http://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&feed=atom&action=historyTeam:Cornell/project/wetlab/nickel - Revision history2024-03-28T21:12:08ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=400887&oldid=prevK.Hui at 03:53, 18 October 20142014-10-18T03:53:17Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li>Fulkerson, J., & Mobley, H. (2000). Membrane Topology of the <i>nixA</i> Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III. Journal of Bacteriology, 1722-1730.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li>Fulkerson, J., & Mobley, H. (2000). Membrane Topology of the <i>nixA</i> Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III. <ins class="diffchange diffchange-inline"><i></ins>Journal of Bacteriology<ins class="diffchange diffchange-inline"></i></ins>, 1722-1730.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene <i>nixA</i>: Synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Molecular Microbiology, 97-109.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene <i>nixA</i>: Synthesis of catalytically active urease in Escherichia coli independent of growth conditions. <ins class="diffchange diffchange-inline"><i></ins>Molecular Microbiology<ins class="diffchange diffchange-inline"></i></ins>, 97-109.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li>Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation. Applied and Environmental Microbiology, 5383-5386.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li>Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation. <ins class="diffchange diffchange-inline"><i></ins>Applied and Environmental Microbiology<ins class="diffchange diffchange-inline"></i></ins>, 5383-5386.</li></div></td></tr>
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</table>K.Huihttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=399187&oldid=prevO.Spassibojko at 03:41, 18 October 20142014-10-18T03:41:15Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell.<sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity,<sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation.<sup>[3]</sup></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell.<sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity,<sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation.<sup>[3]</sup></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460003">BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460006">BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and <del class="diffchange diffchange-inline">GST-</del><i>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460003">BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460006">BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and <i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </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_K1460003 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (<del class="diffchange diffchange-inline">GST-</del><i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel 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_K1460003 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (<i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel 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>Figure 3 shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (figure 4) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and <del class="diffchange diffchange-inline">GST-</del><i>crs5</i> are in fact able to remove nickel ions from water. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure 3 shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (figure 4) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and <i><ins class="diffchange diffchange-inline">GST-</ins>crs5</i> are in fact able to remove nickel ions from water. </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>Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.</div></td></tr>
</table>O.Spassibojkohttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=392296&oldid=prevE.Holmes at 02:53, 18 October 20142014-10-18T02:53:47Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell.<sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity,<sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation.<sup>[3]</sup></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell.<sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity,<sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation.<sup>[3]</sup></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: #ffa; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href=<del class="diffchange diffchange-inline">”http</del>://parts.igem.org/Part:<del class="diffchange diffchange-inline">BBa_K1460003”</del>>BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href=<del class="diffchange diffchange-inline">”http</del>://parts.igem.org/Part:<del class="diffchange diffchange-inline">BBa_K1460006”</del>>BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and GST-<i>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href=<ins class="diffchange diffchange-inline">"http</ins>://parts.igem.org/Part:<ins class="diffchange diffchange-inline">BBa_K1460003"</ins>>BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href=<ins class="diffchange diffchange-inline">"http</ins>://parts.igem.org/Part:<ins class="diffchange diffchange-inline">BBa_K1460006"</ins>>BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and GST-<i>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td></tr>
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</table>E.Holmeshttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=391214&oldid=prevO.Spassibojko at 02:45, 18 October 20142014-10-18T02:45:40Z<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>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell <sup>[1]</sup><del class="diffchange diffchange-inline">. </del>Normally used by <i>H. pylori</i> to allow for urease activity <sup>[2]</sup><del class="diffchange diffchange-inline">, </del><i>nixA</i> also shows promise for purposes of bioaccumulation and remediation<sup>[3]</sup><del class="diffchange diffchange-inline">.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell<ins class="diffchange diffchange-inline">.</ins><sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity<ins class="diffchange diffchange-inline">,</ins><sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation<ins class="diffchange diffchange-inline">.</ins><sup>[3]</sup></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>The first nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460003”>BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460006”>BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and GST-<i>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460003”>BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460006”>BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and GST-<i>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td></tr>
</table>O.Spassibojkohttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=388208&oldid=prevE.Holmes at 02:21, 18 October 20142014-10-18T02:21:38Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After 24 hours of growth, no significant difference in growth was observed between the two strains. What we consistently observed as well, however, is that there is no inhibition of growth of BL21 at high concentrations of nickel (figure 2). Even if <i>nixA</i> is expressed and is actively transporting nickel ions into cells, it is possible that the concentration of nickel is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test nickel concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the nickel (II) chloride that we were using for testing.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After 24 hours of growth, no significant difference in growth was observed between the two strains. What we consistently observed as well, however, is that there is no inhibition of growth of BL21 at high concentrations of nickel (figure 2). Even if <i>nixA</i> is expressed and is actively transporting nickel ions into cells, it is possible that the concentration of nickel is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test nickel concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the nickel (II) chloride that we were using for testing.</div></td></tr>
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</table>E.Holmeshttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=379737&oldid=prevE.Holmes at 01:06, 18 October 20142014-10-18T01:06:13Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Part BBa_K1460003 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 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 Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel 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_K1460003 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 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 Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel 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/nickel&diff=378325&oldid=prevE.Holmes at 00:54, 18 October 20142014-10-18T00:54:34Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>After 24 hours of growth, no significant difference in growth was observed between the two strains. What we consistently observed as well, however, is that there is no inhibition of growth of BL21 at high concentrations of nickel (<del class="diffchange diffchange-inline">right </del>figure). Even if <i>nixA</i> is expressed and is actively transporting nickel ions into cells, it is possible that the concentration of nickel is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test nickel concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the nickel (II) chloride that we were using for testing.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After 24 hours of growth, no significant difference in growth was observed between the two strains. What we consistently observed as well, however, is that there is no inhibition of growth of BL21 at high concentrations of nickel (figure <ins class="diffchange diffchange-inline">2</ins>). Even if <i>nixA</i> is expressed and is actively transporting nickel ions into cells, it is possible that the concentration of nickel is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test nickel concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the nickel (II) chloride that we were using for testing.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">The chart on the left </del>shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (<del class="diffchange diffchange-inline">on the right chart</del>) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and GST-<i>crs5</i> are in fact able to remove nickel ions from water. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Figure 3 </ins>shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (<ins class="diffchange diffchange-inline">figure 4</ins>) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and GST-<i>crs5</i> are in fact able to remove nickel ions from water. </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>Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.</div></td></tr>
</table>E.Holmeshttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=377413&oldid=prevC.Zhang at 00:47, 18 October 20142014-10-18T00:47:06Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The chart on the left shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (on the right chart) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and GST-<del class="diffchange diffchange-inline">YMT </del>are in fact able to remove nickel ions from water. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The chart on the left shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (on the right chart) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and GST-<ins class="diffchange diffchange-inline"><i>crs5</i> </ins>are in fact able to remove nickel ions from water. </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>Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.</div></td></tr>
</table>C.Zhanghttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=377342&oldid=prevC.Zhang at 00:46, 18 October 20142014-10-18T00:46:31Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Part BBa_K1460003 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (GST-<del class="diffchange diffchange-inline">YMT</del>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted by 1/2 with LB + 2 mM Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel 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_K1460003 in pUC57 was co-transformed with part <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (GST-<ins class="diffchange diffchange-inline"><i>crs5</i></ins>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted by 1/2 with LB + 2 mM Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel 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>C.Zhanghttp://2014.igem.org/wiki/index.php?title=Team:Cornell/project/wetlab/nickel&diff=377210&oldid=prevC.Zhang at 00:45, 18 October 20142014-10-18T00:45:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell <sup>[1]</sup>. Normally used by <i>H. pylori</i> to allow for urease activity <sup>[2]</sup>, <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation<sup>[3]</sup>.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>, imports nearby Ni<sup>2+</sup> ions into the cell <sup>[1]</sup>. Normally used by <i>H. pylori</i> to allow for urease activity <sup>[2]</sup>, <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation<sup>[3]</sup>.</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: #ffa; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460003”>BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460006”>BBaK1460006</a> was constructed by inserting our metallothionein construct, the T7 promoter and GST-<del class="diffchange diffchange-inline">YMT </del>metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The first nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460003”>BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href=”http://parts.igem.org/Part:BBa_K1460006”>BBaK1460006</a> was constructed by inserting our metallothionein construct, the <ins class="diffchange diffchange-inline"><i></ins>T7<ins class="diffchange diffchange-inline"></i> </ins>promoter and GST-<ins class="diffchange diffchange-inline"><i>crs5</i> </ins>metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
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</table>C.Zhang