http://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&feed=atom&action=historyTeam:Stanford-Brown/HellCell/Desiccation - Revision history2024-03-28T22:08:07ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=296079&oldid=prevRsharma: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-27T02:26:15Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1: This graph displays the percent of surviving cells after 24 hours of desiccation. The exact survival percentages and standard errors of the mean are displayed above each sample in decimal form. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1: This graph displays the percent of surviving cells after 24 hours of desiccation. The exact survival percentages and standard errors of the mean are displayed above each sample in decimal form. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:<del class="diffchange diffchange-inline">Desiccation_2</del>.<del class="diffchange diffchange-inline">png</del>|800px|center]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:<ins class="diffchange diffchange-inline">Desiccation_graph</ins>.<ins class="diffchange diffchange-inline">jpg</ins>|800px|center]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 2: Displays Figure 1 in a log scale.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 2: Displays Figure 1 in a log scale.</div></td></tr>
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</table>Rsharmahttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=264807&oldid=prevVishesh.jain at 02:10, 4 October 20122012-10-04T02:10:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Liquid cultures of negative control, MntH, betAB, and otsAB transformed NEB5α ''E. coli'' were grown up over night at 37°C. After incubation, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with 300uL of negative control bacteria, another 15 with transformants containing MntH, another 15 with transformants containing betAB, and another 15 with transformants containing otsAB. The petri dishes were allowed to desiccate while covered for 24 hours at 37°C while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri <del class="diffchange diffchange-inline">dish </del>to determine the final density of live cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Liquid cultures of negative control, MntH, betAB, and otsAB transformed NEB5α ''E. coli'' were grown up over night at 37°C. After incubation, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with 300uL of negative control bacteria, another 15 with transformants containing MntH, another 15 with transformants containing betAB, and another 15 with transformants containing otsAB. The petri dishes were allowed to desiccate while covered for 24 hours at 37°C while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri <ins class="diffchange diffchange-inline">dishes </ins>to determine the final density of live cells. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Conclusions'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Conclusions'''</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Based on cell survival after 24 hours of desiccation, it was observed that the bet construct and ots construct <del class="diffchange diffchange-inline">provides significantly </del>desiccation resistance. Additionally, the MntH construct may also <del class="diffchange diffchange-inline">provide </del>slightly <del class="diffchange diffchange-inline">increased </del>desiccation resistance when compared to the negative control, but clearly not to the extent of the bet or ots <del class="diffchange diffchange-inline">construct</del>. This trend can be more clearly observed in Figure 2. Figure 2 clearly displays that the ''E. coli'' MntH construct increases survivability by almost one order of magnitude, while the bet and ots constructs provide almost two orders of magnitude increase in survivability. Thus, all three constructs, Mnth, bet, and ots, provide desiccation resistance. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Based on cell survival after 24 hours of desiccation, it was observed that the bet construct and ots construct <ins class="diffchange diffchange-inline">provide significant </ins>desiccation resistance. Additionally, the MntH construct may also slightly <ins class="diffchange diffchange-inline">increase </ins>desiccation resistance when compared to the negative control, but clearly not to the extent of the bet or ots <ins class="diffchange diffchange-inline">constructs</ins>. This trend can be more clearly observed in Figure 2. Figure 2 clearly displays that the ''E. coli'' MntH construct increases survivability by almost one order of magnitude, while the bet and ots constructs provide almost two orders of magnitude increase in survivability. Thus, all three constructs, Mnth, bet, and ots, provide desiccation resistance. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sources: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sources: </div></td></tr>
</table>Vishesh.jainhttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=260686&oldid=prevKendrickwang: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-03T22:15:17Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Liquid cultures of negative control, MntH, betAB, and otsAB transformed NEB5α ''E. coli'' were grown up over night at <del class="diffchange diffchange-inline">37oC</del>. After incubation, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with <del class="diffchange diffchange-inline">300 uL </del>of negative control bacteria, another 15 with transformants containing MntH, another 15 with transformants containing betAB, and another 15 with transformants containing otsAB. The petri dishes were allowed to desiccate while covered for 24 hours at <del class="diffchange diffchange-inline">37oC </del>while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri dish to determine the final density of live cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Liquid cultures of negative control, MntH, betAB, and otsAB transformed NEB5α ''E. coli'' were grown up over night at <ins class="diffchange diffchange-inline">37°C</ins>. After incubation, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with <ins class="diffchange diffchange-inline">300uL </ins>of negative control bacteria, another 15 with transformants containing MntH, another 15 with transformants containing betAB, and another 15 with transformants containing otsAB. The petri dishes were allowed to desiccate while covered for 24 hours at <ins class="diffchange diffchange-inline">37°C </ins>while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri dish to determine the final density of live cells. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Based on cell survival after 24 hours of desiccation, it <del class="diffchange diffchange-inline">is </del>observed that the bet construct and ots construct <del class="diffchange diffchange-inline">significantly </del>provides desiccation resistance. Additionally, the MntH construct may also provide slightly increased desiccation resistance when compared to the negative control, but clearly not to the extent of the bet or ots construct. This trend can be more clearly observed in Figure 2. Figure 2 clearly displays that the ''E. coli'' MntH construct increases survivability by almost one order of magnitude, while the bet and ots constructs provide almost two orders of magnitude increase in survivability. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Based on cell survival after 24 hours of desiccation, it <ins class="diffchange diffchange-inline">was </ins>observed that the bet construct and ots construct provides <ins class="diffchange diffchange-inline">significantly </ins>desiccation resistance. Additionally, the MntH construct may also provide slightly increased desiccation resistance when compared to the negative control, but clearly not to the extent of the bet or ots construct. This trend can be more clearly observed in Figure 2. Figure 2 clearly displays that the ''E. coli'' MntH construct increases survivability by almost one order of magnitude, while the bet and ots constructs provide almost two orders of magnitude increase in survivability<ins class="diffchange diffchange-inline">. Thus, all three constructs, Mnth, bet, and ots, provide desiccation resistance</ins>. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sources: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sources: </div></td></tr>
</table>Kendrickwanghttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=260432&oldid=prevRsharma: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-03T21:44:24Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Based on cell survival after 24 hours of desiccation, it is observed that the bet construct and ots construct provides desiccation resistance. Additionally, the <del class="diffchange diffchange-inline">mntH </del>construct may also provide slightly increased desiccation resistance<del class="diffchange diffchange-inline">. Figure 1 displays that mntH only slightly provides desiccation resistance</del>, <del class="diffchange diffchange-inline">while </del>bet <del class="diffchange diffchange-inline">and </del>ots <del class="diffchange diffchange-inline">provides significantly more desiccation resistance</del>. This trend can be more clearly observed in Figure 2. Figure 2 clearly displays that the E. coli <del class="diffchange diffchange-inline">mntH </del>construct increases survivability by almost one order of magnitude<del class="diffchange diffchange-inline">. Additionally</del>, <del class="diffchange diffchange-inline">Figure 2 also displays that </del>the bet and ots constructs provide almost two orders of magnitude increase in survivability. <del class="diffchange diffchange-inline">Based on these results, it is reasonable to conclude that the bet and ots constructs provide significant desiccation resistance and that mntH provides slight desiccation resistance. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Based on cell survival after 24 hours of desiccation, it is observed that the bet construct and ots construct <ins class="diffchange diffchange-inline">significantly </ins>provides desiccation resistance. Additionally, the <ins class="diffchange diffchange-inline">MntH </ins>construct may also provide slightly increased desiccation resistance <ins class="diffchange diffchange-inline">when compared to the negative control</ins>, <ins class="diffchange diffchange-inline">but clearly not to the extent of the </ins>bet <ins class="diffchange diffchange-inline">or </ins>ots <ins class="diffchange diffchange-inline">construct</ins>. This trend can be more clearly observed in Figure 2. Figure 2 clearly displays that the <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' MntH </ins>construct increases survivability by almost one order of magnitude, <ins class="diffchange diffchange-inline">while </ins>the bet and ots constructs provide almost two orders of magnitude increase in survivability. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sources: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sources: </div></td></tr>
</table>Rsharmahttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=259524&oldid=prevRsharma: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-03T19:54:34Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>''Saccharomyces cerevisiae'', baker’s yeast, is remarkably resistant to desiccation, and it has been shown that certain osmoprotectant chemicals accumulate in these cells when they desiccate. One of these chemicals is trehalose, a disaccharide that was initially implicated in desiccation resistance because of this accumulation (Calahan, Dunham, DeSevo, and Koshland 2011). Surprisingly, attempts to understand trehalose's role and effectiveness in desiccation resistance have produced conflicting and inconsistent results (Calahan et al. 2011).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>''Saccharomyces cerevisiae'', baker’s yeast, is remarkably resistant to desiccation, and it has been shown that certain osmoprotectant chemicals accumulate in these cells when they desiccate. One of these chemicals is trehalose, a disaccharide that was initially implicated in desiccation resistance because of this accumulation (Calahan, Dunham, DeSevo, and Koshland 2011). Surprisingly, attempts to understand trehalose's role and effectiveness in desiccation resistance have produced conflicting and inconsistent results (Calahan et al. 2011).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Glycine betaine (see Cold) also has been found to play an osmoprotectant role in ''E. coli'' <del class="diffchange diffchange-inline">itself </del>(Cayley, Lewis, and Record 1992). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Glycine betaine (see Cold) also has been found to play an osmoprotectant role in ''E. coli<ins class="diffchange diffchange-inline">,</ins>'' <ins class="diffchange diffchange-inline">balancing the osmolarity of the cytoplasm when the cell is under osmotic stress </ins>(Cayley, Lewis, and Record 1992). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The gram-negative biocontrol agent ''Pantoea agglomerans'' was ineffective at controlling blue mold on fruits because of its low viability during the long periods of dehydration that it experienced during fruit processing (Bonaterra, Camps, and Montesinos 2005). Its effectiveness was significantly increased when it was engineered to accumulate trehalose and glycine betaine during desiccation, and it was hypothesized that these osmoprotectants “operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state and stabilising proteins by water replacement via hydrogen bounding” (Bonaterra, Camps, and Montesinos 2005). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The gram-negative biocontrol agent ''Pantoea agglomerans'' was ineffective at controlling blue mold on fruits because of its low viability during the long periods of dehydration that it experienced during fruit processing (Bonaterra, Camps, and Montesinos 2005). Its effectiveness was significantly increased when it was engineered to accumulate <ins class="diffchange diffchange-inline">both </ins>trehalose and glycine betaine during desiccation, and it was hypothesized that these osmoprotectants “operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state and stabilising proteins by water replacement via hydrogen bounding” (Bonaterra, Camps, and Montesinos 2005). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To see if the osmoprotectants trehalose and glycine betaine can be applied <del class="diffchange diffchange-inline">in </del>conferring desiccation resistance <del class="diffchange diffchange-inline">in </del>''E. coli'', the Hell Cell Squad isolated the genes necessary for biosynthesis of trehalose and glycine betaine <del class="diffchange diffchange-inline">in </del>''E. coli'' and put them into the Test Plasmid. We also tested MntH mutants for desiccation resistance-conferring properties (see Radiation). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To see if the osmoprotectants trehalose and glycine betaine can be applied <ins class="diffchange diffchange-inline">to </ins>conferring desiccation resistance <ins class="diffchange diffchange-inline">to </ins>''E. coli'', the Hell Cell Squad isolated the genes necessary for biosynthesis of trehalose and glycine betaine <ins class="diffchange diffchange-inline">from </ins>''E. coli'' and put them into the Test Plasmid <ins class="diffchange diffchange-inline">(see Test Plasmid)</ins>. We also tested MntH mutants for desiccation resistance-conferring properties (see Radiation<ins class="diffchange diffchange-inline">, MntH is also believed to help provide protection against desiccation</ins>). </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Liquid cultures of negative control, <del class="diffchange diffchange-inline">recA</del>, <del class="diffchange diffchange-inline">dps</del>, and <del class="diffchange diffchange-inline">sdaB </del>transformed NEB5α E. coli were grown up over night at 37oC. After <del class="diffchange diffchange-inline">the proper </del>incubation <del class="diffchange diffchange-inline">period</del>, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with negative control bacteria, another 15 with transformants containing <del class="diffchange diffchange-inline">recA</del>, another 15 with transformants containing <del class="diffchange diffchange-inline">dps</del>, and another 15 with transformants containing <del class="diffchange diffchange-inline">sdaB</del>. The petri dishes were allowed to desiccate while covered for 24 hours at 37oC while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri dish to determine the final density of live cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Liquid cultures of negative control, <ins class="diffchange diffchange-inline">MntH</ins>, <ins class="diffchange diffchange-inline">betAB</ins>, and <ins class="diffchange diffchange-inline">otsAB </ins>transformed NEB5α <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>were grown up over night at 37oC. After incubation, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with <ins class="diffchange diffchange-inline">300 uL of </ins>negative control bacteria, another 15 with transformants containing <ins class="diffchange diffchange-inline">MntH</ins>, another 15 with transformants containing <ins class="diffchange diffchange-inline">betAB</ins>, and another 15 with transformants containing <ins class="diffchange diffchange-inline">otsAB</ins>. The petri dishes were allowed to desiccate while covered for 24 hours at 37oC while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri dish to determine the final density of live cells. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
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</table>Rsharmahttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=259457&oldid=prevRsharma: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-03T19:45:52Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>''Saccharomyces cerevisiae'', baker’s yeast, is remarkably resistant to desiccation, and it has been shown that certain osmoprotectant chemicals accumulate in these cells when they desiccate. One of these chemicals is trehalose, a disaccharide that was initially implicated in desiccation resistance because of this accumulation (Calahan, Dunham, DeSevo, and Koshland 2011). Surprisingly, attempts to understand trehalose's role and effectiveness in desiccation resistance have produced conflicting and inconsistent results (Calahan et al. 2011). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>''Saccharomyces cerevisiae'', baker’s yeast, is remarkably resistant to desiccation, and it has been shown that certain osmoprotectant chemicals accumulate in these cells when they desiccate. One of these chemicals is trehalose, a disaccharide that was initially implicated in desiccation resistance because of this accumulation (Calahan, Dunham, DeSevo, and Koshland 2011). Surprisingly, attempts to understand trehalose's role and effectiveness in desiccation resistance have produced conflicting and inconsistent results (Calahan et al. 2011).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">The gram-negative biocontrol agent ''Pantoea agglomerans'' was ineffective at controlling blue mold on fruits because of its low viability during the long periods of dehydration that it experienced during fruit processing (Bonaterra, Camps, and Montesinos 2005). Its effectiveness was significantly increased when it accumulated trehalose and glycine </del>betaine <del class="diffchange diffchange-inline">during desiccation, and it was hypothesized that these osmoprotectants “operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state and stabilising proteins by water replacement via hydrogen bounding” </del>(<del class="diffchange diffchange-inline">Bonaterra, Camps, and Montesinos 2005</del>)<del class="diffchange diffchange-inline">. Glycine betaine was </del>also found to <del class="diffchange diffchange-inline">have </del>an osmoprotectant role in ''E. coli'' itself (Cayley, Lewis, and Record 1992). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Glycine </ins>betaine (<ins class="diffchange diffchange-inline">see Cold</ins>) also <ins class="diffchange diffchange-inline">has been </ins>found to <ins class="diffchange diffchange-inline">play </ins>an osmoprotectant role in ''E. coli'' itself (Cayley, Lewis, and Record 1992). <ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">To learn more about </del>the <del class="diffchange diffchange-inline">ability </del>of trehalose and glycine betaine and <del class="diffchange diffchange-inline">potentially apply them towards </del>desiccation resistance, the Hell Cell Squad isolated the genes necessary for biosynthesis of trehalose and glycine betaine in ''E. coli'' and put them into the Test Plasmid. We also tested MntH mutants for desiccation resistance-conferring properties (see Radiation). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The gram-negative biocontrol agent ''Pantoea agglomerans'' was ineffective at controlling blue mold on fruits because of its low viability during </ins>the <ins class="diffchange diffchange-inline">long periods </ins>of <ins class="diffchange diffchange-inline">dehydration that it experienced during fruit processing (Bonaterra, Camps, and Montesinos 2005). Its effectiveness was significantly increased when it was engineered to accumulate </ins>trehalose and glycine betaine <ins class="diffchange diffchange-inline">during desiccation, </ins>and <ins class="diffchange diffchange-inline">it was hypothesized that these osmoprotectants “operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state and stabilising proteins by water replacement via hydrogen bounding” (Bonaterra, Camps, and Montesinos 2005). </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">To see if the osmoprotectants trehalose and glycine betaine can be applied in conferring </ins>desiccation resistance <ins class="diffchange diffchange-inline">in ''E. coli''</ins>, the Hell Cell Squad isolated the genes necessary for biosynthesis of trehalose and glycine betaine in ''E. coli'' and put them into the Test Plasmid. We also tested MntH mutants for desiccation resistance-conferring properties (see Radiation). </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td></tr>
</table>Rsharmahttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=259418&oldid=prevRsharma: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-03T19:41:33Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>''Saccharomyces cerevisiae'', baker’s yeast, is remarkably resistant to desiccation, and it has been shown that certain osmoprotectant chemicals accumulate in these cells when <del class="diffchange diffchange-inline">desiccated</del>. One of these chemicals is trehalose, a disaccharide that was initially implicated in desiccation resistance because of <del class="diffchange diffchange-inline">its </del>accumulation (Calahan, Dunham, DeSevo, and Koshland 2011). Surprisingly, <del class="diffchange diffchange-inline">assays of trehalose’s </del>effectiveness in <del class="diffchange diffchange-inline">the </del>resistance <del class="diffchange diffchange-inline">of yeast </del>have produced conflicting and inconsistent results (Calahan et al. 2011). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>''Saccharomyces cerevisiae'', baker’s yeast, is remarkably resistant to desiccation, and it has been shown that certain osmoprotectant chemicals accumulate in these cells when <ins class="diffchange diffchange-inline">they desiccate</ins>. One of these chemicals is trehalose, a disaccharide that was initially implicated in desiccation resistance because of <ins class="diffchange diffchange-inline">this </ins>accumulation (Calahan, Dunham, DeSevo, and Koshland 2011). Surprisingly, <ins class="diffchange diffchange-inline">attempts to understand trehalose's role and </ins>effectiveness in <ins class="diffchange diffchange-inline">desiccation </ins>resistance have produced conflicting and inconsistent results (Calahan et al. 2011). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The gram-negative biocontrol agent ''Pantoea agglomerans'' was ineffective at controlling blue mold on fruits because of the long periods of dehydration that it experienced during <del class="diffchange diffchange-inline">the </del>processing <del class="diffchange diffchange-inline">of the fruit </del>(Bonaterra, Camps, and Montesinos 2005). Its effectiveness was significantly increased when it accumulated trehalose and glycine betaine during desiccation, and it was hypothesized that these osmoprotectants “operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state and stabilising proteins by water replacement via hydrogen bounding” (Bonaterra, Camps, and Montesinos 2005). Glycine betaine was also found to have an osmoprotectant role in ''E. coli'' itself (Cayley, Lewis, and Record 1992). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The gram-negative biocontrol agent ''Pantoea agglomerans'' was ineffective at controlling blue mold on fruits because of <ins class="diffchange diffchange-inline">its low viability during </ins>the long periods of dehydration that it experienced during <ins class="diffchange diffchange-inline">fruit </ins>processing (Bonaterra, Camps, and Montesinos 2005). Its effectiveness was significantly increased when it accumulated trehalose and glycine betaine during desiccation, and it was hypothesized that these osmoprotectants “operate through protection of membrane phospholipids by direct hydrogen bounding with phospolipid head groups maintaining the liquid crystal state and stabilising proteins by water replacement via hydrogen bounding” (Bonaterra, Camps, and Montesinos 2005). Glycine betaine was also found to have an osmoprotectant role in ''E. coli'' itself (Cayley, Lewis, and Record 1992). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To learn more about the ability of trehalose and glycine betaine and potentially apply them towards desiccation resistance, the Hell Cell Squad isolated the genes necessary for biosynthesis of trehalose and glycine betaine in ''E. coli'' and put them into the Test Plasmid. We also tested MntH mutants for desiccation resistance-conferring properties (see Radiation). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To learn more about the ability of trehalose and glycine betaine and potentially apply them towards desiccation resistance, the Hell Cell Squad isolated the genes necessary for biosynthesis of trehalose and glycine betaine in ''E. coli'' and put them into the Test Plasmid. We also tested MntH mutants for desiccation resistance-conferring properties (see Radiation). </div></td></tr>
</table>Rsharmahttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=257365&oldid=prevRsharma at 09:49, 3 October 20122012-10-03T09:49:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="/Team:Stanford-Brown/HellCell/pH">pH</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="/Team:Stanford-Brown/HellCell/pH">pH</a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="/Team:Stanford-Brown/Parts">BioBricks</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="/Team:Stanford-Brown/Parts">BioBricks</a></li></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;"><li><a href="https://docs.google.com/document/d/1Pe9voM2l_nrVJk0hwzJ6z6tCCMn9ckykVNX5nLZ8qGg/edit">Lab Notebook</a></li></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;"><li><a href="/Team:Stanford-Brown/Protocols">Protocols</a></li></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ul></div></td></tr>
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</table>Rsharmahttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=256221&oldid=prevBbajar at 07:42, 3 October 20122012-10-03T07:42:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Stanford-Brown/Templates/Content}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Stanford-Brown/Templates/Content}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Desiccation''' <br> <br> <font size = 2.5> <u> At a glance</u> <br> Extremophiles: ''Pantoea agglomerans'' and ''Saccharomyces cerevisiae'' <br> Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthesis A and B (trehalose synthesis pathway) <br> Consensus: Effective </font> ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Desiccation''' <br> <br> <font size = 2.5> <u> At a glance</u> <br> Extremophiles: ''Pantoea agglomerans'' and ''Saccharomyces cerevisiae'' <br> Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthesis A and B (trehalose synthesis pathway) <br> Consensus: Effective </font> ==</div></td></tr>
</table>Bbajarhttp://2012.igem.org/wiki/index.php?title=Team:Stanford-Brown/HellCell/Desiccation&diff=255660&oldid=prevKendrickwang: /* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe2012-10-03T07:01:59Z<p>/* Desiccation At a glance Extremophiles: Pantoea agglomerans and Saccharomyces cerevisiae Proteins of interest: choline dehydrogenase and betaine aldehyde dehydrogenase (glycine betaine biosynthesis pathway) and Osmoregulatory trehalose synthe</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Assay'''</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;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Liquid cultures of negative control, recA, dps, and sdaB transformed NEB5α E. coli were grown up over night at 37oC. After the proper incubation period, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with negative control bacteria, another 15 with transformants containing recA, another 15 with transformants containing dps, and another 15 with transformants containing sdaB. The petri dishes were allowed to desiccate while covered for 24 hours at 37oC while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri dish to determine the final density of live cells. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Liquid cultures of negative control, recA, dps, and sdaB transformed NEB5α E. coli were grown up over night at 37oC. After the proper incubation period, a dilution spot assay was conducted on each of the cultures to determine the density of live cells. Next, 15 5cm, round petri dishes were filled with negative control bacteria, another 15 with transformants containing recA, another 15 with transformants containing dps, and another 15 with transformants containing sdaB. The petri dishes were allowed to desiccate while covered for 24 hours at 37oC while shaking. After the 24 hours period, each plate was resuspended with 1mL of fresh LB. A dilution spot assay was then conducted on each of the petri dish to determine the final density of live cells. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Kendrickwang