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| <p>Uranium is a key element used in nuclear energy production and is crucial in many other applications. The most stable and relevant uranium ion in aerobic environments is the uranyl cation. Super Uranyl-binding Protein (SUP) has been rationally designed via structural calculations and functional modification to specifically bind uranyl cations. According to the researchers’ results, SUP is thermodynamically stable and offers very high affinity and selectivity for uranyl with a K<SUB>d</SUB> of 7.4 fM and >10,000-fold selectivity over other metal ions<sup>1</sup>. The binding features of SUP are described later in more detail (Fig. 1.).</p> | | <p>Uranium is a key element used in nuclear energy production and is crucial in many other applications. The most stable and relevant uranium ion in aerobic environments is the uranyl cation. Super Uranyl-binding Protein (SUP) has been rationally designed via structural calculations and functional modification to specifically bind uranyl cations. According to the researchers’ results, SUP is thermodynamically stable and offers very high affinity and selectivity for uranyl with a K<SUB>d</SUB> of 7.4 fM and >10,000-fold selectivity over other metal ions<sup>1</sup>. The binding features of SUP are described later in more detail (Fig. 1.).</p> |
| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width: 90% ;" src="https://static.igem.org/mediawiki/2016/c/c9/Fig1_uranium.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width: 100% ;" src="https://static.igem.org/mediawiki/2016/7/76/T--Peking--images_uo2_fig1.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 1. Uranyl-binding affinity and selectivity of SUP. (A) Competition assay of SUP versus total carbonate for uranyl revealing a K<SUB>d</SUB> of 7.4 fM at pH 8.9. (B) Binding selectivity of SUP for uranyl over various other metal ions. | | Fig. 1. Uranyl-binding affinity and selectivity of SUP. (A) Competition assay of SUP versus total carbonate for uranyl revealing a K<SUB>d</SUB> of 7.4 fM at pH 8.9. (B) Binding selectivity of SUP for uranyl over various other metal ions. |
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| <p>It was found that UO<sub>2</sub><sup>2+</sup> is coordinated by five carboxylate oxygen atoms from four amino acid residues in SUP. The hydrogen bonds between the amino acid residues coordinating UO<sub>2</sub><sup>2+</sup> and residues in its second coordination sphere also affects the protein’s uranyl binding ability (Fig. 2.) <sup>2</sup>.</p> | | <p>It was found that UO<sub>2</sub><sup>2+</sup> is coordinated by five carboxylate oxygen atoms from four amino acid residues in SUP. The hydrogen bonds between the amino acid residues coordinating UO<sub>2</sub><sup>2+</sup> and residues in its second coordination sphere also affects the protein’s uranyl binding ability (Fig. 2.) <sup>2</sup>.</p> |
| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width:50% ;" src=" https://static.igem.org/mediawiki/2016/7/72/T--Peking--images_uranyl_adsorption_fig2.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width:45% ;" src="https://static.igem.org/mediawiki/2016/7/72/T--Peking--images_uranyl_adsorption_fig2.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 2. Coordination Environment of UO<sub>2</sub><sup>2+</sup> in SUP. UO<sub>2</sub><sup>2+</sup> is coordinated by five carboxylate oxygen atoms from four amino acid residues of SUP. | | Fig. 2. Coordination Environment of UO<sub>2</sub><sup>2+</sup> in SUP. UO<sub>2</sub><sup>2+</sup> is coordinated by five carboxylate oxygen atoms from four amino acid residues of SUP. |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
− | <p>As mentioned above, we fused SUP to three SpyTags in order to construct the 3A-SUP monomer, so the function of SUP might be affected by these additional modules. To make sure that 3A-SUP could still function when bound in the polymer net, we tested its ability to adsorb UO<sub>2</sub><sup>2+</sup> in various environments.</p> | + | <p>As mentioned above, we fused SUP to three SpyTags in order to construct the 3A-SUP monomer, so the function of SUP might be affected by these additional modules. To make sure that 3A-SUP could still function when bound in the polymer network, we tested its ability to adsorb UO<sub>2</sub><sup>2+</sup> in various environments.</p> |
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| <a id="Methods"></a> | | <a id="Methods"></a> |
| <div class="texttitle">Methods</div> | | <div class="texttitle">Methods</div> |
− | <p>Appropriate volumes of 3/4/6A-SUP and 3B were mixed and incubated for 1 hour. Subsequently, a uranyl solution in TBS buffer was prepared and its pH value adjusted appropriately. After complete cross-linking, the uranyl solution was contacted with the pre-incubated proteins and mixed thoroughly by vortexing (Fig. 3.).</p> | + | <p>Appropriate volume of 3/4/6A-SUP and 3B were mixed and incubated for 1 hour. Subsequently, a uranyl solution in TBS buffer was prepared and its pH value adjusted appropriately. After complete cross-linking, the uranyl solution was contacted with the pre-incubated proteins and mixed thoroughly by vortexing (Fig. 3.).</p> |
| <figure> | | <figure> |
| <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/d/dc/T--Peking--images_uranyl_adsorption_fig3.png" style="width:100%;" alt=""/> | | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/d/dc/T--Peking--images_uranyl_adsorption_fig3.png" style="width:100%;" alt=""/> |
− | <figcaption>Fig. 3. Schematic diagram of uranyl adsorption. | + | <figcaption style="text-align:center;">Fig. 3. Schematic diagram of uranyl adsorption. |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <div class="panel panel-default"> | | <div class="panel panel-default"> |
| <div class="panel-heading panel-title" role="tab" id="heading1"> | | <div class="panel-heading panel-title" role="tab" id="heading1"> |
− | <a role="button" data-toggle="collapse" href="#collapse1" aria-expanded="false" aria-controls="collapse1">1. Uranyl Adsorption onto Fusion Proteins and the Protein Polymer net </a> | + | <a role="button" data-toggle="collapse" href="#collapse1" aria-expanded="false" aria-controls="collapse1">1. Uranyl Adsorption onto Fusion Proteins and the Protein Polymer network </a> |
| </div> | | </div> |
| <div id="collapse1" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="heading1"> | | <div id="collapse1" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="heading1"> |
| <div class="panel-body"> | | <div class="panel-body"> |
− | <p>The adsorption capacities of 10μM proteins and polymer net were measured in TBS buffer against uranyl in equimolar quantities. In very short time, 3A-SUP alone showed an adsorption capacity of up to 87% and cross-linked 3A-SUP+3B could effectively sequester 96% of the total uranyl (Fig. 6.).</p> | + | <p>The adsorption capacities of 10μM proteins and polymer network were measured in TBS buffer against uranyl in equimolar quantities. In very short time, 3A-SUP alone showed an adsorption capacity of up to 87% and cross-linked 3A-SUP+3B could effectively sequester 96% of the total uranyl (Fig. 6.).</p> |
| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width: % ;" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig6.png " alt=""/></p> | + | <p style="text-align:center;"><img style="width:80% ;" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig6.png " alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 6. Adsorption capacities of 3A-SUP and the oligomer mixture 3A-SUP+3B. 3A-SUP alone showed an adsorption capacity of up to 87%. Cross-linked 3A-SUP+3B could effectively sequester 96% of the total uranyl. | | Fig. 6. Adsorption capacities of 3A-SUP and the oligomer mixture 3A-SUP+3B. 3A-SUP alone showed an adsorption capacity of up to 87%. Cross-linked 3A-SUP+3B could effectively sequester 96% of the total uranyl. |
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| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width: 90% ;" src=" https://static.igem.org/mediawiki/2016/1/1e/Fig7%E6%94%B9.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width: 86% ;" src="https://static.igem.org/mediawiki/2016/c/c9/T--Peking--images_uo2_fig7.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
− | Fig. 7. Statistical analysis of adsorption capacities of several kinds of proteins and protein polymer net. ****p<0.0001, ***p<0.001. n=3. Error bars indicate standard deviations. | + | Fig. 7. Statistical analysis of adsorption capacities of several kinds of proteins and protein polymer network. ****p<0.0001, ***p<0.001. n=3. Error bars indicate standard deviations. |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <div class="panel-body"> | | <div class="panel-body"> |
| <p>We next wondered whether the adsorption capacity of 3A-SUP+3B would increase with an increased protein-uranyl ratio. We thus tested the adsorption capacity of 3A-SUP+3B at different protein-uranyl ratios in TBS buffer. In these experiments, the concentration of uranyl ions was kept at 10μM.</p> | | <p>We next wondered whether the adsorption capacity of 3A-SUP+3B would increase with an increased protein-uranyl ratio. We thus tested the adsorption capacity of 3A-SUP+3B at different protein-uranyl ratios in TBS buffer. In these experiments, the concentration of uranyl ions was kept at 10μM.</p> |
− | <p>The 3A-SUP+3B polymer net could sequester 89% and 93% of the total uranyl when the protein to uranyl ratio was one and ten, respectively (Fig. 8.). The standard deviations were calculated from triplicate experiments</p> | + | <p>The 3A-SUP+3B polymer network could sequester 89% and 93% of the total uranyl when the protein to uranyl ratio was one and ten, respectively (Fig. 8.). The standard deviations were calculated from triplicate experiments</p> |
| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width:40% ;" src="https://static.igem.org/mediawiki/2016/5/5c/Fig8%E6%94%B9.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width:48% ;" src="https://static.igem.org/mediawiki/2016/9/9a/T--Peking--images_uo2_fig8.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 8. Adsorption capacity of 3A-SUP+3B at protein-uranyl ratios of 1 and 10. ****p<0.0001, ns means no significant difference. n=3. Error bars indicate standard deviations. | | Fig. 8. Adsorption capacity of 3A-SUP+3B at protein-uranyl ratios of 1 and 10. ****p<0.0001, ns means no significant difference. n=3. Error bars indicate standard deviations. |
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| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width:50% ;" src=" https://static.igem.org/mediawiki/2016/5/5b/Fig9%E6%94%B9.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2016/4/4e/T--Peking--images_uo2_fig9.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 9. Adsorption capacity of 3A-SUP+3B in different conditions. ****p<0.0001, n=3. Error bars indicate standard deviations. | | Fig. 9. Adsorption capacity of 3A-SUP+3B in different conditions. ****p<0.0001, n=3. Error bars indicate standard deviations. |
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| <div id="collapse4" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="heading1"> | | <div id="collapse4" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="heading1"> |
| <div class="panel-body"> | | <div class="panel-body"> |
− | <p>The adsorption capacity of 3A-SUP+3B against 13nM uranyl was tested at a protein-uranyl ratio of 6000:1, and it was able to sequester 48% and 35% of the total uranyl in TBS buffer and artificial seawater, respectively (Fig. 10.). That means that the functional polymer net could not only adsorb uranyl at high concentrations of uranium pollution, but could also bind uranyl at very low concentrations - as low as 13nM - which equals the uranyl concentration in natural seawater.</p> | + | <p>The adsorption capacity of 3A-SUP+3B against 13nM uranyl was tested at a protein-uranyl ratio of 6000:1, and it was able to sequester 48% and 35% of the total uranyl in TBS buffer and artificial seawater, respectively (Fig. 10.). That means that the functional polymer network could not only adsorb uranyl at high concentrations of uranium pollution, but could also bind uranyl at very low concentrations - as low as 13nM - which equals the uranyl concentration in natural seawater.</p> |
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| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width:35% ;" src=" https://static.igem.org/mediawiki/2016/5/5d/Fig10%E6%94%B9.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width:42% ;" src="https://static.igem.org/mediawiki/2016/a/a3/T--Peking--images_uo2_fig10.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig 10. Adsorption capacity of 3A-SUP+3B in low uranyl concentration. **p<0.01, n=3. Error bars indicate standard deviations. | | Fig 10. Adsorption capacity of 3A-SUP+3B in low uranyl concentration. **p<0.01, n=3. Error bars indicate standard deviations. |
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| <a id="discussion"></a> | | <a id="discussion"></a> |
| <div class="texttitle">Discussion</div> | | <div class="texttitle">Discussion</div> |
− | <p>Triple SpyTag-SUP (3A-SUP) was able to adsorb uranyl with great efficiency, and the cross-linked product of 3A-SUP+3B showed an even better adsorption capacity. What’s more, 3A-SUP+3B could also perform its function well in contaminated conditions, and could thus be used to detoxify the environment. Furthermore, we confirmed that the biological functional polymer net could indeed adsorb uranyl ions from seawater, and might even be employed to gather uranium directly from seawater in the future. Thus, our biological functional polymer net has broad potential application fields.</p> | + | <p>Triple SpyTag-SUP (3A-SUP) was able to adsorb uranyl with great efficiency, and the cross-linked product of 3A-SUP+3B showed an even better adsorption capacity. What’s more, 3A-SUP+3B could also perform its function well in contaminated conditions, and could thus be used to detoxify the environment. Furthermore, we confirmed that the biological functional polymer network could indeed adsorb uranyl ions from seawater, and might even be employed to gather uranium directly from seawater in the future. Thus, our biological functional polymer network has broad potential application fields.</p> |
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| <a id="references"></a> | | <a id="references"></a> |