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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 Kd 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 Kd 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> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/a/a2/T--Peking--images_uranyl_adsorption_fig1.png" style="width:100%;" alt=""/> |
| <figcaption>Fig. 1. Uranyl-binding affinity and selectivity of SUP. (A) Competition assay of SUP versus total carbonate for uranyl revealing a Kd of 7.4 fM at pH 8.9. (B) Binding selectivity of SUP for uranyl over various other metal ions. | | <figcaption>Fig. 1. Uranyl-binding affinity and selectivity of SUP. (A) Competition assay of SUP versus total carbonate for uranyl revealing a Kd of 7.4 fM at pH 8.9. (B) Binding selectivity of SUP for uranyl over various other metal ions. |
| </figcaption> | | </figcaption> |
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| <p>It was found that UO22+ is coordinated by five carboxylate oxygen atoms from four amino acid residues in SUP. The hydrogen bonds between the amino acid residues coordinating UO22+ 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 UO22+ is coordinated by five carboxylate oxygen atoms from four amino acid residues in SUP. The hydrogen bonds between the amino acid residues coordinating UO22+ and residues in its second coordination sphere also affects the protein’s uranyl binding ability (Fig.2) <sup>2</sup>.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/7/72/T--Peking--images_uranyl_adsorption_fig2.png" style="width:100%;" alt=""/> |
| <figcaption>Fig. 2. Coordination Environment of UO2<sup>2+</sup> in SUP. UO2<sup>2+</sup> is coordinated by five carboxylate oxygen atoms from four amino acid residues of SUP. | | <figcaption>Fig. 2. Coordination Environment of UO2<sup>2+</sup> in SUP. UO2<sup>2+</sup> is coordinated by five carboxylate oxygen atoms from four amino acid residues of SUP. |
| </figcaption> | | </figcaption> |
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| <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 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> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" 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>Fig. 3. Schematic diagram of uranyl adsorption. |
| </figcaption> | | </figcaption> |
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| <p>The adsorption reaction was allowed to continue for 1 min, after which the mixture was immediately transferred into 10kDa cutoff centrifuge filters and centrifuged for 10 min at 14000g to exclude non-specific protein interference by removing proteins (Fig.4). Finally, 100μL aliquots of the filtrate were collected for further analysis.</p> | | <p>The adsorption reaction was allowed to continue for 1 min, after which the mixture was immediately transferred into 10kDa cutoff centrifuge filters and centrifuged for 10 min at 14000g to exclude non-specific protein interference by removing proteins (Fig.4). Finally, 100μL aliquots of the filtrate were collected for further analysis.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f9/T--Peking--images_uranyl_adsorption_fig4.png" style="width:100%;" alt=""/> |
| <figcaption>Fig. 4. 10kDa cutoff centrifugal filters. The reaction mixtures were immediately transferred into 10kDa cutoff centrifugal filters and centrifuged for 10 min at 14000g to exclude protein interference. | | <figcaption>Fig. 4. 10kDa cutoff centrifugal filters. The reaction mixtures were immediately transferred into 10kDa cutoff centrifugal filters and centrifuged for 10 min at 14000g to exclude protein interference. |
| </figcaption> | | </figcaption> |
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| <p>What makes these results more reliable is that we set up control groups which contained the same concentration of uranyl as the test groups. We used the uranyl concentration of the filtrates from the control groups as the actual uranyl concentration to account for the adsorption of uranyl on centrifuge filters. Two different methods were applied to determine the uranyl concentrations in the filtrate. For higher concentrations (>1μM), we used a modification of the Arsenazo III method<sup>1</sup>. For lower concentrations (<1μM), ICP-MS was employed. </p> | | <p>What makes these results more reliable is that we set up control groups which contained the same concentration of uranyl as the test groups. We used the uranyl concentration of the filtrates from the control groups as the actual uranyl concentration to account for the adsorption of uranyl on centrifuge filters. Two different methods were applied to determine the uranyl concentrations in the filtrate. For higher concentrations (>1μM), we used a modification of the Arsenazo III method<sup>1</sup>. For lower concentrations (<1μM), ICP-MS was employed. </p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/8/80/T--Peking--images_uranyl_adsorption_fig5.png" style="width:100%;" alt=""/> |
| <figcaption>Fig. 5. Uranyl detection assay with Arsenazo III. (A) Mechanism of chromogenic reaction. (B) Standard curve of uranyl concentration. | | <figcaption>Fig. 5. Uranyl detection assay with Arsenazo III. (A) Mechanism of chromogenic reaction. (B) Standard curve of uranyl concentration. |
| </figcaption> | | </figcaption> |
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| <p>The adsorption capacities of 10μM proteins and hydrogel 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 hydrogel 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> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig6.png" style="width:100%;" alt=""/> |
| <figcaption>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. | | <figcaption>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. |
| </figcaption> | | </figcaption> |
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| <p>Furthermore, we measured the adsorption capacities of other kinds of fusion constructs and polymers, such as 4A-SUP (84%), 6A-SUP (87%), 4A-SUP+3B (88%), 6A-SUP+3B (63%) (Fig. 7). All of these were less efficient but they also could sequester at least 60% of the total uranyl. The standard deviations were calculated from triplicate experiments (n=3). These results were promising since different kinds of fusion proteins and protein constructs were able to adsorb uranyl with great efficiency. Among these, 3A-SUP+3B (96%) showed the best adsorption capacity and was consequently used for all further experiments.</p> | | <p>Furthermore, we measured the adsorption capacities of other kinds of fusion constructs and polymers, such as 4A-SUP (84%), 6A-SUP (87%), 4A-SUP+3B (88%), 6A-SUP+3B (63%) (Fig. 7). All of these were less efficient but they also could sequester at least 60% of the total uranyl. The standard deviations were calculated from triplicate experiments (n=3). These results were promising since different kinds of fusion proteins and protein constructs were able to adsorb uranyl with great efficiency. Among these, 3A-SUP+3B (96%) showed the best adsorption capacity and was consequently used for all further experiments.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig7.png" style="width:100%;" alt=""/> |
| <figcaption>Fig. 7. Statistical analysis of adsorption capacities of several kinds of proteins and protein hydrogel. ****p<0.0001, ***p<0.001. n=3. Error bars indicate standard deviations. | | <figcaption>Fig. 7. Statistical analysis of adsorption capacities of several kinds of proteins and protein hydrogel. ****p<0.0001, ***p<0.001. n=3. Error bars indicate standard deviations. |
| </figcaption> | | </figcaption> |
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| <p>The 3A-SUP+3B hydrogel 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 hydrogel 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> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig8.png" style="width:100%;" alt=""/> |
| <figcaption>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. | | <figcaption>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. |
| </figcaption> | | </figcaption> |
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| <p>At the protein-uranyl ratio of 10:1, 3A-SUP+3B could sequester 93%, 67% and 83% of total uranyl in TBS buffer, fresh water and artificial seawater, respectively (Fig.9). The standard deviations were calculated from triplicate experiments.</p> | | <p>At the protein-uranyl ratio of 10:1, 3A-SUP+3B could sequester 93%, 67% and 83% of total uranyl in TBS buffer, fresh water and artificial seawater, respectively (Fig.9). The standard deviations were calculated from triplicate experiments.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig9.png" style="width:100%;" alt=""/> |
| <figcaption>Fig. 9. Adsorption capacity of 3A-SUP+3B in different conditions. ****p<0.0001, n=3. Error bars indicate standard deviations. | | <figcaption>Fig. 9. Adsorption capacity of 3A-SUP+3B in different conditions. ****p<0.0001, n=3. Error bars indicate standard deviations. |
| </figcaption> | | </figcaption> |
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| <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 hydrogel can not only adsorb uranyl at high concentrations of uranium pollution, but can 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 hydrogel can not only adsorb uranyl at high concentrations of uranium pollution, but can also bind uranyl at very low concentrations - as low as 13nM - which equals the uranyl concentration in natural seawater.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--images_uranyl_adsorption_fig10.png" style="width:100%;" alt=""/> |
| <figcaption>Fig 10. Adsorption capacity of 3A-SUP+3B in low uranyl concentration. **p<0.01, n=3. Error bars indicate standard deviations. | | <figcaption>Fig 10. Adsorption capacity of 3A-SUP+3B in low uranyl concentration. **p<0.01, n=3. Error bars indicate standard deviations. |
| </figcaption> | | </figcaption> |