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− | This year’s iGEM team from the University of Toronto has been working on a cell-free method of detecting gold on a paper-based biosensor. Their goal is develop a cheaper, more efficient, and more | + | This year’s iGEM team from the University of Toronto has been working on a cell-free method of detecting gold on a paper-based biosensor. Their goal is develop a cheaper, more efficient, and more environmentally friendly method to detect gold for the mining industry. Their detector is GolS, a transcriptional activator that binds gold ions in solution. Following gold ion sequestration, the protein binds to a specific promoter called the P(golTS) promoter region and activates downstream transcription. |
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IgeM UofT sent us two plasmids of their design. One plasmid (called GolS hereon in, BBa_K2048001) contains the sequence of GolS under regulation of a TetOn promoter and a downstream P(golTS) promoter linked to a LacZɑ sequence, contained in pSB1C3. The other plasmid (called P118A hereon in, BBa_K2048002) contained the same DNA sequences, however the GolS sequence has a point mutation changing a proline to an alanine at position 118. Wild-type GolS in Salmonella enterica is known to bind to other metal ions, such as Cu, but the GolSP118A mutation abolishes specificity for Cu while only slightly decreasing specificity for Au ions1. | IgeM UofT sent us two plasmids of their design. One plasmid (called GolS hereon in, BBa_K2048001) contains the sequence of GolS under regulation of a TetOn promoter and a downstream P(golTS) promoter linked to a LacZɑ sequence, contained in pSB1C3. The other plasmid (called P118A hereon in, BBa_K2048002) contained the same DNA sequences, however the GolS sequence has a point mutation changing a proline to an alanine at position 118. Wild-type GolS in Salmonella enterica is known to bind to other metal ions, such as Cu, but the GolSP118A mutation abolishes specificity for Cu while only slightly decreasing specificity for Au ions1. | ||
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One of the goals of our project this year is to synthesize gold nanoparticles. When we found that iGEM UofT was working with a gold ion sensor, we became excited at the possibilities of collaboration. After some brainstorming, we came up with the idea of characterizing GolS in vivo to add onto iGEM UofT’s current data (mainly in vitro, but some in vivo). | One of the goals of our project this year is to synthesize gold nanoparticles. When we found that iGEM UofT was working with a gold ion sensor, we became excited at the possibilities of collaboration. After some brainstorming, we came up with the idea of characterizing GolS in vivo to add onto iGEM UofT’s current data (mainly in vitro, but some in vivo). | ||
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− | We were informed that UofT had performed an analysis of the ability for GolS and | + | We were informed that UofT had performed an analysis of the ability for GolS and GolS_P118A to react to copper ions in solution, using a CPRG assay, which yielded strange results. We thought we would test their constructs for copper and gold binding, as well as silver and aluminum, in order to offer more data to them for better characterization of GolS and GolSP118A. |
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− | We initially wanted to determine if our DH5ɑ E.coli would express GolS constitutively, so we set up an preliminary reaction of untransformed cells, cells transformed with GolS, and cells transformed with | + | We initially wanted to determine if our DH5ɑ E.coli would express GolS constitutively, so we set up an preliminary reaction of untransformed cells, cells transformed with GolS, and cells transformed with GolS_P118A, in liquid media with X-gal and different concentrations of gold. We were excited to see that, following day, the cells containing gold and X-gal had blue precipitate, while untransformed cells had no blue product. Due to time constraints and lack of material, we were unable to perform a CPRG or ONPG assay with our liquid cultures. However, iGEM UofT informed us that we could send them pictures of our liquid cultures since they were developing a mobile app that could relate color development to GolS activity. We decided then that our liquid X-gal assay would be acceptable. |
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− | We grew untransformed E. coli cells with no antibiotic, E. coli cells transformed with GolS grown in chloramphenicol, and E. coli cells transformed with | + | We grew untransformed E. coli cells with no antibiotic, E. coli cells transformed with GolS grown in chloramphenicol, and E. coli cells transformed with GolS_P118A grown with chloramphenicol to an OD of 3. We diluted our cells into a final volume of 600 µL of M9 media, having an OD of 1.5. We used Au (III) ions at 1 µM and 5 µM, Cu(II) at 1 µM and 10 µM, Ag (I) ions at 1 µM and 5 µM, and Al (III) ions at 1 µM and 5 µM. Pictures were taken at different time points after the addition of ions, shown in the pictures below. Reactions were incubated at 37℃, 300 RPM. M9 indicates just M9 buffer, M9X indicates M9 buffer with 4.8 µL of 20 mg/mL X-gal. All wells with indicated ions contained M9X. |
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<img style="padding:50px;" src="https://static.igem.org/mediawiki/2016/4/4f/T--Concordia--GolS_0hr.jpg" width="80%"><br> | <img style="padding:50px;" src="https://static.igem.org/mediawiki/2016/4/4f/T--Concordia--GolS_0hr.jpg" width="80%"><br> |
Latest revision as of 22:49, 19 October 2016