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Team:StanfordBrownSpelman/Biodegradability
From 2014.igem.org
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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. | 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. | ||
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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). | ||
Revision as of 02:43, 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.
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).
'''JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.'''
We next tried to initiate degradation using a light sensor, which would activate degradation in the darkness, allowing our UAV to have a flight time of one day. We planned to do this using the construct from the UT Austin and UCSF 2004 Coliroid project. However, the strain of E. coli (EnvZ) that we needed to work with to use this construct was resistant to all 4 of the main antibiotics we had in our lab, making the bacteria difficult and expensive to work with.
Finally, we decided to use quorum sensing as a means of creating a time delay for initiation of degradation. Two previous constructs, BBa_I13202 and BBa_T9002, when combined, create a quorum sensing cascade used to initiate expression of GFP. We ligated these two parts together to create our novel part, BBa_K1499500, which we used for our assays. We found that, in lac deficient cells, the quorum sensing construct can be initiated by induction with IPTG. After this induction, GFP expression increases with time, implying that the construct is doing its job. Our data for this assay is shown below.
'''ARYO PLEASE INSERT THE FLOW CYTOMETRY DATA HERE SINCE I DON'T HAVE IT.'''
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.
