Difference between revisions of "Team:Peking/Proof"

 
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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 id="logo" style="max-width:170px"><a class="" href="https://2016.igem.org/Team:Peking"></a></div>
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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="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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                            <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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                                <ul class="dropdown-menu">
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                            <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Results</a></li>
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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/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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                                    <li><a href="https://2016.igem.org/Team:Peking/Collaborations" >Collaborations</a></li>
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                        </ul>
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                                </ul>
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                    </li>
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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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                            <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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                                <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/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/Design" >Design</a></li>
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                            <li><a href="https://2016.igem.org/Team:Peking/Crosslinking" >Crosslinking</a></li>
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                                    <li><a href="https://2016.igem.org/Team:Peking/Crosslinking" >Crosslinking</a></li>
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                            <li><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption" >Uranyl adsorption</a></li>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption" >Uranyl adsorption</a></li>
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                            <li><a href="https://2016.igem.org/Team:Peking/Recovery" >Recovery</a></li>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/Clearance" >Clearance</a></li>
−
                            <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li>
−
                            <li><a href="https://2016.igem.org/Team:Peking/Proof" >Proof and speculation</a></li>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Final Performance</a></li>
−
                        </ul>
+
                                </ul>
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                    </li>
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                            </li>
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                    <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a>
+
                            <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a>
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                            <li><a href="https://2016.igem.org/Team:Peking/Model" >Protein polymerization</a></li>
+
                                    <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/Software" >Software</a></li>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/Model/MassDistribution" > Model of Mass Distribution</a></li>
−
                        </ul>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/Software" >Software</a></li>
−
                    </li>
+
                                </ul>
−
                    <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a>
+
                            </li>
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                        <ul class="dropdown-menu">
+
                            <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a>
−
                            <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li>
+
                                <ul class="dropdown-menu">
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                            <li><a href="https://2016.igem.org/Team:Peking/HP/Gold#311" >Field research</a></li>
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                                    <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li>
−
                            <li><a href="https://2016.igem.org/Team:Peking/HP/Gold#questionnaire" >Questionnaire</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/Gold#consulting" >Consulting</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/Gold#questionnaire" >Other work</a></li>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/consulting" >Consulting</a></li>
−
                        </ul>
+
                                    <li><a href="https://2016.igem.org/Team:Peking/HP/otherHP" >Education&nbsp;&amp;&nbsp;Other</a></li>
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                    </li>
+
                                </ul>
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                    <li class="menu-6"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Safety" >Safety</a>
+
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            <h1> Integrated experiment<span>.</span></h1>
 
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                        <h4><a href="javascript:void(0);" onclick="naver('Background')">Background&nbsp；&amp;&nbsp;Design</a></h4>
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                         <h1>Proof of Concept</h1>
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                         <h4><a href="javascript:void(0);" onclick="naver('Methods')">Methods</a></h4>
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                            <div class="texttitle">Background&nbsp;&amp;&nbsp;Design</div>
 
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                            <p>Since we have successfully demonstrated that the bio-functional hydrogel could assemble itself quickly, capture uranyl efficiently and be enriched conveniently, we proposed to combine the features of these experiments in a single trial. We thus incubated triple SpyTag-SUP together with triple SpyTag-mSA and triple SpyCatcher, and contacted the product of crosslinking with simulated pollution. After adsorption, we used a magnet and biotin-coated beads to collect the hydrogel, together with its contents of adsorbed uranyl (Fig. 1).</p>
 
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                                    <figcaption>Fig. 1.  Conceptual graph of the integrated experiment.
 
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                             <div class="texttitle">Methods</div>
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                            <p>We prepared 10μM uranyl nitrate solutions in TBS buffer, as well as in fresh water (sampled from Weiming Lake) and seawater (sampled from Qinhuang Island) to simulate real-life pollution.</p>
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                            <p>After mixing of 5mg/mL solutions of triple SpyTag-SUP, triple SpyTag-mSA and triple SpyCatcher, each, in a volume ratio of 1:1:2, and subsequent incubation for 1h, we were able to obtain the dual-function crosslinked hydrogel encompassing both uranium adsorption and hydrogel recovery functions.</p>
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                            <p>The protein hydrogel was added into the polluted solution and the mixture shaken on a swing bed for 1min, after which the solution was contacted with a suspension of biotin-coated beads (10mg/ml) at a volume ratio of 5:1 (reaction system: beads), and shaken for 1h to ensure good contact between the protein and the beads. Finally, the beads were immobilized using a magnetic shelf, and the uranyl ion concentration in the supernatant was measured using the Arsenazo III assay.
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                            <h3 class="classic-title">Learn more:</h3>
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                                    <a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:integrating_experiments" aria-expanded="false">Integrating Experiment</a>
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                                <p style="text-align:center;"><a href="https://2016.igem.org/Team:Peking/Crosslinking"><img style="width:  %;" src="https://static.igem.org/mediawiki/2016/b/b3/T--Peking--image_H1.png" alt="" /></p></a>
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                                <p style="font-family:raleway-bold, sans-serif !important;font-size:24px !important; text-align:center; margin-top:20px;">Crosslinking</p>
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                                <p style="font-family:raleway, sans-serif !important; font-weight:100;font-size:18px !important; text-align:justify;">By taking advantage of the covalent crosslinking of SpyTag and SpyCatcher, a polymer network was designed to possess special properties, such as strong mechanical strength, large contact area and stable structure.</p>
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            <a id="Results"></a>
 
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                            <div class="texttitle">Results</div>
 
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                            <p>The statistical analysis of experimental results is shown in Fig.18. Under the circumstances mentioned above, the Uranium Reaper achieved a relatively good adsorption efficiency (51.4% in TBS buffer, 55.8% in seawater and 60.1% in fresh water). </p>
 
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                                <figcaption>
 
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                                    Fig. 2.  The remaining proportion of 10μM uranyl in TBS buffer, fresh and seawater after treatment with our hydrogel. ****p &lt; 0.0001. n=3. Error bars indicate standard deviations.
 
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                                <p style="text-align:center;"><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption"><img style="width:  %;" src="https://static.igem.org/mediawiki/2016/b/b1/T--Peking--image_pr2.png" /></p></a>
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                                <p style="font-family:raleway-bold, sans-serif !important;font-size:24px !important; text-align:center; margin-top:20px;">Uranyl Adsorption</p>
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                                <p style="font-family:raleway, sans-serif !important; font-weight:100;font-size:18px !important; text-align:justify;">Super Uranyl-binding Protein (SUP) is stable and highly selective for uranyl which may cause negative impact on the environment. Triple SpyTag-SUP (3A-SUP) was constructed to adsorb uranyl, and the cross-linked product of 3A-SUP and 3B showed an even better adsorption capacity.</p>
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                                <p style="text-align:center;"><a href="https://2016.igem.org/Team:Peking/Clearance"><img style="width:  %;" src="https://static.igem.org/mediawiki/2016/9/95/T--Peking--image_H3.png" alt=""/></p></a>
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                                <p style="font-family:raleway-bold, sans-serif !important;font-size:24px !important; text-align:center; margin-top:20px;">Clearance</p>
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                                <p style="font-family:raleway, sans-serif !important; font-weight:100;font-size:18px !important; text-align:justify;">The monomeric streptavidin constructed in the polymer network could realize the clearance of Spy Crosslinking Network in the environment by interacting with biotinylated magnetic beads.</p>
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                                <p style="text-align:center;"><a href="https://2016.igem.org/Team:Peking/Secretion"><img style="width:  %;" src="https://static.igem.org/mediawiki/2016/1/1f/T--Peking--image_H2.png" alt=""/></p></a>
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                                <p style="font-family:raleway-bold, sans-serif !important;font-size:24px !important; text-align:center; margin-top:20px;">Secretion</p>
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                                <p style="font-family:raleway, sans-serif !important; font-weight:100;font-size:18px !important; text-align:justify;">To make the polymer network reproductive and economical, a signal peptide library was constructed. We carried out a series of experiments to find an optimal secretion strategy.</p>
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            <a id="Conclusion"></a>
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                        <div class="texttitle">Discussion</div>
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                        <p>In this integrated experiment, the Uranium Reaper showed an adsorption rate of 60%, which corresponds well to the product of the uranyl adsorption rate and hydrogel recovery rate (90% × 70% = 63%). This result has reached the expectations but is still far removed from the efficiencies that would be needed for an industrial process. Consequently, both the uranyl adsorption and hydrogel recovery rate have to be increased to improve the overall efficiency of the Uranium Reaper, which, of course, requires further research and development.</p>
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                    padding-right: 12px;
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                    color: #b4bbbb;
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            footer .copyright span:after {
 
                 content: "|";
 
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                 <ul class="copyright">
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                    <li><a href="Home.html">Home</a>&nbsp;&nbsp;&nbsp;<a href="#">Contact</a></li>
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        }
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                    <span> &copy;2016 PEKING IGEM. All Rights Reserved.</span>
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        </style>
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                    <li style="padding-right:20px"><a href="https://www.facebook.com/pekingigem"><i class="fa fa-facebook"><img style="height:22px; margin-top:4px; margin-bottom:-4px;" src="https://static.igem.org/mediawiki/2016/c/c4/T--Peking--image_homefacebook.png"/></i></a></li>
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        <footer id="page-footer">
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                     <li style="padding-right:20px"><a href="https://twitter.com/iGEM_Peking"><i class="fa fa-twitter"><img style="height:22px; margin-top:4px; margin-bottom:-4px;" src="https://static.igem.org/mediawiki/2016/3/39/T--Peking--image_hometwitter.png"/></i></a></li>
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                    <li style="padding-right:20px"><a href="#"><i class="fa fa-skype"><img style="height:22px; margin-top:4px; margin-bottom:-4px;" src="https://static.igem.org/mediawiki/2016/c/cc/T--Peking--image_homeskype.png"/></i></a></li>
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                        <!--<li>&copy; 2014 Sparrow</li> -->
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                        <li><a href="2016.igem.org/Team:Peking">Home</a>&nbsp;&nbsp;&nbsp;<a href="mailto:pkuigem2016@126.com">Contact</a></li>
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                        <span> &copy;2016 PEKING IGEM. All Rights Reserved.</span>
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                        <li><a href="http://getbootstrap.com/2.3.2/">Based on Bootstrap</a></li>
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                <div id="go-top" style="display: block;"><a title="Back to Top" href="#">Go To Top</a></div>
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            <!-----go-to-top------>
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            <div id="go-top" style="display: block;z-index:1000;"><a title="Back to Top" href="#">Go To Top</a></div>
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Latest revision as of 00:40, 20 October 2016

Proof

Proof of Concept

Crosslinking

By taking advantage of the covalent crosslinking of SpyTag and SpyCatcher, a polymer network was designed to possess special properties, such as strong mechanical strength, large contact area and stable structure.

Uranyl Adsorption

Super Uranyl-binding Protein (SUP) is stable and highly selective for uranyl which may cause negative impact on the environment. Triple SpyTag-SUP (3A-SUP) was constructed to adsorb uranyl, and the cross-linked product of 3A-SUP and 3B showed an even better adsorption capacity.

Clearance

The monomeric streptavidin constructed in the polymer network could realize the clearance of Spy Crosslinking Network in the environment by interacting with biotinylated magnetic beads.

Secretion

To make the polymer network reproductive and economical, a signal peptide library was constructed. We carried out a series of experiments to find an optimal secretion strategy.


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