http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Pgodbole&year=&month=2014.igem.org - User contributions [en]2024-03-28T13:58:02ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/File:Sbs_igem_esteraseassayfinalfinal.jpgFile:Sbs igem esteraseassayfinalfinal.jpg2014-10-17T08:26:53Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem esteraseassayfinalfinal.jpg&quot;</p>
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<div>Larger esterase assay image</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseassayfinal.jpgFile:Sbs igem esteraseassayfinal.jpg2014-10-17T08:24:38Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem esteraseassayfinal.jpg&quot;: Reverted to version as of 08:24, 17 October 2014</p>
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<div>Edited final esterase assay</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseassayfinal.jpgFile:Sbs igem esteraseassayfinal.jpg2014-10-17T08:24:28Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem esteraseassayfinal.jpg&quot;</p>
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<div>Edited final esterase assay</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseassayfinal.jpgFile:Sbs igem esteraseassayfinal.jpg2014-10-17T08:24:01Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem esteraseassayfinal.jpg&quot;</p>
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<div>Edited final esterase assay</div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-17T07:54:29Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</center></h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. </h6><br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center><b>Figure 4. </b>A graph demonstrating the relationship between impact pressure and height of crash.</center></h6><br />
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In the graph above, we measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.<br />
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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.<h6><br />
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<h6>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. </h6><br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><b>Figure 5.</b> Flow-cytometer data & histogram plots from fluorescence testing for quorum sensing construct. </center></h6><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.<br />
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The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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In conjunction with working on controlling the initiation of degradation, we simultaneously worked with our two degradation enzymes, esterase and cellulase. 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.<br />
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<h6><center><b>Figure 6. </b>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene (1.5 kB).</center></h6><br />
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<h6><center><b>Figure 7. </b>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center><b>Figure 8. </b>Pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/File:Slide2.jpgFile:Slide2.jpg2014-10-17T07:53:16Z<p>Pgodbole: </p>
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<div></div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseproteingelfinal.jpgFile:Sbs igem esteraseproteingelfinal.jpg2014-10-17T07:51:15Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem esteraseproteingelfinal.jpg&quot;</p>
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<div>Updated protein gel - correct size</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseproteingel.jpgFile:Sbs igem esteraseproteingel.jpg2014-10-17T07:47:11Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem esteraseproteingel.jpg&quot;</p>
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<div>esterase protein gel labeled</div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-17T07:31:38Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</center></h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center><b>Figure 4. </b>A graph demonstrating the relationship between impact pressure and height of crash.</center></h6><br />
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In the graph above, we measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.<br />
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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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><center><b>Figure 5. </b>Flow-Cytometer Data & Graphs from fluorescence testing for quorum sensing construct. </center></h6><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.<br />
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The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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In conjunction with working on controlling the initiation of degradation, we simultaneously worked with our two degradation enzymes, esterase and cellulase. 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.<br />
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<h6><center><b>Figure 6. </b>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene (1.5 kB).</center></h6><br />
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<h6><center><b>Figure 7. </b>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center><b>Figure 8. </b>Pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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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. </h6><br />
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<div class="small-8 small-centered columns"><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center><b>Figure 4. </b>A graph demonstrating the relationship between impact pressure and height of crash.</center></h6><br />
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In the graph above, we measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.<br />
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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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
<img class="fitAll2" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 5. </b>Flow-Cytometer Data & Graphs from fluorescence testing for quorum sensing construct. </center></h6><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.<br />
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The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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In conjunction with working on controlling the initiation of degradation, we simultaneously worked with our two degradation enzymes, esterase and cellulase. 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.<br />
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<h6><center><b>Figure 6. </b>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene (1.5 kB).</center></h6><br />
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<h6><center><b>Figure 7. </b>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center><b>Figure 8. </b>Pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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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. </h6><br />
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<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center><b>Figure 4. </b>A graph demonstrating the relationship between impact pressure and height of crash.</center></h6><br />
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In the graph above, we measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.<br />
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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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
<img class="fitAll2" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/b/b1/IPTG-POSITIVE_TABLE.png"><br />
<img class="reduceSize" src="https://static.igem.org/mediawiki/2014/1/1d/NEGATIVE_CONTROL_GRAPH.JPG"><br />
<img class="reduceSize" src="https://static.igem.org/mediawiki/2014/1/18/GFP_IPTG_NEGATIVE.JPG"><br />
<img class="reduceSize" src="https://static.igem.org/mediawiki/2014/4/43/GFP_IPTG_POSITIVE.JPG"><br />
<h6><center><b>Figure 5. </b>Flow-Cytometer Data & Graphs from fluorescence testing for quorum sensing construct. </center></h6><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.<br />
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The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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In conjunction with working on controlling the initiation of degradation, we simultaneously worked with our two degradation enzymes, esterase and cellulase. 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.<br />
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<h6><center><b>Figure 6. </b>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene (1.5 kB).</center></h6><br />
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<h6><center><b>Figure 7. </b>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center><b>Figure 8. </b>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_forceplategraph.pngFile:Sbs igem forceplategraph.png2014-10-17T07:20:35Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem forceplategraph.png&quot;: Reverted to version as of 07:18, 17 October 2014</p>
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<div>Force plate pressure graph</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_forceplategraph.pngFile:Sbs igem forceplategraph.png2014-10-17T07:20:19Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem forceplategraph.png&quot;</p>
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<div>Force plate pressure graph</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_forceplategraph.pngFile:Sbs igem forceplategraph.png2014-10-17T07:19:48Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem forceplategraph.png&quot;</p>
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<div>Force plate pressure graph</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_forceplategraph.pngFile:Sbs igem forceplategraph.png2014-10-17T07:18:22Z<p>Pgodbole: uploaded a new version of &quot;File:Sbs igem forceplategraph.png&quot;</p>
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<div>Force plate pressure graph</div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-17T07:15:13Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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<div class="sub4"><h6><a href="http://docs.google.com/document/d/1-xtwn-iB2w-IVkquCmboShdY0sgYeFM80Uoc9zjVmXw/edit?usp=sharing" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="http://docs.google.com/document/d/1-xtwn-iB2w-IVkquCmboShdY0sgYeFM80Uoc9zjVmXw/edit?usp=sharing">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center><b>Figure 4. </b>A graph demonstrating the relationship between impact pressure and height of crash.</center></h6><br />
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In the graph above, we measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.<br />
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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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
<img class="fitAll2" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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<div class="sub4"><h6><a href="http://docs.google.com/document/d/1-xtwn-iB2w-IVkquCmboShdY0sgYeFM80Uoc9zjVmXw/edit?usp=sharing" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="http://docs.google.com/document/d/1-xtwn-iB2w-IVkquCmboShdY0sgYeFM80Uoc9zjVmXw/edit?usp=sharing">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center><b>Figure 3. </b>A graph demonstrating the relationship between impact pressure and height of crash.</center></h6><br />
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In the graph above, we measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.<br />
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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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-17T07:12:01Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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<div class="sub4"><h6><a href="http://docs.google.com/document/d/1-xtwn-iB2w-IVkquCmboShdY0sgYeFM80Uoc9zjVmXw/edit?usp=sharing" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="http://docs.google.com/document/d/1-xtwn-iB2w-IVkquCmboShdY0sgYeFM80Uoc9zjVmXw/edit?usp=sharing">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center><b>Figure 3. </b>A graph showing the relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-17T07:11:13Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the loop.</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>A graph showing the experimentally measured relationship between pressure applied and expression of GFP under control of the ptet promoter.</center></h6><br />
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The graph above shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells. When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process.<br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>Our ideal quorum sensing system works in a loop: quorum sensing will activate a promoter that will trigger the expression of the degradation enzymes, which will activate positive feedback on the entire loop.</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 2. </b>This image shows how we can ideally connect quorum sensing to the initiation of degradation. The system works in a loop: quorum sensing will activate a promoter that will trigger the expression of two degradation enzymes (esterase and cellulase). The production of these enzymes will lead act as signals for quorum sensing, and hence trigger even more expression of degradation enzymes</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<div class="small-8 small-centered columns"><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
<img class="fitAll2" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 2. </b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 1.</b>This image shows how we can ideally connect quorum sensing to the initiation of degradation. The system works in a loop: quorum sensing will activate a promoter that will trigger the expression of two degradation enzymes (esterase and cellulase). The production of these enzymes will lead act as signals for quorum sensing, and hence trigger even more expression of degradation enzymes</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<div class="small-8 small-centered columns"><center><img src=https://static.igem.org/mediawiki/2014/a/ac/Sbs_igem_forceplategraph.png><br><br />
<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
<img class="fitAll2" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/b/b1/IPTG-POSITIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1.</b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center><b>Figure 1.</b>This image shows how we can ideally connect quorum sensing to the initiation of degradation. The system works in a loop: quorum sensing will activate a promoter that will trigger the expression of two degradation enzymes (esterase and cellulase). The production of these enzymes will lead act as signals for quorum sensing, and hence trigger even more expression of degradation enzymes</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
<img class="fitAll2" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1.</b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center> This image shows how we can ideally connect quorum sensing to the initiation of degradation. The system works in a loop: quorum sensing will activate a promoter that will trigger the expression of two degradation enzymes (esterase and cellulase). The production of these enzymes will lead act as signals for quorum sensing, and hence trigger even more expression of degradation enzymes</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-17T07:04:12Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1.</b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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<h6>In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <h6><br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center> This image shows how we can ideally connect quorum sensing to the initiation of degradation. The system works in a loop: quorum sensing will activate a promoter that will trigger the expression of two degradation enzymes (esterase and cellulase). The production of these enzymes will lead act as signals for quorum sensing, and hence trigger even more expression of degradation enzymes</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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Creating a biodegradable unmanned aerial vehicle (UAV) will reduce collateral waste, lightening the footprint of unmanned science missions on planetary environments and micro-ecosystems. Since we are using Bacterial Cellulose Acetate (BCOAc) for the construction of our UAV, we investigated in nature to find organisms that can naturally break down Cellulose Acetate. We found out that Niesseria sicca uses cellulose acetate as its carbon source by producing two genes that break down cellulose acetate. The first gene is Cellulose Acetate Esterase which de-acetylates BCOAc, and the second is Endo-1,4-beta-glucanase, a cellulase that breaks down the glycosidic linkages in cellulose to produce glucose monomers.<br />
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<h6><center><b>Figure 1.</b> This image describes the degradation process, with the esterase enzyme de-acetylating cellulose acetate, and the cellulase enzyme breaking down the cellulose into glucose monomers.</center></h6><br />
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In order to trigger the onset of degradation our UAV at specific conditions, we investigated pressure-sensitive promoters for their abilities to stimulate degradation upon impact and time-sensitive promoters that will control the amount of time the UAV should fly before it degrades. After a series of experimentations, we chose to use quorum sensing as a time delay mechanism to control the length of time our UAV can fly before degradation enzymes are produced. <br />
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<h5><center>Approach & Methods</h5><br />
<h6>We extracted the esterase and cellulase gene sequences from Neisseria Sicca and transformed them in E.coli. Since the genes were synthesized with a T7 promoter, we induced the expression of the proteins by adding IPTG to the cell cultures. The Esterase was successfully purified and its presence was confirmed by observing a band at 43kDa when a protein gel was run. The activity of the Esterase enzyme was tested by soaking in blue stain reagent and testing for intensity of the stain. As for cellulase, we are currently working to express the cellulase in E.coli so as we can purify it and test its activity. </h6><br />
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<h6>In addition to working with the degradation enzymes, we created a mechanism where quorum sensing can cause a needed time delay for degradation so that our UAV does not degrade as soon as it starts to fly. Quorum sensing is a cell to cell communication mechanism, and here we use it to control when and how fast degradation should occur. <br />
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<h6><center> This image shows how we can ideally connect quorum sensing to the initiation of degradation. The system works in a loop: quorum sensing will activate a promoter that will trigger the expression of two degradation enzymes (esterase and cellulase). The production of these enzymes will lead act as signals for quorum sensing, and hence trigger even more expression of degradation enzymes</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T20:11:25Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h5><center>Approach & Methods</h5><br />
<h6>Methods here.</h6> </div></div><br />
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<h6><center><b>Figure 1.</b> Figure caption here.</center></h6><br />
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<h6>More methods here. <br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/ac/Sbs_igem_forceplategraph.png><br><br />
<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h6><center><b>Figure 1.</b> Figure caption here.</center></h6><br />
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<h6>More methods here. <br />
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<div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/ac/Sbs_igem_forceplategraph.png><br><br />
<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is an image of a gel electrophoresis done after running colony PCR with E. coli colonies transformed with our esterase gene. The esterase gene is approximately 1.5 kB, so this gel confirmed our transformation had worked, allowing us to proceed to protein purification.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseassayfinalfinal.jpgFile:Sbs igem esteraseassayfinalfinal.jpg2014-10-16T20:08:24Z<p>Pgodbole: Larger esterase assay image</p>
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<div>Larger esterase assay image</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esterasepcrfinal.jpgFile:Sbs igem esterasepcrfinal.jpg2014-10-16T20:08:01Z<p>Pgodbole: Labeled esterase pcr gel</p>
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<div>Labeled esterase pcr gel</div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_esteraseproteingelfinal.jpgFile:Sbs igem esteraseproteingelfinal.jpg2014-10-16T20:07:38Z<p>Pgodbole: Updated protein gel - correct size</p>
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<div>Updated protein gel - correct size</div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:51:12Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h6><center><b>Figure 1.</b> Figure caption here.</center></h6><br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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. In the future, we can better characterize our esterase protein by doing assays with more replicates, longer time periods, and varying amounts of protein.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:49:01Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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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.<br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick </a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:48:27Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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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.<br />
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<h6>More methods here. <br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBrick</a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:47:01Z<p>Pgodbole: </p>
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<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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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.<br />
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<h6>More methods here. <br />
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<h5><center>Results</h5><br />
<h6><br />
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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/ac/Sbs_igem_forceplategraph.png><br><br />
<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>Image description goes here.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biobricks">BioBrick</a> <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biobricks">BioBrick</a><a href="http://parts.igem.org/Part:BBa_K1499501">(BBa_K1499501)</a>.<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:40:49Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h6>Methods here.</h6> </div></div><br />
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<h6><center><b>Figure 1.</b> Figure caption here.</center></h6><br />
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<h6>More methods here. <br />
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<div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
<h6><br />
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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/ac/Sbs_igem_forceplategraph.png><br><br />
<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<br></br><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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<h6>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.<h6><br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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<h6>We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<h6><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:39:24Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h6>Methods here.</h6> </div></div><br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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 as high a pressure as 30 MPa, based on projections from the data we collected. (see graph below). <br />
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<h6><center>We measured the pressure upon impact of an object similar in shape and weight to our UAV. We took measurements by dropping or throwing the object from various heights and angles. Based on our data, it is projected that even from a drop height of 10m, the pressure upon impact would not even be 1 MPa, making it unlikely that any crash would ever cause 30 MPa of pressure.</center></h6><br />
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<h6>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.<h6><br />
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<h6>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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
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<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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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.<br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T19:35:46Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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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.<br />
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<h6>More methods here. <br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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). <br />
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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.<br />
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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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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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.<br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/File:Sbs_igem_forceplategraph.pngFile:Sbs igem forceplategraph.png2014-10-16T19:34:34Z<p>Pgodbole: Force plate pressure graph</p>
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<div>Force plate pressure graph</div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T09:24:14Z<p>Pgodbole: </p>
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<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
<h6 id="int"><br />
<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h6>Methods here.</h6> </div></div><br />
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<h6><center><b>Figure 1.</b> Figure caption here.</center></h6><br />
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<h6>More methods here. <br />
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<div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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<div id="subheader" class="small-8 small-centered columns"> <h6><br />
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). <br />
<br></br><br />
<br />
JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.'<br />
<br />
<br></br><br />
<br />
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.<br />
<br />
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. <br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h0><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
<br />
<br></br><br />
<br />
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. <br />
<br />
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.<br />
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<h6><center>Image description goes here.</center></h6><br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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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.<br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T09:23:37Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
</h6><br />
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<h6>Methods here.</h6> </div></div><br />
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<h6><center><b>Figure 1.</b> Figure caption here.</center></h6><br />
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<h6>More methods here. <br />
<br />
<div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
<h6><br />
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. </h6><br><br />
</div></div><br />
<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
</div><br />
<div class="row"><br />
<div id="subheader" class="small-8 small-centered columns"> <h6><br />
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). <br />
<br></br><br />
<br />
JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.'<br />
<br />
<br></br><br />
<br />
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.<br />
<br />
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. <br />
</div></div></h6><br />
<br />
<br></br><br />
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<h5><center>Quorum Sensing Data</h5> <br></br><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/f/fc/NEGATIVE_CONTROL_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/3/3f/IPTG-NEGATIVE_TABLE.png"><br />
<img class="fitAll" src="https://static.igem.org/mediawiki/2014/b/b1/IPTG-POSITIVE_TABLE.png"><br />
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<img class="reduceSize" src="https://static.igem.org/mediawiki/2014/1/1d/NEGATIVE_CONTROL_GRAPH.JPG"><br />
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<h6><center><b>Figure 1. Flow-Cytometer Data & Graphs: </b></h6><h0 class="introText"> The three graphs and tables above represent the flow-cytometer data obtained from three samples of lac-deficient E. coli cells. One of the samples was a negative control of LB-cultured E. coli cells (Negative Control), and the other two samples (IPTG-Positive and IPTG-Negative) had the GFP-quorum sensing construct. The IPTG-Positive sample had a ~6X increase in GFP expression over the background IPTG-Negative cells, showing our construct worked correctly.</center></h6><br />
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Flow-cytometry is a laser-based technology that has a wide-range of uses, including multiparametric cell counting and cell sorting. In this study, we utilized the cell counting ability of a flow cytometer in order to determine how many of our cells were expressing GFP after being induced with IPTG (a lac analog). The “X-Median” seen on the three tables above shows the fluorescence intensity of all of the cells that were counted that were not considered dead (Median). <br />
Each sample measured 50,000 cells (Seen under All Events - Count), and then counted all of the cells that were alive (Median - Count), where each sample had between 47,000 and 49,000 live cells. From the data, and as illustrated in the graphs above, we can gather that the fluorescence intensity (X-Median) of the LB only cells was approximately 63 arbitrary fluorescence units (AFU), the IPTG-negative cells was ~17,000 AFU, and the intensity of the IPTG-positive cells was ~107,000 AFU, meaning that the IPTG-positive quorum sensing cells increased fluorescence intensity over 6X the background intensity (IPTG-negative cells).<br />
These results indicate that our quorum sensing construct works and greatly increases GFP expression when activated through IPTG-induction. The IPTG-negative cells also expressed some non-negligible degree of GFP, which suggests that the construct could still be improved further. However, the successful proof of concept of our quorum sensing construct is incredibly promising and allows us to proceed toward the next step.<br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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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.<br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_AcetateTeam:StanfordBrownSpelman/Cellulose Acetate2014-10-16T08:55:01Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Acetate">Biomaterials</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA">BioBricks</a></h7></div><br />
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The body of the UAV is designed to consist a styrofoam-like filler consisting of fungal mycelia, coated with a cellulose acetate covering. The skin will be biologically waterproofed.<br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Material_Waterproofing" target="_blank">Material Waterproofing, </a><br />
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Biosensors can be linked to the cellulose acetate skin (see <br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Amberless_Hell_Cell" target="_blank">Amberless Hell Cell), </a><br />
through a biological cross-linker<br />
(see<br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker" target="_blank">Cellulose Cross Linker.) </a><br />
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Cellulose acetate is a biodegradable thermoplastic polymer used for a variety of industrial applications.[1] The monomer of cellulose acetate is glucose with one or more of its available hydroxyl groups replaced with acetyl groups. Cellulose acetate is industrially produced by treating cellulose, typically from wood or cotton, with acetic anhydride and sulfuric acid at high temperatures.[1] Our aim is to engineer bacterial cells to produce industrial-grade cellulose acetate biologically, allowing this plastic to be produced anywhere that bacterial colonies can be grown (i. e. in space). This material could then be used as a basis or coating for a biodegradable UAV. Many species of bacteria produce cellulose fibers; however, <i>Gluconacetobacter hansenii</i> has been identified as species producing the highest yield of cellulose. [2] Another strain of bacteria, the SBW25 isolate of the species <i>Pseudomonas fluorescens</i>, produces a biofilm containing cellulose fibers with a small degree of acetylation (.14 acetyl groups per glucose monomer).[3] Industrial-grade cellulose acetate must have at least 1.71 acetyl groups per glucose monomer. [4] In order to engineer a bacterium to efficiently produce cellulose acetate, our strategy is to transform <i>G. hansenii</i> with the four genes, wssF, wssG, wssH, and wssI, that have been identified [3] as being involved in cellulose acetylation in <i>P. fluorescens</i>, and to use directed evolution to further increase percent acetylation of the polymer.<br />
<br />
</p>In addition, we seek to create a streptavidin/cellulose-binding-domain fusion protein which will have the capacity to both cross-link bacterial cellulose acetate polymers (improving material properties) and allow the modular addition of cells (e.g. biosensors). This will be accomplished through the expression on the cells of a biotinylated membrane protein. This will allow biological sensors to be added directly to our cellulose acetate fibers, allowing bacterial sensors to be attached directly to the body of our UAV. <br />
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<h5><center>Approach & Methods</h5><br />
<h6>The first step in working towards producing our building material was to grow cultures of cellulose producing bacteria. After these cultures grew for 1-2 weeks, we removed the produced cellulose sheet from the culture to test various methods of drying. We experimented with drying the sheet in an oven, to produce an extreme thin layer of cellulose. We also wrapped fungal mycelium, which we intend to be the body of our UAV, with wet cellulose, and allowed the cellulose to dry on its own. This will provide the platform for us to alter the biomaterial for flight, by making it waterproof, for example.</h6> <br />
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<h6><center>Figure 1: Production of dried cellulose. a) A wet cellulose sheet, soaking in 50% alcohol solution. b) The cellulose was placed between two acrylic gel casters and left in a 75ºC oven for 2 days. c) A thin, dry cellulose sheet. d) Fungal mycelium wrapped in dry cellulose.</center></h6><br />
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<h6>One alteration we intend to make in order to produce a functional UAV is to draw circuits on the biomaterial to conduct electricity. In order to produce a biodegradable circuit, we worked with a company called <a href="http://agic.cc" target="_blank">AgiC</a>, which prints circuits out of silver nano particles (see our <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Building a UAV</a> page for a full circuit). By taking silver ink and painting it on to our bacterial cellulose, we were able to test the conductive capabilities of our building material.</h6> <br />
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<h6><center>Figure 2: Making cellulose electrically conductive. a) The silver ink used to paint cellulose. b) Silver nano particles painted onto cellulose covered mycelium. c) Positive Control: Aluminum foil has a resistance of 0.5 ohms. d) Negative Control: Unaltered cellulose has no resistance, and thus no conductivity. e) Experimental: Cellulose painted with silver nano particles has a resistance of 1.6 ohms. </center></h6><br />
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<br><br><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone"><b>Building a Biological UAV</b></a></center><br><br />
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Our team modeled, prototyped, and collaborated with Ecovative Design to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew! <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Check our more about the material, design, and construction aspects of our biomaterial project here.</a><br />
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<div class="sub5">1. Fischer S <i>et al.</i> (2008) Properties and Applications of Cellulose Acetate. <i>Macromol. Symp.</i> 262: 89-96. DOI: <a href="http://blogs.unpad.ac.id/evyerna/files/2010/11/ca.pdf" target="_blank">10.1002/masy.200850210</a></div><br />
<div class="sub5">2. Ross P <i>et al.</i> (1991) Cellulose Biosynthesis and Function in Bacteria. <i>Microbiological Reviews</i> 55: 35-58. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2030672" target="_blank">2030672 </a></div><br />
<div class="sub5">3. Spiers AJ <i>et al.</i> (2003) Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. <i>Molecular Microbiology</i> 50: 15-27. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/14507360" target="_blank">14507360 </a></div><br />
<div class="sub5">4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013. </div><br />
<div class="sub5">5. Hall PE <i>et al.</i> (1992) Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. <i>Plasmid</i> 28: 194-200. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1461938" target="_blank">1461938 </a></div><br />
<div class="sub5">6. Close TJ <i>et al.</i> (1984) Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. <i>Plasmid</i> 12: 111-118. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/6095350" target="_blank">6095350 </a></div><br />
<div class="sub5">7. Spiers AJ <i>et al.</i> (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in <i>Cellulose - Medical, Pharmaceutical, and Electronic Applications</i>. DOI: <a href="http://cdn.intechopen.com/pdfs-wm/45637.pdf" target="_blank">10.5772/53736</a></div><br />
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</body></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_AcetateTeam:StanfordBrownSpelman/Cellulose Acetate2014-10-16T08:50:22Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Acetate">Biomaterials</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA">BioBricks</a></h7></div><br />
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The body of the UAV is designed to consist a styrofoam-like filler consisting of fungal mycelia, coated with a cellulose acetate covering. The skin will be biologically waterproofed.<br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Material_Waterproofing" target="_blank">Material Waterproofing, </a><br />
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Biosensors can be linked to the cellulose acetate skin (see <br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Amberless_Hell_Cell" target="_blank">Amberless Hell Cell), </a><br />
through a biological cross-linker<br />
(see<br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker" target="_blank">Cellulose Cross Linker.) </a><br />
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Cellulose acetate is a biodegradable thermoplastic polymer used for a variety of industrial applications.[1] The monomer of cellulose acetate is glucose with one or more of its available hydroxyl groups replaced with acetyl groups. Cellulose acetate is industrially produced by treating cellulose, typically from wood or cotton, with acetic anhydride and sulfuric acid at high temperatures.[1] Our aim is to engineer bacterial cells to produce industrial-grade cellulose acetate biologically, allowing this plastic to be produced anywhere that bacterial colonies can be grown (i. e. in space). This material could then be used as a basis or coating for a biodegradable UAV. Many species of bacteria produce cellulose fibers; however, <i>Gluconacetobacter hansenii</i> has been identified as species producing the highest yield of cellulose. [2] Another strain of bacteria, the SBW25 isolate of the species <i>Pseudomonas fluorescens</i>, produces a biofilm containing cellulose fibers with a small degree of acetylation (.14 acetyl groups per glucose monomer).[3] Industrial-grade cellulose acetate must have at least 1.71 acetyl groups per glucose monomer. [4] In order to engineer a bacterium to efficiently produce cellulose acetate, our strategy is to transform <i>G. hansenii</i> with the four genes, wssF, wssG, wssH, and wssI, that have been identified [3] as being involved in cellulose acetylation in <i>P. fluorescens</i>, and to use directed evolution to further increase percent acetylation of the polymer.<br />
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</p>In addition, we seek to create a streptavidin/cellulose-binding-domain fusion protein which will have the capacity to both cross-link bacterial cellulose acetate polymers (improving material properties) and allow the modular addition of cells (e.g. biosensors). This will be accomplished through the expression on the cells of a biotinylated membrane protein. This will allow biological sensors to be added directly to our cellulose acetate fibers, allowing bacterial sensors to be attached directly to the body of our UAV. <br />
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<h5><center>Approach & Methods</h5><br />
<h6>The first step in working towards producing our building material was to grow cultures of cellulose producing bacteria. After these cultures grew for 1-2 weeks, we removed the produced cellulose sheet from the culture to test various methods of drying. We experimented with drying the sheet in an oven, to produce an extreme thin layer of cellulose. We also wrapped fungal mycelium, which we intend to be the body of our UAV, with wet cellulose, and allowed the cellulose to dry on its own. This will provide the platform for us to alter the biomaterial for flight, by making it waterproof, for example.</h6> <br />
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<h6><center>Figure 1: Production of dried cellulose. a) A wet cellulose sheet, soaking in 50% alcohol solution. b) The cellulose was placed between two acrylic gel casters and left in a 75ºC oven for 2 days. c) A thin, dry cellulose sheet. d) Fungal mycelium wrapped in dry cellulose.</center></h6><br />
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<h6>One alteration we intend to make in order to produce a functional UAV is to draw circuits on the biomaterial to conduct electricity. In order to produce a biodegradable circuit, we worked with a company called AgiC, which prints circuits out of silver nano particles (see our Building a UAV page for a full circuit). By taking silver ink and painting it on to our bacterial cellulose, we were able to test the conductive capabilities of our building material.</h6> <br />
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<h6><center>Figure 2: Making cellulose electrically conductive. a) The silver ink used to paint cellulose. b) Silver nano particles painted onto cellulose covered mycelium. c) Positive Control: Aluminum foil has a resistance of 0.5 ohms. d) Negative Control: Unaltered cellulose has no resistance, and thus no conductivity. e) Experimental: Cellulose painted with silver nano particles has a resistance of 1.6 ohms. </center></h6><br />
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<h6>More methods here. <br />
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<div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div><br />
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<h5><center>Results</h5><br />
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Results go here.<br />
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<br><br><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone"><b>Building a Biological UAV</b></a></center><br><br />
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Our team modeled, prototyped, and collaborated with Ecovative Design to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew! <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Check our more about the material, design, and construction aspects of our biomaterial project here.</a><br />
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<h5><center>References</h5><br />
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<div class="sub5">1. Fischer S <i>et al.</i> (2008) Properties and Applications of Cellulose Acetate. <i>Macromol. Symp.</i> 262: 89-96. DOI: <a href="http://blogs.unpad.ac.id/evyerna/files/2010/11/ca.pdf" target="_blank">10.1002/masy.200850210</a></div><br />
<div class="sub5">2. Ross P <i>et al.</i> (1991) Cellulose Biosynthesis and Function in Bacteria. <i>Microbiological Reviews</i> 55: 35-58. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2030672" target="_blank">2030672 </a></div><br />
<div class="sub5">3. Spiers AJ <i>et al.</i> (2003) Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. <i>Molecular Microbiology</i> 50: 15-27. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/14507360" target="_blank">14507360 </a></div><br />
<div class="sub5">4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013. </div><br />
<div class="sub5">5. Hall PE <i>et al.</i> (1992) Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. <i>Plasmid</i> 28: 194-200. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1461938" target="_blank">1461938 </a></div><br />
<div class="sub5">6. Close TJ <i>et al.</i> (1984) Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. <i>Plasmid</i> 12: 111-118. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/6095350" target="_blank">6095350 </a></div><br />
<div class="sub5">7. Spiers AJ <i>et al.</i> (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in <i>Cellulose - Medical, Pharmaceutical, and Electronic Applications</i>. DOI: <a href="http://cdn.intechopen.com/pdfs-wm/45637.pdf" target="_blank">10.5772/53736</a></div><br />
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</body></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/Building_The_DroneTeam:StanfordBrownSpelman/Building The Drone2014-10-16T08:37:40Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Prototyping a Biological UAV</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="data">Materials &amp; Designs</a> ● <a href="#" id="methods">Parts &amp; Downloads</a> ● <a href="#" id="links">References</a></h7></div><br />
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Our team modeled, prototyped, and collaborated with <a href="http://www.ecovativedesign.com">Ecovative Design</a> to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew!<br />
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<li><img src="https://static.igem.org/mediawiki/2014/f/ff/SBSiGEM2014BTD1.jpg"></li><h6>Harvesting a pure bacterial cellulose sheet.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/6/66/SBSiGEM2014BTD2.JPG"></li><h6>Experimenting with cellulose material shape.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/e/e6/SBSiGEM2014BTD4.JPG"></li><h6>Layering cellulose to create thicker leather, see here at the back of the hood.</h6><br />
<li><img src="https://static.igem.org/mediawiki/2014/d/d3/SBSiGEM2014BTD5.jpg"></li><h6>Mycelium drone chassis, modeled and 3D-designed by our team, produced by Ecocative.</h6><br />
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<li><img src="https://static.igem.org/mediawiki/2014/c/c5/SBSiGEM2014BTD8.JPG"></li><h6>Spreading a cellulose sheet out to dry.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/5/57/SBSiGEM2014_Cellulose_Circuit.jpg"></li><h6>Our team colored with <a href="http://agic.cc" target="_blank">AgiC</a> to print circuits onto our cellulose-based biomaterials in order to prototype how fully biodegradable circuitry might function on a biological UAV. See our <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Acetate">Biomaterials</a> page for details on the conductivity of this circuitry.</h6><br />
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<h5><center>Starting Small, Ending Big</h5><br />
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We began by experimenting with producing cellulose in sheets and cellulose acetate non-biologically. Seeing that primarily cellulose materials are extremely strong and tough, but tear easily and becomes soggy when wet, we sought to increase the durability of the cellulose by grinding it into pieces to create a cellulose paste (that became spreadable into sheets like paper made from wood pulp) and stretching and twisting it into ropes to add strength. A few of our material samples follow: </h6><br />
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<div class="small-7 small-centered columns"><br><center><img src="https://static.igem.org/mediawiki/2014/3/38/SBSiGEM2014_Cellulose_Screw.jpg"><br><br />
<h6><center>A spiral rope made by waving together several cellulose sheets and dehydrating them.</center></h6><br />
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<h6><center>A piece of cellulose leather generated by laying multiple sheets of cellulose together in perpendicular orientations.</center></h6><br />
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<div id="subheader" class="small-8 small-centered columns"><h6><br />
While experimenting with cellulose-based materials, we also explored traditional starch bioplastics to compare material functionality. Here is an example of a starch bioplastic that we produced synthetically in the lab:</h6><br />
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<h6><center>Common starch bioplastic, which is more voluminous but less strong than bacterial cellulose. Starch bioplastics, like bacterial cellulose materials, suffer disintegration when wet.</center></h6><br />
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Realizing that cellulose acetate is tough but thin, our team was in need of a building material that was durable and lightweight. So, we reached out to Evocative Design, a pioneering fungal-mycelium-based biomaterial company, to prototype a mycelium form that could serve as the chassis of our vehicle. Evocative shipped us mycelium samples (pictured below), that we skinned in bacterial cellulose.</h6><br />
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<h6><center>6" by 6" by 1" sample of Ecovative's lightweight mycelium-based biomaterial.</center></h6><br />
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<h6><center>A piece of fungal mycelium skinned in bacterial cellulose.</center></h6><br />
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Thanks to Evocative, we were able to construct a prototype biological unmanned aerial vehicle!<br><br>But we didn't stop there. Our team was enthusiastic about drone design and so we developed concept UAV designs meant to inspire future scientists and designers to think outside the box about how a future, partially living vehicle might look. Pseudo-natural and pseudo-industrial, our drone design references the traditional biological architecture of birds while embracing industrial additive manufacturability.<br><br>All 3D printable files for this concept drone are available in the downloads section. Images of our work follow:</h6><br />
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We succeeded in producing multiple viable chassis designs for mycelium UAV concept prototypes. You can download our basic chassis designs here. If you would like to receive a copy of the designs for our more involved, final UAV concept (pictured above), then please reach out to us! We would love to share our work! In the meantime, download and check out our other models below:<br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/6/66/Mycelium_Drone_1.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/6/66/Mycelium_Drone_1.SLDPRT.zip">Mycelium_Drone_1.SLDPRT: Download a 3D printable STL file for a secondary drone chassis mold concept. Modeled in house and ready for printing, molding, and biomaterial casting.</a></div><br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/c/cd/Mycelium_Drone_2.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/c/cd/Mycelium_Drone_2.SLDPRT.zip">Mycelium_Drone_2.SLDPRT: Download a 3D printable STL file for a third drone chassis. Shaped like a paper airplane, this mold can be used to make testable mycelium drone prototypes.</a></div><br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/0/02/Mycelium_Quad_1.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/0/02/Mycelium_Quad_1.SLDPRT.zip">Mycelium_Quad_1.SLDPRT: Download a 3D printable STL file for our mycelium quad chassic (pictured at top of page), which can accept four motors and serve as the foundation for a DIY biomaterial drone prototype.</a></div><br />
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Interested in what we've been working on and want to find more relevant information? Check out some of the following sites, companies, and people who either aided us in our production of biomaterials or collaborated with us in working to produce a viable biological unmanned aerial vehicle.<br />
<div class="sub5"><a href="http://www.ecovativedesign.com">● Ecovative — Fungal-mycelium-based biomaterial production company</a></div><br />
<div class="sub5"><a href="http://issuu.com/miriamribul/docs/miriam_ribul_recipes_for_material_a">● Miriam Ribul's <i>Recipes for Material Activism</i> documents bioplastic production with household ingredients</a></div><br />
<div class="sub5"><a href="http://cookingobjects.com/images">● Cooking Objects — Understanding objects, objectivity, and our relationship with sustainably produced, biodegradable household objects</a></div><br />
<div class="sub5"><a href="http://www.nasa.gov/centers/ames/spaceshop/about/index.html#.VD8Hq4f9pbw">● The NASA Space shop, providing resources and tools for rapid prototyping at the NASA Ames Research Center</a></div><br />
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Here is a collection of speculative work that stimulated our team to think about synthetic biology, the future, and the role of personal unmanned aerial vehicles or biological devices in an evolving world of DIY craft, government surveillance, and channelled creativity.<br />
<div class="sub5"><a href="http://www.fabrica.it/projects/drone">● Frabrica — <i>Drone</i>, speculative fictions in the age of the drone</a></div><br />
<div class="sub5"><a href="http://www.dronesurvivalguide.org">● Drone Survival Guide — a poster series highlighting the uneasy relationship between the public and drones</a></div><br />
<div class="sub5"><a href="http://www.huffingtonpost.com/2013/04/03/anti-drone-hoodie-adam-harvey-surveillance_n_3007064.html#slide=2295806">● Anti-Drone hoodie</a></div><br />
<div class="sub5"><a href="http://diydrones.com">● DIY Drones — a growing online community of makers committed to building unmanned aircraft</a></div><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/Building_The_DroneTeam:StanfordBrownSpelman/Building The Drone2014-10-16T08:36:44Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Prototyping a Biological UAV</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="data">Materials &amp; Designs</a> ● <a href="#" id="methods">Parts &amp; Downloads</a> ● <a href="#" id="links">References</a></h7></div><br />
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Our team modeled, prototyped, and collaborated with <a href="http://www.ecovativedesign.com">Ecovative Design</a> to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew!<br />
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<li><img src="https://static.igem.org/mediawiki/2014/6/66/SBSiGEM2014BTD2.JPG"></li><h6>Experimenting with cellulose material shape.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/e/e6/SBSiGEM2014BTD4.JPG"></li><h6>Layering cellulose to create thicker leather, see here at the back of the hood.</h6><br />
<li><img src="https://static.igem.org/mediawiki/2014/d/d3/SBSiGEM2014BTD5.jpg"></li><h6>Mycelium drone chassis, modeled and 3D-designed by our team, produced by Ecocative.</h6><br />
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<li><img src="https://static.igem.org/mediawiki/2014/c/c5/SBSiGEM2014BTD8.JPG"></li><h6>Spreading a cellulose sheet out to dry.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/5/57/SBSiGEM2014_Cellulose_Circuit.jpg"></li><h6>Our team colored with <a href="http://agic.cc" target="_blank">AgiC</a> to print circuits onto our cellulose-based biomaterials in order to prototype how fully biodegradable circuitry might function on a biological UAV. See our <a href="http://agic.cc" target="_blank">AgiC</a> page for details on the conductivity of this circuitry.</h6><br />
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<h5><center>Starting Small, Ending Big</h5><br />
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We began by experimenting with producing cellulose in sheets and cellulose acetate non-biologically. Seeing that primarily cellulose materials are extremely strong and tough, but tear easily and becomes soggy when wet, we sought to increase the durability of the cellulose by grinding it into pieces to create a cellulose paste (that became spreadable into sheets like paper made from wood pulp) and stretching and twisting it into ropes to add strength. A few of our material samples follow: </h6><br />
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<h6><center>A spiral rope made by waving together several cellulose sheets and dehydrating them.</center></h6><br />
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<h6><center>A piece of cellulose leather generated by laying multiple sheets of cellulose together in perpendicular orientations.</center></h6><br />
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While experimenting with cellulose-based materials, we also explored traditional starch bioplastics to compare material functionality. Here is an example of a starch bioplastic that we produced synthetically in the lab:</h6><br />
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<h6><center>Common starch bioplastic, which is more voluminous but less strong than bacterial cellulose. Starch bioplastics, like bacterial cellulose materials, suffer disintegration when wet.</center></h6><br />
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Realizing that cellulose acetate is tough but thin, our team was in need of a building material that was durable and lightweight. So, we reached out to Evocative Design, a pioneering fungal-mycelium-based biomaterial company, to prototype a mycelium form that could serve as the chassis of our vehicle. Evocative shipped us mycelium samples (pictured below), that we skinned in bacterial cellulose.</h6><br />
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<div class="small-7 small-centered columns"><br><center><img src="https://static.igem.org/mediawiki/2014/5/57/SBSiGEM2014_Mycelium_Sample2.jpg"><br><br />
<h6><center>6" by 6" by 1" sample of Ecovative's lightweight mycelium-based biomaterial.</center></h6><br />
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<div class="small-7 small-centered columns"><br><center><img src="https://static.igem.org/mediawiki/2014/1/1b/SBSiGEM2014_Skinned_Mycelium.jpg"><br><br />
<h6><center>A piece of fungal mycelium skinned in bacterial cellulose.</center></h6><br />
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Thanks to Evocative, we were able to construct a prototype biological unmanned aerial vehicle!<br><br>But we didn't stop there. Our team was enthusiastic about drone design and so we developed concept UAV designs meant to inspire future scientists and designers to think outside the box about how a future, partially living vehicle might look. Pseudo-natural and pseudo-industrial, our drone design references the traditional biological architecture of birds while embracing industrial additive manufacturability.<br><br>All 3D printable files for this concept drone are available in the downloads section. Images of our work follow:</h6><br />
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<h6><center>Concept UAV Design</center></h6><br />
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<h6><center>Biological UAV Concept, Exploded View</center></h6><br />
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We succeeded in producing multiple viable chassis designs for mycelium UAV concept prototypes. You can download our basic chassis designs here. If you would like to receive a copy of the designs for our more involved, final UAV concept (pictured above), then please reach out to us! We would love to share our work! In the meantime, download and check out our other models below:<br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/6/66/Mycelium_Drone_1.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/6/66/Mycelium_Drone_1.SLDPRT.zip">Mycelium_Drone_1.SLDPRT: Download a 3D printable STL file for a secondary drone chassis mold concept. Modeled in house and ready for printing, molding, and biomaterial casting.</a></div><br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/c/cd/Mycelium_Drone_2.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/c/cd/Mycelium_Drone_2.SLDPRT.zip">Mycelium_Drone_2.SLDPRT: Download a 3D printable STL file for a third drone chassis. Shaped like a paper airplane, this mold can be used to make testable mycelium drone prototypes.</a></div><br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/0/02/Mycelium_Quad_1.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/0/02/Mycelium_Quad_1.SLDPRT.zip">Mycelium_Quad_1.SLDPRT: Download a 3D printable STL file for our mycelium quad chassic (pictured at top of page), which can accept four motors and serve as the foundation for a DIY biomaterial drone prototype.</a></div><br />
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Interested in what we've been working on and want to find more relevant information? Check out some of the following sites, companies, and people who either aided us in our production of biomaterials or collaborated with us in working to produce a viable biological unmanned aerial vehicle.<br />
<div class="sub5"><a href="http://www.ecovativedesign.com">● Ecovative — Fungal-mycelium-based biomaterial production company</a></div><br />
<div class="sub5"><a href="http://issuu.com/miriamribul/docs/miriam_ribul_recipes_for_material_a">● Miriam Ribul's <i>Recipes for Material Activism</i> documents bioplastic production with household ingredients</a></div><br />
<div class="sub5"><a href="http://cookingobjects.com/images">● Cooking Objects — Understanding objects, objectivity, and our relationship with sustainably produced, biodegradable household objects</a></div><br />
<div class="sub5"><a href="http://www.nasa.gov/centers/ames/spaceshop/about/index.html#.VD8Hq4f9pbw">● The NASA Space shop, providing resources and tools for rapid prototyping at the NASA Ames Research Center</a></div><br />
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Here is a collection of speculative work that stimulated our team to think about synthetic biology, the future, and the role of personal unmanned aerial vehicles or biological devices in an evolving world of DIY craft, government surveillance, and channelled creativity.<br />
<div class="sub5"><a href="http://www.fabrica.it/projects/drone">● Frabrica — <i>Drone</i>, speculative fictions in the age of the drone</a></div><br />
<div class="sub5"><a href="http://www.dronesurvivalguide.org">● Drone Survival Guide — a poster series highlighting the uneasy relationship between the public and drones</a></div><br />
<div class="sub5"><a href="http://www.huffingtonpost.com/2013/04/03/anti-drone-hoodie-adam-harvey-surveillance_n_3007064.html#slide=2295806">● Anti-Drone hoodie</a></div><br />
<div class="sub5"><a href="http://diydrones.com">● DIY Drones — a growing online community of makers committed to building unmanned aircraft</a></div><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/Building_The_DroneTeam:StanfordBrownSpelman/Building The Drone2014-10-16T08:35:36Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Prototyping a Biological UAV</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="data">Materials &amp; Designs</a> ● <a href="#" id="methods">Parts &amp; Downloads</a> ● <a href="#" id="links">References</a></h7></div><br />
<h6 id="int"><br />
Our team modeled, prototyped, and collaborated with <a href="http://www.ecovativedesign.com">Ecovative Design</a> to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew!<br />
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<li><img src="https://static.igem.org/mediawiki/2014/f/ff/SBSiGEM2014BTD1.jpg"></li><h6>Harvesting a pure bacterial cellulose sheet.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/6/66/SBSiGEM2014BTD2.JPG"></li><h6>Experimenting with cellulose material shape.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/e/e6/SBSiGEM2014BTD4.JPG"></li><h6>Layering cellulose to create thicker leather, see here at the back of the hood.</h6><br />
<li><img src="https://static.igem.org/mediawiki/2014/d/d3/SBSiGEM2014BTD5.jpg"></li><h6>Mycelium drone chassis, modeled and 3D-designed by our team, produced by Ecocative.</h6><br />
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<li><img src="https://static.igem.org/mediawiki/2014/c/c5/SBSiGEM2014BTD8.JPG"></li><h6>Spreading a cellulose sheet out to dry.</h6><br><br />
<li><img src="https://static.igem.org/mediawiki/2014/5/57/SBSiGEM2014_Cellulose_Circuit.jpg"></li><h6>Our team colored with <a href="http://agic.cc" target="_blank">AgiC</a> to print circuits onto our cellulose-based biomaterials in order to prototype how fully biodegradable circuitry might function on a biological UAV. See our biomaterials page for details on the conductivity of this circuitry.</h6><br />
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<h5><center>Starting Small, Ending Big</h5><br />
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We began by experimenting with producing cellulose in sheets and cellulose acetate non-biologically. Seeing that primarily cellulose materials are extremely strong and tough, but tear easily and becomes soggy when wet, we sought to increase the durability of the cellulose by grinding it into pieces to create a cellulose paste (that became spreadable into sheets like paper made from wood pulp) and stretching and twisting it into ropes to add strength. A few of our material samples follow: </h6><br />
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<h6><center>A spiral rope made by waving together several cellulose sheets and dehydrating them.</center></h6><br />
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<h6><center>A piece of cellulose leather generated by laying multiple sheets of cellulose together in perpendicular orientations.</center></h6><br />
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While experimenting with cellulose-based materials, we also explored traditional starch bioplastics to compare material functionality. Here is an example of a starch bioplastic that we produced synthetically in the lab:</h6><br />
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<h6><center>Common starch bioplastic, which is more voluminous but less strong than bacterial cellulose. Starch bioplastics, like bacterial cellulose materials, suffer disintegration when wet.</center></h6><br />
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Realizing that cellulose acetate is tough but thin, our team was in need of a building material that was durable and lightweight. So, we reached out to Evocative Design, a pioneering fungal-mycelium-based biomaterial company, to prototype a mycelium form that could serve as the chassis of our vehicle. Evocative shipped us mycelium samples (pictured below), that we skinned in bacterial cellulose.</h6><br />
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<div class="small-7 small-centered columns"><br><center><img src="https://static.igem.org/mediawiki/2014/5/57/SBSiGEM2014_Mycelium_Sample2.jpg"><br><br />
<h6><center>6" by 6" by 1" sample of Ecovative's lightweight mycelium-based biomaterial.</center></h6><br />
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<div class="small-7 small-centered columns"><br><center><img src="https://static.igem.org/mediawiki/2014/1/1b/SBSiGEM2014_Skinned_Mycelium.jpg"><br><br />
<h6><center>A piece of fungal mycelium skinned in bacterial cellulose.</center></h6><br />
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Thanks to Evocative, we were able to construct a prototype biological unmanned aerial vehicle!<br><br>But we didn't stop there. Our team was enthusiastic about drone design and so we developed concept UAV designs meant to inspire future scientists and designers to think outside the box about how a future, partially living vehicle might look. Pseudo-natural and pseudo-industrial, our drone design references the traditional biological architecture of birds while embracing industrial additive manufacturability.<br><br>All 3D printable files for this concept drone are available in the downloads section. Images of our work follow:</h6><br />
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<h6><center>Concept UAV Design</center></h6><br />
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<h6><center>Biological UAV Concept, Exploded View</center></h6><br />
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<h5><center>Designed Parts &amp; Downloads</h5><br />
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We succeeded in producing multiple viable chassis designs for mycelium UAV concept prototypes. You can download our basic chassis designs here. If you would like to receive a copy of the designs for our more involved, final UAV concept (pictured above), then please reach out to us! We would love to share our work! In the meantime, download and check out our other models below:<br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/6/66/Mycelium_Drone_1.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/6/66/Mycelium_Drone_1.SLDPRT.zip">Mycelium_Drone_1.SLDPRT: Download a 3D printable STL file for a secondary drone chassis mold concept. Modeled in house and ready for printing, molding, and biomaterial casting.</a></div><br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/c/cd/Mycelium_Drone_2.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/c/cd/Mycelium_Drone_2.SLDPRT.zip">Mycelium_Drone_2.SLDPRT: Download a 3D printable STL file for a third drone chassis. Shaped like a paper airplane, this mold can be used to make testable mycelium drone prototypes.</a></div><br />
<div class="sub4"><a href="https://static.igem.org/mediawiki/2014/0/02/Mycelium_Quad_1.SLDPRT.zip"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/0/02/Mycelium_Quad_1.SLDPRT.zip">Mycelium_Quad_1.SLDPRT: Download a 3D printable STL file for our mycelium quad chassic (pictured at top of page), which can accept four motors and serve as the foundation for a DIY biomaterial drone prototype.</a></div><br />
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Interested in what we've been working on and want to find more relevant information? Check out some of the following sites, companies, and people who either aided us in our production of biomaterials or collaborated with us in working to produce a viable biological unmanned aerial vehicle.<br />
<div class="sub5"><a href="http://www.ecovativedesign.com">● Ecovative — Fungal-mycelium-based biomaterial production company</a></div><br />
<div class="sub5"><a href="http://issuu.com/miriamribul/docs/miriam_ribul_recipes_for_material_a">● Miriam Ribul's <i>Recipes for Material Activism</i> documents bioplastic production with household ingredients</a></div><br />
<div class="sub5"><a href="http://cookingobjects.com/images">● Cooking Objects — Understanding objects, objectivity, and our relationship with sustainably produced, biodegradable household objects</a></div><br />
<div class="sub5"><a href="http://www.nasa.gov/centers/ames/spaceshop/about/index.html#.VD8Hq4f9pbw">● The NASA Space shop, providing resources and tools for rapid prototyping at the NASA Ames Research Center</a></div><br />
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<h5><center>Drone Futures</h5><br />
<h6><br />
Here is a collection of speculative work that stimulated our team to think about synthetic biology, the future, and the role of personal unmanned aerial vehicles or biological devices in an evolving world of DIY craft, government surveillance, and channelled creativity.<br />
<div class="sub5"><a href="http://www.fabrica.it/projects/drone">● Frabrica — <i>Drone</i>, speculative fictions in the age of the drone</a></div><br />
<div class="sub5"><a href="http://www.dronesurvivalguide.org">● Drone Survival Guide — a poster series highlighting the uneasy relationship between the public and drones</a></div><br />
<div class="sub5"><a href="http://www.huffingtonpost.com/2013/04/03/anti-drone-hoodie-adam-harvey-surveillance_n_3007064.html#slide=2295806">● Anti-Drone hoodie</a></div><br />
<div class="sub5"><a href="http://diydrones.com">● DIY Drones — a growing online community of makers committed to building unmanned aircraft</a></div><br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_AcetateTeam:StanfordBrownSpelman/Cellulose Acetate2014-10-16T08:25:24Z<p>Pgodbole: </p>
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<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA">BioBricks</a></h7></div><br />
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The body of the UAV is designed to consist a styrofoam-like filler consisting of fungal mycelia, coated with a cellulose acetate covering. The skin will be biologically waterproofed.<br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Material_Waterproofing" target="_blank">Material Waterproofing, </a><br />
<br />
Biosensors can be linked to the cellulose acetate skin (see <br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Amberless_Hell_Cell" target="_blank">Amberless Hell Cell), </a><br />
through a biological cross-linker<br />
(see<br />
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker" target="_blank">Cellulose Cross Linker.) </a><br />
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Cellulose acetate is a biodegradable thermoplastic polymer used for a variety of industrial applications.[1] The monomer of cellulose acetate is glucose with one or more of its available hydroxyl groups replaced with acetyl groups. Cellulose acetate is industrially produced by treating cellulose, typically from wood or cotton, with acetic anhydride and sulfuric acid at high temperatures.[1] Our aim is to engineer bacterial cells to produce industrial-grade cellulose acetate biologically, allowing this plastic to be produced anywhere that bacterial colonies can be grown (i. e. in space). This material could then be used as a basis or coating for a biodegradable UAV. Many species of bacteria produce cellulose fibers; however, <i>Gluconacetobacter hansenii</i> has been identified as species producing the highest yield of cellulose. [2] Another strain of bacteria, the SBW25 isolate of the species <i>Pseudomonas fluorescens</i>, produces a biofilm containing cellulose fibers with a small degree of acetylation (.14 acetyl groups per glucose monomer).[3] Industrial-grade cellulose acetate must have at least 1.71 acetyl groups per glucose monomer. [4] In order to engineer a bacterium to efficiently produce cellulose acetate, our strategy is to transform <i>G. hansenii</i> with the four genes, wssF, wssG, wssH, and wssI, that have been identified [3] as being involved in cellulose acetylation in <i>P. fluorescens</i>, and to use directed evolution to further increase percent acetylation of the polymer.<br />
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</p>In addition, we seek to create a streptavidin/cellulose-binding-domain fusion protein which will have the capacity to both cross-link bacterial cellulose acetate polymers (improving material properties) and allow the modular addition of cells (e.g. biosensors). This will be accomplished through the expression on the cells of a biotinylated membrane protein. This will allow biological sensors to be added directly to our cellulose acetate fibers, allowing bacterial sensors to be attached directly to the body of our UAV. <br />
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<br><br><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone"><b>Building a Biological UAV</b></a></center><br><br />
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Our team modeled, prototyped, and collaborated with Ecovative Design to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew! <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Check our more about the material, design, and construction aspects of our biomaterial project here.</a><br />
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<div class="sub5">1. Fischer S <i>et al.</i> (2008) Properties and Applications of Cellulose Acetate. <i>Macromol. Symp.</i> 262: 89-96. DOI: <a href="http://blogs.unpad.ac.id/evyerna/files/2010/11/ca.pdf" target="_blank">10.1002/masy.200850210</a></div><br />
<div class="sub5">2. Ross P <i>et al.</i> (1991) Cellulose Biosynthesis and Function in Bacteria. <i>Microbiological Reviews</i> 55: 35-58. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2030672" target="_blank">2030672 </a></div><br />
<div class="sub5">3. Spiers AJ <i>et al.</i> (2003) Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. <i>Molecular Microbiology</i> 50: 15-27. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/14507360" target="_blank">14507360 </a></div><br />
<div class="sub5">4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013. </div><br />
<div class="sub5">5. Hall PE <i>et al.</i> (1992) Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. <i>Plasmid</i> 28: 194-200. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1461938" target="_blank">1461938 </a></div><br />
<div class="sub5">6. Close TJ <i>et al.</i> (1984) Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. <i>Plasmid</i> 12: 111-118. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/6095350" target="_blank">6095350 </a></div><br />
<div class="sub5">7. Spiers AJ <i>et al.</i> (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in <i>Cellulose - Medical, Pharmaceutical, and Electronic Applications</i>. DOI: <a href="http://cdn.intechopen.com/pdfs-wm/45637.pdf" target="_blank">10.5772/53736</a></div><br />
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</body></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T08:20:16Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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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.<br />
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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. </h6><br><br />
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<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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). <br />
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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.'<br />
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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.<br />
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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. <br />
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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.<br />
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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. <br />
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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.<br />
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<h6><center>This is the SDS-page gel that was run after purifying the esterase protein from bacteria. The band is at 43 kDa, which is the expected size of the esterase protein.</center></h6><br />
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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.<br />
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<h6><center>This image shows the results from our staining assay on cellulose acetate. The pieces of cellulose acetate were soaked in esterase enzyme for varying amounts of time before their level of degradation was tested using the blue cellulose-binding dye.</center></h6><br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T08:17:13Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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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.<br />
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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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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). <br />
<h6><br />
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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.'<br />
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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.<br />
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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. <br />
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'''ARYO PLEASE INSERT THE FLOW CYTOMETRY DATA HERE SINCE I DON'T HAVE IT.'''<br />
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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.<br />
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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. <br />
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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.<br />
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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.<br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T08:11:07Z<p>Pgodbole: </p>
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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.<br />
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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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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). <br />
<h6><br />
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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.'<br />
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<br />
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.<br />
<br />
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. <br />
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'''ARYO PLEASE INSERT THE FLOW CYTOMETRY DATA HERE SINCE I DON'T HAVE IT.'''<br />
<br />
<br></br><br />
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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.<br />
<br />
<br></br><br />
<br />
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. <br />
<br />
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.<br />
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INSERT GEL IMAGES FOR ESTERASE COLONY PCR AND PROTEIN GEL HERE.<br />
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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.<br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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</html></div>Pgodbolehttp://2014.igem.org/Team:StanfordBrownSpelman/BiodegradabilityTeam:StanfordBrownSpelman/Biodegradability2014-10-16T08:09:47Z<p>Pgodbole: </p>
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<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Biodegradability">Biodegradability</a></h3><br />
<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#B">BioBricks</a></h7></div><br />
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<center><iframe width="420" height="315" src="//www.youtube.com/embed/PgdF8xIAh8c" frameborder="0" allowfullscreen></iframe><br><br></center><br />
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.<br />
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<h6>Methods here.</h6> </div></div><br />
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<h6>More methods here. <br />
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<h5><center>Results</h5><br />
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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. </h6><br><br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/a/a5/Ptet_promoter_testing.png><br><br />
<h6><center>This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process</center></h6><br />
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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). <br />
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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.'<br />
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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.<br />
<br />
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. <br />
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<br />
'''ARYO PLEASE INSERT THE FLOW CYTOMETRY DATA HERE SINCE I DON'T HAVE IT.'''<br />
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<br></br><br />
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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.<br />
<br />
<br></br><br />
<br />
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. <br />
<br />
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.<br />
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<div class="small-7 small-centered columns"><br><center><img src=https://static.igem.org/mediawiki/2014/1/1b/Esterase_colony_PCR.png><br><br />
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INSERT GEL IMAGES FOR ESTERASE COLONY PCR AND PROTEIN GEL HERE.<br />
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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.<br />
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We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).<br />
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