> |
| <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li> | | <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li> |
| <li><a href="https://2016.igem.org/Team:Peking/Proof" >Proof of Concept</a></li> | | <li><a href="https://2016.igem.org/Team:Peking/Proof" >Proof of Concept</a></li> |
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| </div> | | </div> |
| <div class="col-md-8"> | | <div class="col-md-8"> |
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| <h3 style="text-align:center"> Dairy</h3> | | <h3 style="text-align:center"> Dairy</h3> |
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| <div class="panel-heading"> | | <div class="panel-heading"> |
| <h4 class="panel-title"> | | <h4 class="panel-title"> |
− | <a data-toggle="collapse" href="#collapse8">Week 8 (8/14/2016-8/24/2016)</a> | + | <a data-toggle="collapse" href="#collapse8">Week 8 (8/14/2016-8/20/2016)</a> |
| </h4> | | </h4> |
| </div> | | </div> |
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| <li><a>3A-msa in the form of inclusion body</a>: 2.273 mg/ml</li> | | <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> | | <li><a>3A</a>: 29.768 mg/ml</li> |
− | <li><a>3A-sup</a>: 20.349 mg/ml</li>
| |
| </ul> | | </ul> |
| <li><b>Uranyl Absorption:</b></li> | | <li><b>Uranyl Absorption:</b></li> |
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| <div class="panel-body"> | | <div class="panel-body"> |
| <ul> | | <ul> |
− | <li><b>Parts Construction:</b></li> | + | <li><b>Protein Secretion:</b></li> |
| <ul> | | <ul> |
− | <li><a>3SpyTag (A) </a>assemble</li> | + | <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> |
− | <li><a>SUP</a> PCR</li>
| + | </ul> |
− | <li><a>pET28a backbone </a>PCR</li>
| + | <li><b>Protein Purification:</b></li> |
− | <li><a>SpyCatcher (B) </a>PCR</li>
| + | <ul> |
− | <li><a>Monomeric Streptavidin (mSA)</a> PCR</li> | + | <li><a>3A-sup</a>: 20.349 mg/ml</li> |
| </ul> | | </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> |
| + | |
| + | |
| + | <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> | | <li><b>Protein Secretion:</b></li> |
| <ul> | | <ul> |
− | <li><a href="http://parts.igem.org/Part:BBa_J33201" target="_blank">BBa_J33201</a> (arsR)</li> | + | <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> |
− | <li>Plasmids were isolated using a miniprep kit.</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>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> |
| + | </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> | | </ul> |
| <li><b>Protein Purification:</b></li> | | <li><b>Protein Purification:</b></li> |
| <ul> | | <ul> |
− | <li><a href="http://parts.igem.org/Part:BBa_J33201" target="_blank">BBa_J33201</a> (arsR)</li> | + | <li>Purified the cell lysate as well as medium of OmpA SUP using Ni-NTA chromatography.</li> |
− | <li>Plasmids were isolated using a miniprep kit.</li> | + | </ul> |
| + | <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> | | </ul> |
| <li><b>Uranyl Absorption:</b></li> | | <li><b>Uranyl Absorption:</b></li> |
| <ul> | | <ul> |
− | <li>Water from Weiming lake was collected and simulated sea water was prepared.</li> | + | <li> We repeated experiments on adsorption in different water conditions(boiled and without CO2).</li> |
− | <li>We tested the adsorption capacity of 3A-SUP+3B in different conditions, including TBS buffer, Weiming lake and simulated sea water. Besides, we changed the protein-uranyl ratio from 1:1 to 10:1 to determine whether the adsorption capacity increased. Next, we decreased the uranyl concentration to 5uM. What’s more, we decreased the uranyl concentration to 13nM and increased the protein-uranyl ratio to 6000:1</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> | | </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> | | <li><b>Retrivability:</b></li> |
| <ul> | | <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. We can get the quantitative data as Fig.2 shown.</li> | + | <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> | | </ul> |
| + | <li><b>Protein Secretion:</b></li> |
| + | <ul> |
| + | |
| + | </ul> |
| + | <li><b>Protein Purification:</b></li> |
| + | <ul> |
| + | |
| + | </ul> |
| + | <li><b>Secretion examination: </b></li> |
| + | <ul> |
| + | |
| + | </ul> |
| + | <li><b>Uranyl Absorption:</b></li> |
| + | <ul> |
| + | |
| + | </ul> |
| + | <li><b>Retrivability:</b></li> |
| + | <ul> |
| + | <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> |
| + | </ul> |
| </ul> | | </ul> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
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