http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&feed=atom&action=historyTeam:StanfordBrownSpelman/Amberless Hell Cell - Revision history2024-03-28T17:56:54ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=398245&oldid=prevRaman2004 at 03:34, 18 October 20142014-10-18T03:34:38Z<p></p>
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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">More results here</del>. <del class="diffchange diffchange-inline"> </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">We determined that the <i>D. radiodurans</i> UV damage endonuclease uvsE gene can confer extra radiation resistance to <i>E. coli</i>. After confirming its efficacy, we used mutagenesis PCR to introduce stop codons at two different sites on the uvsE gene. Sequencing showed that our mutagenesis protocol for TTG(leucine)->TAG(stop) produces 70-95% of plasmids with the desired mutation. We are currently working on assembling this uvsE-stop codon gene with the supP tRNA to show that radiation resistance will only occur in Amberless cells when Codon Security is employed</ins>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h5><center>Conclusions</h5></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h5><center>Conclusions</h5></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">Conclusion 1</del>.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>● <ins class="diffchange diffchange-inline">We showed that Codon Security prevents DH5-alpha from expressing a construct containing stop codons. Despite having the supP tRNA in the construct, the cells do not survive transformation or fail to produce the protein of interest with mutated supP due to the toxicity of having a UAG-reading tRNA</ins>.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>● <del class="diffchange diffchange-inline">Conclusion 2</del>.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>● <ins class="diffchange diffchange-inline">We have created an orthogonal synthetic biology system such that genes only properly translate in the Amberless chassis</ins>.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>● <del class="diffchange diffchange-inline">Conclusion 3</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>● <ins class="diffchange diffchange-inline">We isolated a radiation resistance gene from <i>D. radiodurans</i>, uvsE, and have for the first time shown its radiation resistance effects in <i>E. coli</i></ins>.</div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=395828&oldid=prevRaman2004 at 03:19, 18 October 20142014-10-18T03:19:53Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure 8.</b> Comparison of </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure 8.</b> Comparison of <ins class="diffchange diffchange-inline">radiation resistance genes by measuring the fraction of colonies present after exposure to different amounts of UVC radiation. SOD was a resistance gene with unknown radiation resistance qualities. MntH Mut3 was a version of MntH with a UAG stop codon for leucine. Only uvsE-transformed cells showed an indication of radiation resistance. </ins></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">More results here</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">After determining that uvsE was a new gene that could confer increased radiation resistance to DH5-alpha cells, we repeated the experiment with three replicates of a DH5-alpha negative control and DH5-alpha transformed with uvsE</ins>. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure 9.</b> <del class="diffchange diffchange-inline">Figure caption here</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure 9.</b> <ins class="diffchange diffchange-inline">UvsE confers significant radiation resistance to DH5-alpha cells after exposure to UVC. Data were determined to be significant to p<.05 and p<.01 using a two-tailed Student's t-test</ins>.</div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=394774&oldid=prevRaman2004 at 03:12, 18 October 20142014-10-18T03:12:32Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In parallel with demonstrating the Codon Security concept, we worked on applying it to radiation resistance genes to create the Amberless Hell Cell chassis. The goal of this part of the project was to confer resistances to Amberless Cells so that they could survive in harsh environments encountered in UAV or space applications. At the same time, we wanted to prove that applying Codon Security, we could ensure that using these "super-strains" of Amberless in the environment would not pose a risk of horizontal gene transfer into other bacteria. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In parallel with demonstrating the Codon Security concept, we worked on applying it to radiation resistance genes to create the Amberless Hell Cell chassis. The goal of this part of the project was to confer resistances to Amberless Cells so that they could survive in harsh environments encountered in UAV or space applications. At the same time, we wanted to prove that applying Codon Security, we could ensure that using these "super-strains" of Amberless in the environment would not pose a risk of horizontal gene transfer into other bacteria. <br><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We first isolated 5 radiation resistance genes by PCR from <i>D. radiodurans</i> genomic DNA: <a href="http://parts.igem.org/Part:BBa_K1499200" target="_blank">uvsE</a> [3], <a href="http://parts.igem.org/Part:BBa_K1499201" target="_blank">uracil glycosylase 1</a>, <a href="http://parts.igem.org/Part:BBa_K1499202" target="_blank">uracil glycosylase 2</a> [4], MntH, and DpsMP1. Of these, the last two were previous Stanford-Brown 2012 biobricks that we re-isolated for sequence fidelity. The uvsE and MntH were chosen for <del class="diffchange diffchange-inline">further </del>testing <del class="diffchange diffchange-inline">because of their </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We first isolated 5 radiation resistance genes by PCR from <i>D. radiodurans</i> genomic DNA: <a href="http://parts.igem.org/Part:BBa_K1499200" target="_blank">uvsE</a> [3], <a href="http://parts.igem.org/Part:BBa_K1499201" target="_blank">uracil glycosylase 1</a>, <a href="http://parts.igem.org/Part:BBa_K1499202" target="_blank">uracil glycosylase 2</a> [4], <ins class="diffchange diffchange-inline"><a href="http://partsregistry.org/Part:BBa_K847005" target="_blank"></ins>MntH<ins class="diffchange diffchange-inline"></a></ins>, and <ins class="diffchange diffchange-inline"><a href="http://partsregistry.org/Part:BBa_K847002" target="_blank"></ins>DpsMP1<ins class="diffchange diffchange-inline"></a></ins>. Of these, the last two were previous Stanford-Brown 2012 biobricks that we re-isolated for sequence fidelity. The uvsE and MntH were chosen for <ins class="diffchange diffchange-inline">initial </ins>testing<ins class="diffchange diffchange-inline">. </ins></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure 8.</b> <del class="diffchange diffchange-inline">Figure caption here</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure 8.</b> <ins class="diffchange diffchange-inline">Comparison of </ins></div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=394450&oldid=prevRaman2004 at 03:10, 18 October 20142014-10-18T03:10:11Z<p></p>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><h5><center>Results Part 2 - Amberless Hell Cell</h5></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;"><h6></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;">In parallel with demonstrating the Codon Security concept, we worked on applying it to radiation resistance genes to create the Amberless Hell Cell chassis. The goal of this part of the project was to confer resistances to Amberless Cells so that they could survive in harsh environments encountered in UAV or space applications. At the same time, we wanted to prove that applying Codon Security, we could ensure that using these "super-strains" of Amberless in the environment would not pose a risk of horizontal gene transfer into other bacteria. <br><br></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;"></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;">We first isolated 5 radiation resistance genes by PCR from <i>D. radiodurans</i> genomic DNA: <a href="http://parts.igem.org/Part:BBa_K1499200" target="_blank">uvsE</a> [3], <a href="http://parts.igem.org/Part:BBa_K1499201" target="_blank">uracil glycosylase 1</a>, <a href="http://parts.igem.org/Part:BBa_K1499202" target="_blank">uracil glycosylase 2</a> [4], MntH, and DpsMP1. Of these, the last two were previous Stanford-Brown 2012 biobricks that we re-isolated for sequence fidelity. The uvsE and MntH were chosen for further testing because of their </ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Lajoie, MJ <i>et al.</i> (2013) Genomically Recoded Organisms Impart New Biological Functions. <i>Science</i> 342: 357-60. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24136966" target="_blank">24136966</a>.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Lajoie, MJ <i>et al.</i> (2013) Genomically Recoded Organisms Impart New Biological Functions. <i>Science</i> 342: 357-60. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24136966" target="_blank">24136966</a>.<br><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. Thorbjarnardóttir, S <i>et al.</i> (1985) Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene. <i>J. Bacteriol.</i> 161: 219–22. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2981802" target="_blank">2981802</a>.</a></<del class="diffchange diffchange-inline">div</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. Thorbjarnardóttir, S <i>et al.</i> (1985) Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene. <i>J. Bacteriol.</i> 161: 219–22. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2981802" target="_blank">2981802</a><ins class="diffchange diffchange-inline">.<br><br></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">3. Earl, AM <i>et al</ins>.</<ins class="diffchange diffchange-inline">i> (2002) Genetic evidence that the uvsE gene product of <i>Deinococcus radiodurans</i> R1 is </ins>a <ins class="diffchange diffchange-inline">UV damage endonuclease. <i</ins>><ins class="diffchange diffchange-inline">J. Bacteriol.</ins></<ins class="diffchange diffchange-inline">i> 184(4):1003-9. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/11807060" target="_blank">11807060</a>.<br><br</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">4. Sandigursky, M <i>et al.</i> (2004) Multiple uracil-DNA glycosylase activities in <i>Deinococcus radiodurans</i>. <i>DNA Repair (Amst)</i> 3(2):163-9. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/14706350" target="_blank">14706350</a>.</ins></div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=392123&oldid=prevRaman2004 at 02:52, 18 October 20142014-10-18T02:52:35Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <h5><center>Results</h5></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <h5><center>Results <ins class="diffchange diffchange-inline">Part 1 - Codon Security</ins></h5></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We first tested the <b>Codon Security</b> hypothesis with the GFP test plasmid. We cloned the synthesized GFP+tRNA construct into pSB1C3. We initially tried to transform it into DH5-alpha cells, but effeciency was very low. Sequencing of the growing colonies showed interesting deletions or mutations in the tRNA portion of the construct, giving some evidence supporting our hypothesis that the tRNA was able to be expressed but toxic to the cells. We then BioBricked the GFP and tRNA portions separately. The GFP with 2 stop codons but no tRNA (GFP-2S) was stably transformed into DH5-alpha and amberless cells, and as expected, neither cell-type had fluorescent colonies. The tRNA biobrick could not be cloned in DH5-alpha cells; all colonies came back with mutations in the tRNA, further supporting the idea that non-amberless cells cannot tolerate the supP tRNA. We could only biobrick the tRNA in the Amberless chassis.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We first tested the <b>Codon Security</b> hypothesis with the GFP test plasmid. We cloned the synthesized GFP+tRNA construct into pSB1C3. We initially tried to transform it into DH5-alpha cells, but effeciency was very low. Sequencing of the growing colonies showed interesting deletions or mutations in the tRNA portion of the construct, giving some evidence supporting our hypothesis that the tRNA was able to be expressed but toxic to the cells. We then BioBricked the GFP and tRNA portions separately. The GFP with 2 stop codons but no tRNA (GFP-2S) was stably transformed into DH5-alpha and amberless cells, and as expected, neither cell-type had fluorescent colonies. The tRNA biobrick could not be cloned in DH5-alpha cells; all colonies came back with mutations in the tRNA, further supporting the idea that non-amberless cells cannot tolerate the supP tRNA. We could only biobrick the tRNA in the Amberless chassis.<br><br></div></td></tr>
</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=391821&oldid=prevRaman2004 at 02:50, 18 October 20142014-10-18T02:50:10Z<p></p>
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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">More results here</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Figure 7 shows that the Amberless cells only produce the full aeBlue-3S protein. Any truncated products would appear at 7, 13, and 20 kDa depending on the stop codon. DH5-alpha do not produce any protein, or a possible alternative is that truncated products degrade quickly in the cell and do not appear in the gel. These data strongly indicate that the supP tRNA enables protein expression through UAG stop codons only in Amberless cells and serve as a proof of concept for our Codon Security strategy</ins>. </div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=390371&oldid=prevRaman2004 at 02:39, 18 October 20142014-10-18T02:39:13Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure 7.</b> <del class="diffchange diffchange-inline">Figure caption here</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure 7.</b> <ins class="diffchange diffchange-inline">The aeBlue-3S construct has an N-terminal FLAG-tag that can be detected by Western blot using an anti-FLAG antibody. Lane A has twice the cell lysate concentration of lane B. The band at approximately 30kDa is near the expected molecular weight of the complete aeBlue product (28kDa), meaning that Amberless cells, but not DH5-alpha cells, express the complete protein. </ins></div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=387962&oldid=prevRaman2004 at 02:19, 18 October 20142014-10-18T02:19:43Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h6><b>Figure 7.</b> Figure caption here</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h6><b>Figure 7.</b> Figure caption here</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure <del class="diffchange diffchange-inline">7</del>.</b> Figure caption here</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure <ins class="diffchange diffchange-inline">8</ins>.</b> Figure caption here</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure <del class="diffchange diffchange-inline">8</del>.</b> Figure caption here.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure <ins class="diffchange diffchange-inline">9</ins>.</b> Figure caption here.</div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=385006&oldid=prevRaman2004 at 01:52, 18 October 20142014-10-18T01:52:25Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure 6.</b> Plates after transforming (at Day 0) DH5-alpha and Amberless cells with aeBlue-3S+tRNA. At Day 1, after overnight incubation at 37C, there are no DH5-alpha colonies, but there are over 200 Amberless colonies. At Day 5, 4 days of room temperature incubation, Amberless colonies express the aeBlue protein and colonies appear in DH5-alpha plate, although possibly because of degradation of Chlor antibiotic. Side-by-side comparison of cell pellets shows that only Amberless express the blue protein <del class="diffchange diffchange-inline">despite the fact that both cells received the same construct</del>.</h6></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure 6.</b> Plates after transforming (at Day 0) DH5-alpha and Amberless cells with aeBlue-3S+tRNA. At Day 1, after overnight incubation at 37C, there are no DH5-alpha colonies, but there are over 200 Amberless colonies. At Day 5, 4 days of room temperature incubation, Amberless colonies express the aeBlue protein and colonies appear in DH5-alpha plate, although possibly because of degradation of Chlor antibiotic. Side-by-side comparison of cell pellets shows that only Amberless <ins class="diffchange diffchange-inline">strongly </ins>express the blue protein.</h6></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>More results here. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">As seen in Figure 6, we observed that the sequence-verified aeBlue-3S+tRNA construct could only be transformed into and expressed in Amberless cells. The kinetics of blue protein accumulation in the Amberless cells were relatively slow compared to GFP, with first visible signs at Day 2. We plan to determine the kinetics more thoroughly in an future experiment. Additionally, higher temperatures of incubation were less conducive to protein production; room temperature to 30C was the optimal temperature. The cells were removed from the each plate at Day 5 and lysed for Western blotting. </ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h6><b>Figure 7.</b> Figure caption here</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>More results here.</div></td></tr>
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</table>Raman2004http://2014.igem.org/wiki/index.php?title=Team:StanfordBrownSpelman/Amberless_Hell_Cell&diff=384078&oldid=prevRaman2004 at 01:44, 18 October 20142014-10-18T01:44:01Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h6><b>Figure 6.</b> <del class="diffchange diffchange-inline">caption here</del></h6></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h6><b>Figure 6.</b> <ins class="diffchange diffchange-inline">Plates after transforming (at Day 0) DH5-alpha and Amberless cells with aeBlue-3S+tRNA. At Day 1, after overnight incubation at 37C, there are no DH5-alpha colonies, but there are over 200 Amberless colonies. At Day 5, 4 days of room temperature incubation, Amberless colonies express the aeBlue protein and colonies appear in DH5-alpha plate, although possibly because of degradation of Chlor antibiotic. Side-by-side comparison of cell pellets shows that only Amberless express the blue protein despite the fact that both cells received the same construct.</ins></h6></div></td></tr>
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</table>Raman2004