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Team:British Columbia/Results
From 2014.igem.org
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.
Figure 3. Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in wild type Caulobacter cells.
Figure 4.Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite.Peptides are expressed in fucose knockout cells.
