http://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&feed=atom&action=historyTeam:Cornell/project/wetlab/mercury - Revision history2024-03-29T00:33:59ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=400733&oldid=prevK.Hui at 03:52, 18 October 20142014-10-18T03:52:13Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li>Lund, P., & Brown, N. (1987). Role of the <i>merT</i> and <i>merP</i> gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene, 207-214.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li>Lund, P., & Brown, N. (1987). Role of the <i>merT</i> and <i>merP</i> gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. <ins class="diffchange diffchange-inline"><i></ins>Gene<ins class="diffchange diffchange-inline"></i></ins>, 207-214.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li>Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. Journal of Health Science, 379-383.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li>Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. <ins class="diffchange diffchange-inline"><i></ins>Journal of Health Science<ins class="diffchange diffchange-inline"></i></ins>, 379-383.</li></div></td></tr>
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</table>K.Huihttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=399044&oldid=prevO.Spassibojko at 03:40, 18 October 20142014-10-18T03:40:11Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells). To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (<del class="diffchange diffchange-inline">GST-</del><i>crs5</i> in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells). To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (<i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i> in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and <del class="diffchange diffchange-inline">GST-</del><i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and <i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (<del class="diffchange diffchange-inline">GST-</del><i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (<i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>There was no statistically significant difference between BL21 wild type and BL21 engineered to express <i>merT/merP</i> and <del class="diffchange diffchange-inline">GST-</del><i>crs5</i> in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>There was no statistically significant difference between BL21 wild type and BL21 engineered to express <i>merT/merP</i> and <i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i> in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments. </div></td></tr>
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</table>O.Spassibojkohttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=398841&oldid=prevO.Spassibojko at 03:38, 18 October 20142014-10-18T03:38:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.<sup>[1]</sup> The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.<sup>[2]</sup> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.<sup>[1]</sup> The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.<sup>[2]</sup> </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the <del class="diffchange diffchange-inline">GST-</del><i>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the <i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. </div></td></tr>
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</table>O.Spassibojkohttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=397395&oldid=prevC.Zhang at 03:28, 18 October 20142014-10-18T03:28:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells). To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-<del class="diffchange diffchange-inline">YMT </del>in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells). To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-<ins class="diffchange diffchange-inline"><i>crs5</i> </ins>in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td></tr>
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</table>C.Zhanghttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=394296&oldid=prevG.Livermore at 03:08, 18 October 20142014-10-18T03:08:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells<ins class="diffchange diffchange-inline">)</ins>. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td></tr>
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</table>G.Livermorehttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=392621&oldid=prevE.Holmes at 02:56, 18 October 20142014-10-18T02:56:17Z<p></p>
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</table>E.Holmeshttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=391024&oldid=prevO.Spassibojko at 02:44, 18 October 20142014-10-18T02:44:18Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Construct Design</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Construct Design</h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.<ins class="diffchange diffchange-inline"><sup>[1]</sup> </ins>The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.<ins class="diffchange diffchange-inline"><sup>[2]</sup> </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-<i>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-<i>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. </div></td></tr>
</table>O.Spassibojkohttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=388112&oldid=prevE.Holmes at 02:20, 18 October 20142014-10-18T02:20:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br><br></ins><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.</div></td></tr>
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</table>E.Holmeshttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=380607&oldid=prevE.Holmes at 01:13, 18 October 20142014-10-18T01:13:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted <del class="diffchange diffchange-inline">by 1/2 </del>with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted <ins class="diffchange diffchange-inline">in half </ins>with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.</div></td></tr>
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</table>E.Holmeshttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/mercury&diff=380361&oldid=prevA.Chakravorty at 01:11, 18 October 20142014-10-18T01:11:28Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Results</h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part <del class="diffchange diffchange-inline"><a href="http://parts.igem.org/Part:</del>BBa_K1460004<del class="diffchange diffchange-inline">">BBa_K1460004</a> </del>in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). </div></td></tr>
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</table>A.Chakravorty