Team:British Columbia/ProjectBiomining

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            <h1>Biomining </h1>
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<h1>Biomining</h1>
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<img id="bio_phages" src="https://static.igem.org/mediawiki/2014/a/ab/Phages-01.png" style="width:720px"/>
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<img id="bio_peps" src="https://static.igem.org/mediawiki/2014/d/dd/Bio-mining_peps-01.png" style="width:540px"/>
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<img id="coil_coil" src="https://static.igem.org/mediawiki/2014/8/80/Coil-coil.png" style="width:820px"/>
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Our access to easily extractable copper is gradually diminishing as demands for copper continues to grow worldwide. To meet these demands, non-traditional, metallurgically challenging deposits are expected to become more prevalent, thereby forcing us to deal with more complex, and lower-grade ores containing higher levels of impurities.  Arsenic-challenged deposits is a concern for the copper mining industry as arsenic produces hazardous fumes and oxide dusts during the smelting process. Smelting with arsenic therefore poses a significant risk to the health of the workers and to the environment. Safe removal and disposal of stabilized arsenic is often difficult and costly.  The mining industry has developed several methods for dealing with arsenic impurities, which includes precipitation with scorodite and pressure hydrometallurgical procedures (150C and 1380kPa) for processing high concentration of arsenic while extracting copper in parallel. However, many procedures requires an enormous amount of energy or additional acidic chemicals to help selectively separate arensic-containing minerals in the ore mixtures. With stricter legislation in place, the mining industry is facing increasing pressure to progressively reduce the amount of allowable arsenic concentrations for smelters. Consequently, fines and penalties are set for arsenic concentrations exceeding 0.2%, while ores past 0.5% arsenic concentrations are rejected by smelters.
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                  </p>
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                  <p>
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There is increasing precedence for removing arsenic-containing minerals such as enargite (Cu3AsS4)from relevant minerals, chalcopyrite (CuFeS2), during the flotation process. However, separation is often conflicted as enargite posesses similar floation properties as valuable minerals such
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It would be more economically and environmentally beneficial to remove the minerals containing arsenic at an earlier stage such as during flotation. Their separation is nevertheless difficult as they generally have similar flotation behaviour to the valuable minerals with which they are associated. This is the case in separating arsenopyrite (FeAsS) from pyrite, or removing enargite (Cu3AsS4) and tennantite (Cu12As4S13) from covellite (CuS), chalcocite (Cu2S) and chalcopyrite (CuFeS2). Apart from arsenopyrite, the amount of literature dealing with the separation of arsenic minerals is scarce. One of the potential separation methods relies on the selective oxidation of sulfide minerals due to differences in their electrochemical properties (e.g., Tolley et al., 1996, Byrne et al., 1995, Kydros et al., 1993, Wang et al., 1992, Beattie and Poling, 1988, Guongming and Hongen, 1989 and Chander, 1985). Oxidation can promote the adsorption of collectors, such as xanthate, at low to moderate levels of oxidation, or prevent their adsorption at high levels of oxidation by creating a physical barrier of oxidation products for their diffusion to the mineral surface.
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<h1>System Design</h1>
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              </p>
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<h2>A Separation Problem</h2>
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          <p>
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<img id="bio_phages" src="https://static.igem.org/mediawiki/2014/3/30/Writing.png" style="float: right; margin: 10px 0px 0px 25px;" />
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Chocolate cake macaroon bear claw lollipop. Carrot cake chocolate cake tiramisu jujubes chocolate cake cake pastry jelly beans gummies. Powder tootsie roll ice cream candy lemon drops pastry. Biscuit sweet sweet roll brownie chupa chups sugar plum applicake fruitcake tootsie roll. Danish topping chocolate bar brownie jelly bonbon oat cake halvah. Oat cake sesame snaps jujubes oat cake. Candy lollipop cake gummi bears topping danish. Lemon drops muffin halvah chocolate bar croissant croissant brownie cotton candy. Tart cake macaroon.
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Jelly beans bear claw marshmallow pastry topping tootsie roll halvah. Unerdwear.com icing caramels dessert dessert jujubes. Soufflé jelly beans biscuit chocolate unerdwear.com. Bonbon pastry sugar plum tiramisu cotton candy sweet. Tiramisu topping candy canes caramels tart cheesecake halvah cupcake croissant. Carrot cake cake jujubes jujubes cotton candy ice cream gummies sugar plum icing.
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Our access to easily extractable copper is gradually diminishing as demands for copper continues to grow worldwide. To meet these demands, non-traditional, metallurgically challenging deposits are expected to become more prevalent, thereby forcing us to deal with more complex, and lower-grade ores containing higher levels of impurities.  Arsenic-challenged deposits is a concern for the copper mining industry as arsenic produces hazardous fumes and oxide dusts during the smelting process. Smelting with arsenic therefore poses a significant risk to the health of the workers and to the environment. Furthermore, safe removal and disposal of stabilized arsenic is often difficult and costly.  With stricter legislation in place, the mining industry is facing increasing pressure to progressively reduce the amount of allowable arsenic concentrations for smelters. Consequently, fines and penalties are set for arsenic concentrations exceeding 0.2 %, while ores past 0.5 % arsenic concentrations are rejected by smelters. Therefore, there is increasing precedence for removing arsenic-containing minerals such as enargite (Cu3AsS4) from relevant sulfide minerals, such as chalcopyrite (CuFeS2), during the flotation process. However, separation is often conflicted with shared flotation properties between enargite and the associated valuable minerals. The mining industry has developed several methods for dealing with arsenic impurities, which includes precipitation with scorodite and pressure hydrometallurgical procedures (150 °C and 1380 kPa) for processing high concentration of arsenic while extracting copper in parallel. However, many procedures requires an enormous amount of energy or additional acidic chemicals to help selectively separate arensic-containing minerals in the ore mixtures.
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</p>
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    </div>
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<h2>Our solution: Mineral Binding</h2>
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<p>
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We, the UBC iGEM team, feel that separation and enrichment can be done in other ways that does not rely on energy or chemically-intensive methods. Our synbio solution involves the use of small surface heptapeptides and octapeptides, which have previously been demonstrated in M13 bacteriophage by Curtis <i>et al.</i> to selectively bind to chalcopyrite(3). Three peptides, labelled WSD-1 (TPTTYKV), WSD-2 (DSQKTNPS), and WSD-3 (DPIKHTSG), have been identified for binding. However, operating with bacteriophage is not feasible for large scale operations in mining as it is difficult to scale up titers to compensate for the smaller surface area available for binding in bacteriophage. Our idea is to operate these peptides in bacteria which have a larger surface area and are much more responsive to stimuli. These peptides must be accessible on the surface. Therefore, we have chosen <i>Caulobacter crescentus</i> as our chassis given that it contains a S-protein layer in which we can express our peptides. A developed kit for protein secretion and display of peptides on the cell surface S-layer in <i>Caulobacter crescentus</i> (via cloning to the S-layer gene sequence) can be found within
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<a href="http://ubc.flintbox.com/public/project/1487/">Caulobacter S-layer Kits</a>.
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<img id="bio_phages" src="https://static.igem.org/mediawiki/2014/a/ab/Phages-01.png" width="600px"/>
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<br/>
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<p><b>Figure 1.</b> Curtis <i>et al.</i> screened a random heptapeptide library through a series of ‘biopanning’ assays for peptides that could be expressed on M13 bacteriophage and selectively bind to chalcopyrite. These peptide-containing bacteriophage have the potential to be utilized to separate chalcopyrite from enargite in ore slurries.
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</p>
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</center>
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<center>
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<img id="bio_peps" src="https://static.igem.org/mediawiki/2014/d/dd/Bio-mining_peps-01.png" style="width:600px"/>
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<br/>
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<p><b>Figure 2.</b> Our project adapts these chalcopyrite-binding peptides (WSD-1 (TPTTYKV), WSD-2 (DSQKTNPS), and WSD-3 (DPIKHTSG)) by cloning them into the S-protein layer of <i>Caulobacter crescentus</i>.
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</p>
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</center>
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<h2>Our solution: Mineral Separation</h2>
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<p>
 +
As a proof of concept, we have chosen to express these peptides to selectively bind to chalcopyrite, which will then fall out of solution by aggregation using two interacting coiled-coil peptides expressed on the S-layer of <i>Caulobacter crescentus</i>. These coiled-coil peptides (K-coil: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189012">BBa_K1189012</a> , E-coil: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189013">BBa_K1189013</a>, K-coil-His: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189010">BBa_K1189010</a>, and E-coil-His: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189011">BBa_K1189011</a> )were constructed by iGEM Calgary 2013. In this model, <i>Caulobacter</i> A will contain chalcopyrite-binding peptides bound to chalcopyrite elusively while holding a K-coil interaction peptide. By addition of <i>Caulobacter</i> B which holds the E-coil interaction peptide, <i>Caulobacter</i> A will coalesce with <i>Caulobacter</i> B by E-coil and K-coil interactions, forming a greater mass that will selectively sink in a froth floation solution. Alternatively, we have considered floating bound chalcopyrite using gas vesicle proteins (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_I750016">BBa_I750016</a>) developed by iGEM Groningen 2009.  
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</p>
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<center>
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<img id="coil_coil" src="https://static.igem.org/mediawiki/2014/8/80/Coil-coil.png" style="width:600px"/>
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<br/>
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<p><b>Figure 3.</b> K-coil and E-coil coiled-coil interaction peptides are expressed on the S-protein layer in two strains of <i>Caulobacter crescentus</i>.
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</p>
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</center>
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<center>
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<img id="overview" src="https://static.igem.org/mediawiki/2014/7/71/Biomining_overview.png" style="width:600px"/>
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<br/>
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<p><b>Figure 4.</b> <i>Caulobacter</i> A will contain chalcopyrite-binding peptides bound to chalcopyrite elusively, while holding a K-coil interaction peptide. Addition of <i>Caulobacter</i> B which holds the E-coil interaction peptide will create an interacting link with <i>Caulobacter</i> A, forming a mass that will fall out of solution
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</p>
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</center>
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<h2>Binding Assay</h2>
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<p>
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To observe whether our peptides are capable of binding to chalcopyrite, we incubated 40 μm and 100 μm size chalcopyrite with our peptide expressing <i>Caulobacter crescentus</i> which were then passed through a 40 μm size cell strainer and subsequently washed with buffer and eluted with elution solutions. The elutions were then plated on YPD plate.  Colonies grown on YPD plate suggest that the <i>Caulobacter crescentus</i> were bound to the chalcopyrite as they were present after the wash and elution steps.
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</p>
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<center>
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<img id="overview" src="https://static.igem.org/mediawiki/2014/2/2b/Diagram4.jpg" style="width:900px"/>
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<br/>
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<p><b>Figure 5.</b> Figure 5 Binding assay by incubating peptide-containing <i>Caulobacter crescentus</i> with chalcopyrite and subsequent wash and elution steps to remove unbound bacteria.
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</p>
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</center>
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<h1> Results </h1>
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<h2> Biomining precipitation assays </h2>
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<p>
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Before attempting to demonstrate binding with peptide inserted into the S-Layer, we tested binding with WT cells. After having observed binding, we then hypothesized that the fucose layer in <i>Caulobacter</i> was potentially involved in binding. Since there was a fucos knockout was available, we ere able to confirm fucos binding. Moreover, this observation demonstrated that a precipitation assay was feasible when mixing cells with Chalcopyrite.
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</p>
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<center>
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<video width="920" controls>
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  <source src="https://static.igem.org/mediawiki/2014/a/a1/UBC_iGEM_chalcopyrite_biomining_1.mp4" type="video/mp4">
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Your browser does not support the video tag.
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</video>
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</center>
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<p>
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<center>
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<b>Figure 6.</b> Wild type and fucose knockout <i>Caulobacter</i> cells mixed with Chalcopyrite.
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</center>
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</p>
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<p>
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As a preliminary test, we observed increased Chalcopyrite precipitation with <i>Caulobacter</i> cells expressing a Chalcopyrite binding peptide in the S-layer. While non-quantitative, the data showed noticeable differences in precipitate settle when the peptide expressed in various locations in the S-layer. See the video below.  
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</p>
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<center>
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<video width="920" controls>
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  <source src="https://static.igem.org/mediawiki/2014/5/59/UBC_iGEM_chalcopyrite_biomining_2.mp4" type="video/mp4">
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Your browser does not support the video tag.
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</video>
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</center>
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<p>
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<b>Figure 7.</b>Various peptides from Curtis <i>et al.</i> are displayed in different positions in the S-layer of <i>Caulobacter</i> cells. The cells were mixed with calcopyrite and precipitate settling was observed.
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</p>
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      <h1>Biomining protein assays</h1>
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<p>
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In an attempt to support the above observation, we incubated <i>Caulobacter</i> cells with Chalcopyrite and separated the Chalcopyrite by centrifugation. The supernatant and cells adhered to Chalcopyrite (eluted after several buffer washes) were assayed for protein concentration as a read out for biomass. For fucose containing cells and cells expressing Chalcopyrite binding peptides in the S-layer, we expected protein concentration to decrease in the supernatant and increase on Chalcopyrite as compared to controls.  
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</p>
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<center>
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<img id="overview" src="https://static.igem.org/mediawiki/2014/6/6e/Diagram_biopan_v2_%281%29.png" style="width:900px"/>
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<br/>
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<p><b>Figure 8.</b> Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in wild type <i>Caulobacter</i> cells.
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</p>
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</center>
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<center>
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<img id="overview" src="https://static.igem.org/mediawiki/2014/0/0b/Diagram_bioming2.png" style="width:900px"/>
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<br/>
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<p><b>Figure 9.</b> Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in fucose knockout cells.
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</p>
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Latest revision as of 08:07, 26 November 2014

2014 UBC iGEM

Biomining

System Design

A Separation Problem

Our access to easily extractable copper is gradually diminishing as demands for copper continues to grow worldwide. To meet these demands, non-traditional, metallurgically challenging deposits are expected to become more prevalent, thereby forcing us to deal with more complex, and lower-grade ores containing higher levels of impurities. Arsenic-challenged deposits is a concern for the copper mining industry as arsenic produces hazardous fumes and oxide dusts during the smelting process. Smelting with arsenic therefore poses a significant risk to the health of the workers and to the environment. Furthermore, safe removal and disposal of stabilized arsenic is often difficult and costly. With stricter legislation in place, the mining industry is facing increasing pressure to progressively reduce the amount of allowable arsenic concentrations for smelters. Consequently, fines and penalties are set for arsenic concentrations exceeding 0.2 %, while ores past 0.5 % arsenic concentrations are rejected by smelters. Therefore, there is increasing precedence for removing arsenic-containing minerals such as enargite (Cu3AsS4) from relevant sulfide minerals, such as chalcopyrite (CuFeS2), during the flotation process. However, separation is often conflicted with shared flotation properties between enargite and the associated valuable minerals. The mining industry has developed several methods for dealing with arsenic impurities, which includes precipitation with scorodite and pressure hydrometallurgical procedures (150 °C and 1380 kPa) for processing high concentration of arsenic while extracting copper in parallel. However, many procedures requires an enormous amount of energy or additional acidic chemicals to help selectively separate arensic-containing minerals in the ore mixtures.

Our solution: Mineral Binding

We, the UBC iGEM team, feel that separation and enrichment can be done in other ways that does not rely on energy or chemically-intensive methods. Our synbio solution involves the use of small surface heptapeptides and octapeptides, which have previously been demonstrated in M13 bacteriophage by Curtis et al. to selectively bind to chalcopyrite(3). Three peptides, labelled WSD-1 (TPTTYKV), WSD-2 (DSQKTNPS), and WSD-3 (DPIKHTSG), have been identified for binding. However, operating with bacteriophage is not feasible for large scale operations in mining as it is difficult to scale up titers to compensate for the smaller surface area available for binding in bacteriophage. Our idea is to operate these peptides in bacteria which have a larger surface area and are much more responsive to stimuli. These peptides must be accessible on the surface. Therefore, we have chosen Caulobacter crescentus as our chassis given that it contains a S-protein layer in which we can express our peptides. A developed kit for protein secretion and display of peptides on the cell surface S-layer in Caulobacter crescentus (via cloning to the S-layer gene sequence) can be found within Caulobacter S-layer Kits.


Figure 1. Curtis et al. screened a random heptapeptide library through a series of ‘biopanning’ assays for peptides that could be expressed on M13 bacteriophage and selectively bind to chalcopyrite. These peptide-containing bacteriophage have the potential to be utilized to separate chalcopyrite from enargite in ore slurries.


Figure 2. Our project adapts these chalcopyrite-binding peptides (WSD-1 (TPTTYKV), WSD-2 (DSQKTNPS), and WSD-3 (DPIKHTSG)) by cloning them into the S-protein layer of Caulobacter crescentus.

Our solution: Mineral Separation

As a proof of concept, we have chosen to express these peptides to selectively bind to chalcopyrite, which will then fall out of solution by aggregation using two interacting coiled-coil peptides expressed on the S-layer of Caulobacter crescentus. These coiled-coil peptides (K-coil: BBa_K1189012 , E-coil: BBa_K1189013, K-coil-His: BBa_K1189010, and E-coil-His: BBa_K1189011 )were constructed by iGEM Calgary 2013. In this model, Caulobacter A will contain chalcopyrite-binding peptides bound to chalcopyrite elusively while holding a K-coil interaction peptide. By addition of Caulobacter B which holds the E-coil interaction peptide, Caulobacter A will coalesce with Caulobacter B by E-coil and K-coil interactions, forming a greater mass that will selectively sink in a froth floation solution. Alternatively, we have considered floating bound chalcopyrite using gas vesicle proteins (BBa_I750016) developed by iGEM Groningen 2009.


Figure 3. K-coil and E-coil coiled-coil interaction peptides are expressed on the S-protein layer in two strains of Caulobacter crescentus.


Figure 4. Caulobacter A will contain chalcopyrite-binding peptides bound to chalcopyrite elusively, while holding a K-coil interaction peptide. Addition of Caulobacter B which holds the E-coil interaction peptide will create an interacting link with Caulobacter A, forming a mass that will fall out of solution

Binding Assay

To observe whether our peptides are capable of binding to chalcopyrite, we incubated 40 μm and 100 μm size chalcopyrite with our peptide expressing Caulobacter crescentus which were then passed through a 40 μm size cell strainer and subsequently washed with buffer and eluted with elution solutions. The elutions were then plated on YPD plate. Colonies grown on YPD plate suggest that the Caulobacter crescentus were bound to the chalcopyrite as they were present after the wash and elution steps.


Figure 5. Figure 5 Binding assay by incubating peptide-containing Caulobacter crescentus with chalcopyrite and subsequent wash and elution steps to remove unbound bacteria.

Results

Biomining precipitation assays

Before attempting to demonstrate binding with peptide inserted into the S-Layer, we tested binding with WT cells. After having observed binding, we then hypothesized that the fucose layer in Caulobacter was potentially involved in binding. Since there was a fucos knockout was available, we ere able to confirm fucos binding. Moreover, this observation demonstrated that a precipitation assay was feasible when mixing cells with Chalcopyrite.


Figure 6. Wild type and fucose knockout Caulobacter cells mixed with Chalcopyrite.

As a preliminary test, we observed increased Chalcopyrite precipitation with Caulobacter cells expressing a Chalcopyrite binding peptide in the S-layer. While non-quantitative, the data showed noticeable differences in precipitate settle when the peptide expressed in various locations in the S-layer. See the video below.


Figure 7.Various peptides from Curtis et al. are displayed in different positions in the S-layer of Caulobacter cells. The cells were mixed with calcopyrite and precipitate settling was observed.

Biomining protein assays

In an attempt to support the above observation, we incubated Caulobacter cells with Chalcopyrite and separated the Chalcopyrite by centrifugation. The supernatant and cells adhered to Chalcopyrite (eluted after several buffer washes) were assayed for protein concentration as a read out for biomass. For fucose containing cells and cells expressing Chalcopyrite binding peptides in the S-layer, we expected protein concentration to decrease in the supernatant and increase on Chalcopyrite as compared to controls.


Figure 8. Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in wild type Caulobacter cells.


Figure 9. Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in fucose knockout cells.

© 2014 UBC iGEM