Team:British Columbia/Results

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      <h1>Results</h1>
 
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       <h1>Biomining precipitation assays</h1>
       <h1>Biomining precipitation assays</h1>
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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 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.  
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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 Caulobacter was potentially involved in binding. Since there was a fucose knockout was available, we ere able to confirm fucose binding. Moreover, this observation demonstrated that a precipitation assay was feasible when mixing cells with chalcopyrite.  
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'''Figure 1.''' Wild type and fucos knockout ''Caulobacter'' cells mixed with Chalcopyrite.  
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'''Figure 1.''' Wild type and fucose knockout ''Caulobacter'' cells mixed with Chalcopyrite.  
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      <h1>Biomining protein assays</h1>
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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.
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'''Figure 3.''' Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in wild type ''Caulobacter'' cells.
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https://static.igem.org/mediawiki/2014/0/0b/Diagram_bioming2.png
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'''Figure 4.'''Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite.Peptides are expressed in fucose knockout cells.
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https://static.igem.org/mediawiki/2014/2/2b/Diagram4.jpg
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Latest revision as of 03:26, 18 October 2014

2014 UBC iGEM

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 fucose knockout was available, we ere able to confirm fucose binding. Moreover, this observation demonstrated that a precipitation assay was feasible when mixing cells with chalcopyrite.

Figure 1. 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 2. 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.

Diagram_biopan_v2_%281%29.png

Figure 3. Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in wild type Caulobacter cells.


Diagram_bioming2.png

Figure 4.Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite.Peptides are expressed in fucose knockout cells.

Diagram4.jpg

© 2014 UBC iGEM

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