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| − | <div class="texttitle">Methods</div>
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| − | <a id="Results"></a>
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| − | <div class="texttitle">Results</div>
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| − | <a id="Monomer"></a>
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| − | <a role="button" data-toggle="collapse" href="#collapse1" aria-expanded="false" aria-controls="collapse1">Evaluation of the Efficiency of Triple SpyTag- mSA (3A-mSA) Monomer Retrieval</a>
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| − | <a role="button" data-toggle="collapse" href="#collapse2" aria-expanded="false" aria-controls="collapse2">Evaluation of the Efficiency of Protein Hydrogel Retrieval</a>
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| − | <p>We demonstrated that the modules used to construct the 3A-mSA fusion protein remained functional. However, we still did not know if the polymerized hydrogel containing the mSA modules could be retrieved. We thus mixed three kinds of monomers together and waited for 1 hour to let them crosslink as far as possible. To evaluate the biotin binding efficiency of the Spy Crosslinking Network, the resulting hydrogel was incubated with biotin-coated magnetic beads at 37°C for 1 hour under constant agitation at 1500 rpm in a shaker. The corresponding sample list is shown in Fig. 4.</p>
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| − | <figcaption>Fig. 4. Sample list for the evaluation of the efficiency of protein hydrogel recovery with biotin-coated magnetic beads
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| − | <p>The Bradford assay was used to measure the concentration of remaining unbound protein and the results showed that unbound protein remaining after contacting with the beads was much less than in the controls (Fig.5A). Indeed, a marked decrease of protein concentration was visible when incubating the reaction system with beads for 1h, with only 22.1% of protein remaining unbound (Fig.5B). This meant that the biotin-coated beads could be combined with the mSA module while retaining their functionality, even when incorporated into the protein hydrogel.</p>
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| − | <figcaption>Fig. 5. Recovery of protein hydrogel using biotin-coated beads. (A) SDS-PAGE analysis of Spy Crosslinking Network contacted with beads. Lane 1: Spy Crosslinking Network incubated without beads as control group. Lane 2: Spy Crosslinking Network incubated with beads as experimental group. (B) Statistical analysis of the binding efficiency of the Spy Crosslinking network. ***p < 0.001. n=3. Error bars indicate standard deviations.
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| − | <p>To assess if other protein modules in the hydrogel could be linked via the mSA module, we did another experiment to test the change of fluorescence intensity of an mRFP module incubated with or without beads. The experimental procedures were the same as above. The excitation wavelength used for mRFP was 584 nm, and the emission peak was 607 nm. The fluorescence intensity was measured on a plate reader, and the results are shown in Fig. 6.</p>
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| − | <figcaption>Fig 6. The remaining unbound proportion of fluorescence intensity of a protein hydrogel comprising RFP and mSA modules after contacting with biotin-coated beads. ****p < 0.0001. Error bars indicate standard deviations.
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| − | <p>The fluorescence intensity of control groups corresponded to about 12.8 AU while in the experimental groups this value was 3.19 AU. Only 24.9% of total fluorescence remained unbound, indicating that a majority of 3A-RFP modules could be assembled into the hydrogel, and that the 3A-RFP module can be retrieved via binding of the hydrogel via the 3A-mSA module. These results indicated that if we use 3A-SUP in this hydrogel, the biotin-coated beads can also be used to remove it, together with the bound uranyl cations.</p>
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| − | </div><!-- results end-->
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| − | <a id="Conclusion"></a>
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| − | <div class="texttitle">Conclusion</div>
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| − | <p>We found that monomeric streptavidin, or mSA, which is able to tightly bind biotin, is a great candidate to retrieve the Spy Crosslinking Network and thus Uranium Reaper from the environment. The results demonstrated that the designs were effective. Not only could the monomeric triple SpyTag- mSA be recovered, but the hydrogel which contained mSA modules showed the same behavior, with a retrieval efficiency of about 80%. In further implementations using the 3A-mSA, we can thus exchange the 3A-mRFP, as illustrated in fig.4., for a 3A-SUP module, which could adsorb uranyl, in order to sequester uranium from the environment. In this way the special biological functional hydrogel would solve the pollution problem.</p>
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