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Team:StanfordBrownSpelman/Biodegradability
From 2014.igem.org
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<center><img src=https://static.igem.org/mediawiki/2014/e/e9/Ptet_with_tet_repressor.jpg></div> | <center><img src=https://static.igem.org/mediawiki/2014/e/e9/Ptet_with_tet_repressor.jpg></div> | ||
On top of testing the ptet promoter, we analyzed the impact a crash would have on our UAV by using a force plate and we found it unlikely that the impact of the crash would ever reach such a high pressure (see graph below). | On top of testing the ptet promoter, we analyzed the impact a crash would have on our UAV by using a force plate and we found it unlikely that the impact of the crash would ever reach such a high pressure (see graph below). | ||
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'''JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.''' | '''JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.''' | ||
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Since we know that the quorum sensing construct is functional and inducible with IPTG. We can now work towards replacing the GFP gene with the genes for our degradation enzymes, allowing us to control degradation by applying IPTG at different time points. | Since we know that the quorum sensing construct is functional and inducible with IPTG. We can now work towards replacing the GFP gene with the genes for our degradation enzymes, allowing us to control degradation by applying IPTG at different time points. | ||
| + | In conjunction with working on controlling the initiation of degradation, we simultaneously worked with two degradation enzymes, esterase and cellulase. Both of these genes are isolated from the organism Neisseria sicca; the esterase is designed to de-acetylate cellulose acetate (our building material), and the cellulase breaks down the leftover cellulose into glucose monomers. | ||
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| + | After successfully transforming the esterase gene into E. coli, and confirming via colony PCR, we grew up a large culture of transformed cells and used this to extract and purify the esterase protein. The protein gel confirmed that we had isolated the esterase protein, which is present near 43 kDa on the gel. | ||
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| + | INSERT GEL IMAGES FOR ESTERASE COLONY PCR AND PROTEIN GEL HERE. | ||
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| + | Once we had isolated our protein we were able to do functional assays with the esterase enzyme. By using a cellulose-binding dye that selectively binds to cellulose and not cellulose acetate, we were able to test whether or not the esterase enzyme was effective in de-acetylating commercial grade cellulose acetate. We soaked the cellulose acetate in the esterase protein at its optimal temperature of 30ºC and tested with the stain at multiple points. The results of our assay (shown below) demonstrate that over time the protein was working to degrade the cellulose acetate, as the blue stain intensity increased over time. | ||
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| + | [[File:celluloseacetateassay.jpg]] | ||
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| + | We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501). | ||
Revision as of 08:51, 13 October 2014
Biodegradability
Creating a biodegradable drone will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and microecosystems. Since we are using BCOAc for the construction of our drone, we plan on transforming E. coli with two genes obtained from Niesseria sicca, which synthesizes enzymes capable of degrading BCOAc; the first gene is an esterase which deacetylates the BCOAc, and the second is endo-1,4-beta-glucanase, a cellulase which speeds BC degradation. In order to trigger the onset and spread of degradation, we are investigating pressure-sensitive promoters (to simulate impact) and time-sensitive promoters linked to bacterial quorum sensing machinery. Quorum sensing allows the signal for degradation to spread to surrounding cells, enabling the complete breakdown of our biomaterials from a single point of impact.
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Results
Our goal for this project was not only to isolate biodegradation enzymes but also to control the release of these enzymes, so that our UAV would not degrade uncontrollably. In order to control the initiation of biodegradation, we first considered using a pressure sensor. This would allow the UAV to begin degrading after the impact of a crash. The 2008 Tokyo Tech iGem team found that the ptet promoter was pressure sensitive and increased its activity 3-fold after undergoing 30 MPa of pressure. We got their construct from the distribution kit and tested its functionality by monitoring GFP expression. We found that this promoter is always turned on even at very low pressures (The cells were bright green even with no pressure at all). When we introduced a repressor for the Ptet promoter, we found out that the repressor is too strong, and even at high pressures, the Ptet promoter will still be repressed. The photo and graphs below show our testing of the Ptet promoter in the presence and absence of the Tet repressor.
