Difference between revisions of "Team:Peking/Proof"

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                      <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/Retrievability" >Collection</a></li>
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                      <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li>
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                      <li><a href="https://2016.igem.org/Team:Peking/Proof" >Proof and speculation</a></li>
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                      <li><a href="https://2016.igem.org/Team:Peking/Model" >Protein polymerization</a></li>
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            <h1>Proof and Speculation<span>.</span></h1>
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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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                    <li class="menu-1"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking" >Home</a></li>
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                    <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#" > Achievements</a>
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                        <ul class="dropdown-menu">
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                            <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Results</a></li>
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                            <li><a href="https://2016.igem.org/Team:Peking/Basic_Part" >Parts</a></li>
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                            <li><a href="https://2016.igem.org/Team:Peking/Collaborations" >Collaborations</a></li>
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                        </ul>
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                    </li>
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                    <li class="dropdown menu-3"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a>
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                        <ul class="dropdown-menu">
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Description" >Overview</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Crosslinking" >Crosslinking</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption" >Uranyl adsorption</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Recovery" >Recovery</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>
 +
                        </ul>
 +
                    </li>
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                    <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a>
 +
                        <ul class="dropdown-menu">
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Model" >Protein polymerization</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/Software" >Software</a></li>
 +
                        </ul>
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                    </li>
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                    <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a>
 +
                        <ul class="dropdown-menu">
 +
                            <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/HP/Gold#311" >Field research</a></li>
 +
                            <li><a href="https://2016.igem.org/Team:Peking/HP/Gold#questionnaire" >Questionnaire</a></li>
 +
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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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                        <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>
 +
                            <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>
 +
                            <figure>
 +
                                <img class="featurette-image" src="" style="width:100%;" alt=""/>
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                                    <figcaption>Fig. 1.  Conceptual graph of the integrated experiment.
 +
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            <a id="Methods"></a>
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                            <div class="texttitle">Methods</div>
 +
                            <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>
 +
                            <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>
 +
                            <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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                            <div class="texttitle">Results</div>
 +
                            <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>
 +
                            <figure>
 +
                                <img class="featurette-image" src="" style="width:100%;" alt=""/>
 +
                                <figcaption>
 +
                                    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>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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Revision as of 15:57, 14 October 2016

Proof

Integrated experiment.

Background & Design

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).

Fig. 1. Conceptual graph of the integrated experiment.


Methods

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.

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.

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.

Learn more:



Results

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).

Fig. 2. The remaining proportion of 10μM uranyl in TBS buffer, fresh and seawater after treatment with our hydrogel. ****p < 0.0001. n=3. Error bars indicate standard deviations.


Discussion

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.