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| − | <h1>Designs<span>.</span></h1>
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| − | <p class="title1" style="text-align:center">On this page, we describe our design work in details to show how Uranium Reaper is able to solve real world problems, such as uranium pollution and other heavy metal pollution, in an elegant way. </p>
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| − | <h4>Design</h4>
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| − | <ul>
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| − | <li><a href="#Crosslinking">Crosslinking</a></li>
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| − | <li><a href="#Uranyl">Uranyl<sub>_</sub>Adsorption</a></li>
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| − | <li><a href="#Collection">Recovery</a></li>
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| − | <li><a href="#Secretion">Secretion</a></li>
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| − | <p>In view of existing as well as potential future uranium pollution, the current complicated, time-consuming, inefficient and expensive treatment methods seem woefully out-of-date. To address these drawbacks, the Peking iGEM team put forward the Uranyl Reaper project, which aims to develop an innovative method to deal with uranium pollution.</p>
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| − | <p>In order to achieve this goal, we designed an extraordinary biological adsorption hydrogel which is produced by engineered bacteria and self-assembles completely autonomously. Based on the isopeptide bond formed between SpyCatcher and SpyTag, two designed crosslinking peptides, we developed trimeric SpyTag and SpyCatcher modules which can form a covalently-linked hydrogel which offers high mechanical strength and high stability in diverse challenging environments.</p>
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| − | <p>This hydrogel further gains a capacity to adsorb uranium in aqueous environments with high efficiency via the addition of a super uranyl binding protein (SUP) module. The SUP could also be replaced with other functional elements such as additional metal binding proteins, or the monomerized streptavidin (mSA) module which can mediate the recovery of the self-assembled hydrogel through biotin-avidin interactions, in order to achieve multi-functionality and modularization.</p>
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| − | <p>We utilized a series of signal peptides to ensure efficient secretion. A schematic graph of the whole project is shown in Fig. 1.</p>
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| − | <img class="design_img" src="#" alt=""/>
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| − | <p><b>Figure 1. Schematic diagram of the Uranium Reaper project.</b></p>
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| − | <a id="Crosslinking"></a>
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| − | <br/>
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| − | <h3 class="classic-title"><span>Local Concentration Enhancement - Crosslinking</span> </h3>
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| − | <p>To unite at least two functions in a single protein hydrogel - uranium adsorption and recovery - we needed a protein hydrogel which contains several functional modules. Inspired by the autocatalytic formation of the isopeptide bond between a Lys and an Asp residue in the CnaB2 protein from S. pyogenes, researchers engineered a pair of complementary truncations of CnaB2, called SpyTag and SpyCatcher<sup>1</sup>. These engineered peptides were able to form isopeptide bonds with each other spontaneously. In order to use the SpyTag-SpyCatcher system as scaffold, we fused three SpyTag and three SpyCatcher modules, respectively. the results indicated that the crosslinking reaction between the Triple SpyCatcher and fusion proteins could be completed in 1 hour.</p>
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| − | <a id="Uranyl"></a>
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| − | <br/>
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| − | <h3 class="classic-title"><span>Reaping the ions - Uranyl Adsorption</span></h3>
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| − | <p>Considering that uranium mainly exists as uranyl ion and its carbonate in aqueous solution, we applied the Super Uranyl Binding Protein (SUP), which was designed specifically to bind uranyl via structural calculation and functional modification. SUP is thermodynamically stable and has a very high affinity and selectivity for uranyl ions, with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions<sup>2</sup>. In our experiments, SUP retained its high uranyl affinity after being fused with the triple SpyTag protein, as well as in the covalently cross-linked protein hydrogel. Impressively, in a trial where the concentration of uranyl was set at 10μM, the hydrogel with the SUP module was able to sequester nearly 90% of total uranyl ions within just one minute.
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| − | </p>
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| − | <a id="Collection"></a>
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| − | <br/>
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| − | <h3 class="classic-title"><span>Retrieving the network - Recovery</span></h3>
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| − | <p>How to collect the protein hydrogel together with the adsorbed uranyl from the environment? We focused on monomerized streptavidin (mSA), which binds biotin with a very low Kd value of less than 1nM<sup>3</sup>, and added it as a module to the hydrogel in the form of a triple SpyTag-mSA fusion protein.</p>
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| − | <p>We next ligated biotin molecules to amino-coated magnetic beads, and when we used a magnet to immobilize these beads, the protein hydrogel full of bound uranyl ions could be retrieved with an efficiency of up to 70%.</p>
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| − | <a id="Secretion"></a>
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| − | <br/>
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| − | <h3 class="classic-title"><span>Simplification of protein purification - Secretion</span></h3>
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| − | <p>To reduce costs and simplify the time-consuming procedures necessary to purify proteins from bacteria, we added secretion modules. Whether in E. coli or B. subtilis, secretory proteins usually contain signal peptides that are essential for their export from the cytoplasm. Which signal peptide would best promote the secretion of our constructs was inscrutable, so a library was built in order to screen for the most suitable candidates. We developed two assays (Western Blot and FIAsH fluorescence) to detect successful secretion. Finally, signal sequences derived from OmpA, LtIIB and PhoA were found to be efficient mediators for the secretion of our recombinant proteins. The fusion proteins could be brought to their working concentration by concentrating them directly in the culture supernatants, and the resulting crude concentrates could be used directly for the ultimate goal - the Uranium Reaper.
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| − | <h3>References:</h3>
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| − | <p>[1] Zakeri, B., et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences, 2012. 109(12): p. E690-E697.</p>
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| − | <p>[2] Lu, Z. et al. A protein engineered to bind uranyl selectively and with femtomolar affinity. Nature chemistry 1856, 236-241 (2014).</p>
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| − | <p>[3] Sau-Ching, W. et al. Engineering monomeric streptavidin and its ligands with infinite affinity in binding but reversibility in interaction. Proteins 77, 404-412 (2009).</p>
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| | <p><b>Figure 1. Schematic diagram of the Uranium Reaper project.</b></p> | | <p><b>Figure 1. Schematic diagram of the Uranium Reaper project.</b></p> |
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| | <a id="Crosslinking"></a> | | <a id="Crosslinking"></a> |
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| | <h3 class="classic-title anchor"><span>Local Concentration Enhancement - Crosslinking</span> </h3> | | <h3 class="classic-title anchor"><span>Local Concentration Enhancement - Crosslinking</span> </h3> |
| | <p>To unite at least two functions in a single protein hydrogel - uranium adsorption and recovery - we needed a protein hydrogel which contains several functional modules. Inspired by the autocatalytic formation of the isopeptide bond between a Lys and an Asp residue in the CnaB2 protein from S. pyogenes, researchers engineered a pair of complementary truncations of CnaB2, called SpyTag and SpyCatcher<sup>1</sup>. These engineered peptides were able to form isopeptide bonds with each other spontaneously. In order to use the SpyTag-SpyCatcher system as scaffold, we fused three SpyTag and three SpyCatcher modules, respectively. the results indicated that the crosslinking reaction between the Triple SpyCatcher and fusion proteins could be completed in 1 hour.</p> | | <p>To unite at least two functions in a single protein hydrogel - uranium adsorption and recovery - we needed a protein hydrogel which contains several functional modules. Inspired by the autocatalytic formation of the isopeptide bond between a Lys and an Asp residue in the CnaB2 protein from S. pyogenes, researchers engineered a pair of complementary truncations of CnaB2, called SpyTag and SpyCatcher<sup>1</sup>. These engineered peptides were able to form isopeptide bonds with each other spontaneously. In order to use the SpyTag-SpyCatcher system as scaffold, we fused three SpyTag and three SpyCatcher modules, respectively. the results indicated that the crosslinking reaction between the Triple SpyCatcher and fusion proteins could be completed in 1 hour.</p> |
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| | <a id="Uranyl"></a> | | <a id="Uranyl"></a> |
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| | <h3 class="classic-title anchor"><span>Reaping the ions - Uranyl Adsorption</span></h3> | | <h3 class="classic-title anchor"><span>Reaping the ions - Uranyl Adsorption</span></h3> |
| | <p>Considering that uranium mainly exists as uranyl ion and its carbonate in aqueous solution, we applied the Super Uranyl Binding Protein (SUP), which was designed specifically to bind uranyl via structural calculation and functional modification. SUP is thermodynamically stable and has a very high affinity and selectivity for uranyl ions, with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions<sup>2</sup>. In our experiments, SUP retained its high uranyl affinity after being fused with the triple SpyTag protein, as well as in the covalently cross-linked protein hydrogel. Impressively, in a trial where the concentration of uranyl was set at 10μM, the hydrogel with the SUP module was able to sequester nearly 90% of total uranyl ions within just one minute. | | <p>Considering that uranium mainly exists as uranyl ion and its carbonate in aqueous solution, we applied the Super Uranyl Binding Protein (SUP), which was designed specifically to bind uranyl via structural calculation and functional modification. SUP is thermodynamically stable and has a very high affinity and selectivity for uranyl ions, with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions<sup>2</sup>. In our experiments, SUP retained its high uranyl affinity after being fused with the triple SpyTag protein, as well as in the covalently cross-linked protein hydrogel. Impressively, in a trial where the concentration of uranyl was set at 10μM, the hydrogel with the SUP module was able to sequester nearly 90% of total uranyl ions within just one minute. |
| | </p> | | </p> |
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| | <a id="Collection"></a> | | <a id="Collection"></a> |
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| | <h3 class="classic-title anchor"><span>Retrieving the network - Recovery</span></h3> | | <h3 class="classic-title anchor"><span>Retrieving the network - Recovery</span></h3> |
| | <p>How to collect the protein hydrogel together with the adsorbed uranyl from the environment? We focused on monomerized streptavidin (mSA), which binds biotin with a very low Kd value of less than 1nM<sup>3</sup>, and added it as a module to the hydrogel in the form of a triple SpyTag-mSA fusion protein.</p> | | <p>How to collect the protein hydrogel together with the adsorbed uranyl from the environment? We focused on monomerized streptavidin (mSA), which binds biotin with a very low Kd value of less than 1nM<sup>3</sup>, and added it as a module to the hydrogel in the form of a triple SpyTag-mSA fusion protein.</p> |
| | <p>We next ligated biotin molecules to amino-coated magnetic beads, and when we used a magnet to immobilize these beads, the protein hydrogel full of bound uranyl ions could be retrieved with an efficiency of up to 70%.</p> | | <p>We next ligated biotin molecules to amino-coated magnetic beads, and when we used a magnet to immobilize these beads, the protein hydrogel full of bound uranyl ions could be retrieved with an efficiency of up to 70%.</p> |
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| | <a id="Secretion"></a> | | <a id="Secretion"></a> |
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| | <h3 class="classic-title anchor"><span>Simplification of protein purification - Secretion</span></h3> | | <h3 class="classic-title anchor"><span>Simplification of protein purification - Secretion</span></h3> |
| | <p>To reduce costs and simplify the time-consuming procedures necessary to purify proteins from bacteria, we added secretion modules. Whether in E. coli or B. subtilis, secretory proteins usually contain signal peptides that are essential for their export from the cytoplasm. Which signal peptide would best promote the secretion of our constructs was inscrutable, so a library was built in order to screen for the most suitable candidates. We developed two assays (Western Blot and FIAsH fluorescence) to detect successful secretion. Finally, signal sequences derived from OmpA, LtIIB and PhoA were found to be efficient mediators for the secretion of our recombinant proteins. The fusion proteins could be brought to their working concentration by concentrating them directly in the culture supernatants, and the resulting crude concentrates could be used directly for the ultimate goal - the Uranium Reaper. | | <p>To reduce costs and simplify the time-consuming procedures necessary to purify proteins from bacteria, we added secretion modules. Whether in E. coli or B. subtilis, secretory proteins usually contain signal peptides that are essential for their export from the cytoplasm. Which signal peptide would best promote the secretion of our constructs was inscrutable, so a library was built in order to screen for the most suitable candidates. We developed two assays (Western Blot and FIAsH fluorescence) to detect successful secretion. Finally, signal sequences derived from OmpA, LtIIB and PhoA were found to be efficient mediators for the secretion of our recombinant proteins. The fusion proteins could be brought to their working concentration by concentrating them directly in the culture supernatants, and the resulting crude concentrates could be used directly for the ultimate goal - the Uranium Reaper. |
