http://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&feed=atom&action=historyTeam:Virginia/Results - Revision history2024-03-29T05:04:02ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=362381&oldid=prevEmcmillen at 16:36, 22 November 20132013-11-22T16:36:05Z<p></p>
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</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=362379&oldid=prevEmcmillen at 16:34, 22 November 20132013-11-22T16:34:43Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Conclusions:</b></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Conclusions:</b></p></div></td></tr>
</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=361669&oldid=prevEmcmillen at 03:56, 29 October 20132013-10-29T03:56:26Z<p></p>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p><b>Note:</b></p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p> It is clearly visible in the graphs of the number of cells versus the different concentrations of IPTG that there are no error bars. An initial set of data was initially collected before the regional competition and then the experiment was repeated using grown up glycerol stock (cells with the final construct plasmid) approximately a month later. The glycerol stocks that were made were frozen and refrozen several times when taking samples and this may have led to questionable changes to the membrane and stability of the cells. The results that were obtained and that were to be used to make the error bars and provide additional data were dismissed as unreliable as the growth and results obtained were scattered and showed no observable trends. </ins></div></td></tr>
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</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=360916&oldid=prevCLangguth at 03:37, 29 October 20132013-10-29T03:37:26Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><span>Results</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><span>Results</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Setup</b></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Setup</b></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">After the Biobrick plasmids had all been made, their efficacy and functioning was tested. The first step in that process was to determine the relative growth rate of the XL1-Blue cells that all the plasmid constructs were tested in. The growth curve and its associated data can be seen below and the relative growth was approximately as anticipated for this <del class="diffchange diffchange-inline">strand </del>of E.coli. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">After the Biobrick plasmids had all been made, their efficacy and functioning was tested. The first step in that process was to determine the relative growth rate of the XL1-Blue cells that all the plasmid constructs were tested in. The growth curve and its associated data can be seen below and the relative growth was approximately as anticipated for this <ins class="diffchange diffchange-inline">strain </ins>of E.coli. </p></div></td></tr>
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</table>CLangguthhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=359633&oldid=prevEmcmillen at 02:53, 29 October 20132013-10-29T02:53:49Z<p></p>
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</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=359559&oldid=prevEmcmillen at 02:51, 29 October 20132013-10-29T02:51:48Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Conclusions:</b></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Conclusions:</b></p></div></td></tr>
</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=357891&oldid=prevEmcmillen at 01:55, 29 October 20132013-10-29T01:55:07Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p><a href="https://2013.igem.org/Team:Virginia/Results">Results</a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p><a href="https://2013.igem.org/Team:Virginia/Results">Results</a></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p><a href="https://2013.igem.org/Team:Virginia/Modeling">Modeling</a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p><a href="https://2013.igem.org/Team:Virginia/Modeling">Modeling</a></p></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"> <p><a href="https://2013.igem.org/Team:Virginia/Software">Software</a></p></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p><a href="https://2013.igem.org/Team:Virginia/Chassis_Improvements">Chassis Improvements</a></p></span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p><a href="https://2013.igem.org/Team:Virginia/Chassis_Improvements">Chassis Improvements</a></p></span></div></td></tr>
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</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=357637&oldid=prevCLangguth at 01:45, 29 October 20132013-10-29T01:45:24Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">All the 1-hour samples appeared to show evidence for minicells being present and in the samples with IPTG having been added to the cell culture, both the total cell count observed and the apparent minicell cell count seemed to be relatively high. This indicates that optimal duration of IPTG exposure for minicell formation with the plasmid may be on the shorter side. If minicells were going to be produced to be loaded with pharmaceuticals or other chemicals, then this minimal exposure time would be optimal. For industrial purposes, shortening the length of a phase of production leads to not only quicker production of a potential product, but also saving money if the process is being done under conditions that would require more energy to maintain phase conditions. These results also suggest that finding a minimum value for the concentration of IPTG needed for the same approximate yield of minicells would likely be possible (this is indicated by the leveling out that can be seen in the graph in Figure 9).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">All the 1-hour samples appeared to show evidence for minicells being present and in the samples with IPTG having been added to the cell culture, both the total cell count observed and the apparent minicell cell count seemed to be relatively high. This indicates that optimal duration of IPTG exposure for minicell formation with the plasmid may be on the shorter side. If minicells were going to be produced to be loaded with pharmaceuticals or other chemicals, then this minimal exposure time would be optimal. For industrial purposes, shortening the length of a phase of production leads to not only quicker production of a potential product, but also saving money if the process is being done under conditions that would require more energy to maintain phase conditions. These results also suggest that finding a minimum value for the concentration of IPTG needed for the same approximate yield of minicells would likely be possible (this is indicated by the leveling out that can be seen in the graph in Figure 9).</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">The 14-hour samples yielded results that are supported by previous modeling and hypotheses made after expert consultation. The undesirable rod cells are the most common cell type at the higher IPTG concentration. The mechanism by which this occurs is not completely understood though. Some have suggested that because the protein degradation rate of the GFP is negligible and the protein degradation rate of FtsZ is greatly exceeded by the protein production rate in the presence of the activated plasmid, that there is an accumulated increasing concentration of these proteins in later cell generations. Others suggest that the IPTG levels inside the cells are not able to reach their highest levels until many hours have passed and previous generations of cell have passed down more IPTG saturated cell contents. The diffusivity of the IPTG across the cellular membrane was not modeled and should be considered in future models and experimentation. In order to gain a better understanding of the mechanism causing the rod formation, HA tagged FtsZ is currently being made in order to allow for FtsZ quantification by Western Blotting. Regardless of the mechanism by which these events occurred, both rods and the desired spherical fluorescent minicells were obtained from these samples after the 14 hour experimentation period. These were then purified by centrifugation and treatment with antibiotic to result in the mix of dead parental cells and minicells that can be seen in Figure 10. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">The 14-hour samples yielded results that are supported by previous modeling and hypotheses made after expert consultation. The undesirable rod cells are the most common cell type at the higher IPTG concentration. The mechanism by which this occurs is not completely understood though. Some have suggested that because the protein degradation rate of the GFP is negligible and the protein degradation rate of FtsZ is greatly exceeded by the protein production rate in the presence of the activated plasmid, that there is an accumulated increasing concentration of these proteins in later cell generations. Others suggest that the IPTG levels inside the cells are not able to reach their highest levels until many hours have passed and previous generations of cell have passed down more IPTG saturated cell contents. The diffusivity of the IPTG across the cellular membrane was not modeled and should be considered in future models and experimentation. In order to gain a better understanding of the mechanism causing the rod formation, HA tagged FtsZ is currently being made in order to allow for FtsZ quantification by Western Blotting. Regardless of the mechanism by which these events occurred, both rods and the desired spherical fluorescent minicells were obtained from these samples after the 14 hour experimentation period. These were then purified by centrifugation and treatment with antibiotic to result in the mix of dead parental cells and minicells that can be seen in Figure 10. </p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">There were two methods tested for minicell purification, including a sucrose gradient protocol and a differential centrifugation and filtration protocol. Each method confers different advantages in cost and time. Although both protocols are very effective in achieving minicell purity, the differential centrifugation and filtration protocol typically yielded more minicells with greater overall purity, including the removal of cell debris and free endotoxins.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">There were two methods tested for minicell purification, including a sucrose gradient protocol and a differential centrifugation and filtration protocol. Each method confers different advantages in cost and time. Although both protocols are very effective in achieving minicell purity, the differential centrifugation and filtration protocol typically yielded more minicells with greater overall purity, including the removal of cell debris and free endotoxins<ins class="diffchange diffchange-inline">. Following differential centrifugation of our minicell culture for 10 minutes at 2000 xg, purity of the sample was increased from an average particle size of 1.449 µm to 1.241 µm. Therefore, 82% of the parent cells were removed. However, it should be noted that this is a conservative estimate based on the limitations of the Beckman Coulter Multisizer, which does not size particles under 0.4 µm</ins>.</p></div></td></tr>
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</table>CLangguthhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=343382&oldid=prevEmcmillen at 05:04, 28 October 20132013-10-28T05:04:40Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">The IPTG concentration can be varied when inducing minicell production in our E.coli cells and their minicell count can be determined through our purification and quantification protocols. This, however, excludes FtsZ production from the equation. In order to determine the quantity of FtsZ protein produced, we plan to take our generated minicell samples after purification, after the E.coli have stopped producing additional FtsZ, and run them through a detailed Western blot protocol that will be able to properly quantify the amount of FtsZ protein produced from our given samples (produced under varying IPTG concentrations) through Li-cor imaging.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">The IPTG concentration can be varied when inducing minicell production in our E.coli cells and their minicell count can be determined through our purification and quantification protocols. This, however, excludes FtsZ production from the equation. In order to determine the quantity of FtsZ protein produced, we plan to take our generated minicell samples after purification, after the E.coli have stopped producing additional FtsZ, and run them through a detailed Western blot protocol that will be able to properly quantify the amount of FtsZ protein produced from our given samples (produced under varying IPTG concentrations) through Li-cor imaging.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/7/73/Rainbowbrick_edited.png"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><div id="rainbow"></ins><img src="https://static.igem.org/mediawiki/2013/7/73/Rainbowbrick_edited.png"<ins class="diffchange diffchange-inline">></div</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">In order to make FtsZ protein that can be detected through Western blotting. We are in the process of developing two additional biobricks. These biobricks will include the IPTG-inducible promoter, RBS, FtsZ protein with an HA tag inserted into either the N or C terminus (one biobrick for each HA tag in the two given locations), and a terminator. This biobrick is similar to our final construct with the exceptions of the added HA tag to FtsZ and the exclusion of GFP. The protocol for using these biobricks to induce minicell formation is not expected to differ from the protocol used with the original construct. However, these new biobricks have the added benefit of detectability by an Anti-HA primary antibody through Western blotting and will allow us to better analyze the relationship between promoter, protein, and minicells.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">In order to make FtsZ protein that can be detected through Western blotting. We are in the process of developing two additional biobricks. These biobricks will include the IPTG-inducible promoter, RBS, FtsZ protein with an HA tag inserted into either the N or C terminus (one biobrick for each HA tag in the two given locations), and a terminator. This biobrick is similar to our final construct with the exceptions of the added HA tag to FtsZ and the exclusion of GFP. The protocol for using these biobricks to induce minicell formation is not expected to differ from the protocol used with the original construct. However, these new biobricks have the added benefit of detectability by an Anti-HA primary antibody through Western blotting and will allow us to better analyze the relationship between promoter, protein, and minicells.</p></div></td></tr>
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</table>Emcmillenhttp://2013.igem.org/wiki/index.php?title=Team:Virginia/Results&diff=341686&oldid=prevEmcmillen at 23:55, 27 October 20132013-10-27T23:55:25Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>FtsZ Quantification and Its Importance</b></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>FtsZ Quantification and Its Importance</b></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> <p style="text-indent: 5em;">A critical step towards fully understanding FtsZ’s role in the expression of the minicell phenotype is to develop a quantitative relationship between the production of FtsZ protein and the formation of minicells. For our biobrick in particular, the production of FtsZ protein is controlled by the concentration of IPTG added. Therefore, in order to characterize the efficiency of our biobrick, we must map the connection between these three factors.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> <p style="text-indent: 5em;">A critical step towards fully understanding FtsZ’s role in the expression of the minicell phenotype is to develop a quantitative relationship between the production of FtsZ protein and the formation of minicells. For our biobrick in particular, the production of FtsZ protein is controlled by the concentration of IPTG added. Therefore, in order to characterize the efficiency of our biobrick, we must map the connection between these three factors.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/<del class="diffchange diffchange-inline">thumb</del>/<del class="diffchange diffchange-inline">1/10/Blackiptg.png</del>/800px-<del class="diffchange diffchange-inline">Blackiptg</del>.png"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/<ins class="diffchange diffchange-inline">5</ins>/<ins class="diffchange diffchange-inline">59</ins>/800px-<ins class="diffchange diffchange-inline">Blackiptg_edited</ins>.png"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">The IPTG concentration can be varied when inducing minicell production in our E.coli cells and their minicell count can be determined through our purification and quantification protocols. This, however, excludes FtsZ production from the equation. In order to determine the quantity of FtsZ protein produced, we plan to take our generated minicell samples after purification, after the E.coli have stopped producing additional FtsZ, and run them through a detailed Western blot protocol that will be able to properly quantify the amount of FtsZ protein produced from our given samples (produced under varying IPTG concentrations) through Li-cor imaging.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">The IPTG concentration can be varied when inducing minicell production in our E.coli cells and their minicell count can be determined through our purification and quantification protocols. This, however, excludes FtsZ production from the equation. In order to determine the quantity of FtsZ protein produced, we plan to take our generated minicell samples after purification, after the E.coli have stopped producing additional FtsZ, and run them through a detailed Western blot protocol that will be able to properly quantify the amount of FtsZ protein produced from our given samples (produced under varying IPTG concentrations) through Li-cor imaging.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/<del class="diffchange diffchange-inline">6</del>/<del class="diffchange diffchange-inline">6e</del>/<del class="diffchange diffchange-inline">Rainbowbrick</del>.png"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/<ins class="diffchange diffchange-inline">7</ins>/<ins class="diffchange diffchange-inline">73</ins>/<ins class="diffchange diffchange-inline">Rainbowbrick_edited</ins>.png"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">In order to make FtsZ protein that can be detected through Western blotting. We are in the process of developing two additional biobricks. These biobricks will include the IPTG-inducible promoter, RBS, FtsZ protein with an HA tag inserted into either the N or C terminus (one biobrick for each HA tag in the two given locations), and a terminator. This biobrick is similar to our final construct with the exceptions of the added HA tag to FtsZ and the exclusion of GFP. The protocol for using these biobricks to induce minicell formation is not expected to differ from the protocol used with the original construct. However, these new biobricks have the added benefit of detectability by an Anti-HA primary antibody through Western blotting and will allow us to better analyze the relationship between promoter, protein, and minicells.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><p style="text-indent: 5em;">In order to make FtsZ protein that can be detected through Western blotting. We are in the process of developing two additional biobricks. These biobricks will include the IPTG-inducible promoter, RBS, FtsZ protein with an HA tag inserted into either the N or C terminus (one biobrick for each HA tag in the two given locations), and a terminator. This biobrick is similar to our final construct with the exceptions of the added HA tag to FtsZ and the exclusion of GFP. The protocol for using these biobricks to induce minicell formation is not expected to differ from the protocol used with the original construct. However, these new biobricks have the added benefit of detectability by an Anti-HA primary antibody through Western blotting and will allow us to better analyze the relationship between promoter, protein, and minicells.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/7/70/FtsZ_PCR_Confirmation_Edited.png"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2013/7/70/FtsZ_PCR_Confirmation_Edited.png"></div></td></tr>
</table>Emcmillen