http://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&feed=atom&action=historyGalactose dose response of Gal1 Promoter in pRS415 - Revision history2024-03-29T09:31:10ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=144762&oldid=prevJoseph: Removing all content from page2010-10-25T14:09:11Z<p>Removing all content from page</p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><h3>Aim</h3></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction was achieved at concentrations above 0.5% (data not shown). We wanted to examine the dose responsive behaviour of the GAL1 promoter across a full range of concentrations. Therefore the dose response experiments were repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. </del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><h3>Protocol</h3></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and raffinose (2 %) as the carbon source. <br><br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">2. The following evening this cell culture was sub-cultured into a flask containing pre-warmed SD medium (50 mls) with 2% raffinose, and one of a range of concentrations of galactose between 0.05% and 2% of galactose, to achieve an optical density at 600nm of 0.6 by 9.00 am the following morning. <br></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">3. Samples were washed into PBS, and diluted 1/20 in preparation for FACS analysis.</del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><h3>Results</h3></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 488 nm, and an emission filter of 510 nm, </p></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[[Image: Gal-facs3.jpg|300 px]]</del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><br><br></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of sensitivity to the inducing agent, with concentrations as low as 0.01% having significant inducing potential. </del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><h3>Conclusion</h3></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. </del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></del></div></td><td colspan="2"> </td></tr>
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</table>Josephhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=144421&oldid=prevJoseph at 13:35, 25 October 20102010-10-25T13:35:40Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image: Gal-facs3.jpg|300 px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image: Gal-facs3.jpg|300 px]]</div></td></tr>
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</table>Josephhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138816&oldid=prevI.stansfield at 23:26, 24 October 20102010-10-24T23:26:12Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><b>[[https://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b></ins></div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138778&oldid=prevI.stansfield at 23:21, 24 October 20102010-10-24T23:21:24Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h1>Measurement of <del class="diffchange diffchange-inline">induction </del>of the GAL1 promoter <del class="diffchange diffchange-inline">over time in </del>construct GAL1p-(Npep-GFP)</h1></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h1>Measurement of <ins class="diffchange diffchange-inline">dose responsiveness </ins>of the GAL1 promoter <ins class="diffchange diffchange-inline">to galactose using </ins>construct GAL1p-(Npep-GFP)</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138763&oldid=prevI.stansfield at 23:19, 24 October 20102010-10-24T23:19:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 488 nm, and <del class="diffchange diffchange-inline">a </del>emission filter of 510 nm, </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 488 nm, and <ins class="diffchange diffchange-inline">an </ins>emission filter of 510 nm, </p></div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138760&oldid=prevI.stansfield at 23:19, 24 October 20102010-10-24T23:19:17Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction was achieved at concentrations above 0.5% (data not shown). We wanted to examine the dose responsive behaviour of the GAL1 promoter across a full range of concentrations. Therefore the dose response experiments were repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction was achieved at concentrations above 0.5% (data not shown). We wanted to examine the dose responsive behaviour of the GAL1 promoter across a full range of concentrations. Therefore the dose response experiments were repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. </div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138754&oldid=prevI.stansfield at 23:18, 24 October 20102010-10-24T23:18:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of <del class="diffchange diffchange-inline">480 </del>nm, and a emission filter of <del class="diffchange diffchange-inline">480 </del>nm, </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of <ins class="diffchange diffchange-inline">488 </ins>nm, and a emission filter of <ins class="diffchange diffchange-inline">510 </ins>nm, </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. <ins class="diffchange diffchange-inline">The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. </ins></div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138708&oldid=prevI.stansfield at 23:15, 24 October 20102010-10-24T23:15:14Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><del class="diffchange diffchange-inline">The aim of this experiment </del>was to <del class="diffchange diffchange-inline">test </del>the <del class="diffchange diffchange-inline">response </del>of the GAL1 promoter <del class="diffchange diffchange-inline">in the presence </del>of <del class="diffchange diffchange-inline">galactose over time, by measuring </del>the <del class="diffchange diffchange-inline">expression </del>of <del class="diffchange diffchange-inline">GFP</del>, <del class="diffchange diffchange-inline">the downstream gene</del>. <del class="diffchange diffchange-inline">Construct Gal1p-(Npep-GFP) was used in the experiments described here </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction </ins>was <ins class="diffchange diffchange-inline">achieved at concentrations above 0.5% (data not shown). We wanted </ins>to <ins class="diffchange diffchange-inline">examine </ins>the <ins class="diffchange diffchange-inline">dose responsive behaviour </ins>of the GAL1 promoter <ins class="diffchange diffchange-inline">across a full range </ins>of <ins class="diffchange diffchange-inline">concentrations. Therefore </ins>the <ins class="diffchange diffchange-inline">dose response experiments were repeated using lower concentrations </ins>of <ins class="diffchange diffchange-inline">this inducing agent. We have therefore tested media containing: 0.05%</ins>, <ins class="diffchange diffchange-inline">0</ins>.<ins class="diffchange diffchange-inline">1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. </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 class="diffchange diffchange-inline"></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Protocol</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and <del class="diffchange diffchange-inline">Raffinose </del>(2 %) as the carbon source. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and <ins class="diffchange diffchange-inline">raffinose </ins>(2 %) as the carbon source. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. The following evening <del class="diffchange diffchange-inline">861 µl of </del>this cell culture <del class="diffchange diffchange-inline">were </del>sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.<del class="diffchange diffchange-inline">3 </del>by <del class="diffchange diffchange-inline">10am </del>the following morning. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. The following evening this cell culture <ins class="diffchange diffchange-inline">was </ins>sub-cultured into a flask containing pre-warmed SD medium (50 mls) <ins class="diffchange diffchange-inline">with 2% raffinose, and one of a range of concentrations of galactose between 0.05% and 2% of galactose, </ins> to achieve an optical density at 600nm of 0.<ins class="diffchange diffchange-inline">6 </ins>by <ins class="diffchange diffchange-inline">9.00 am </ins>the following morning. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>3. <del class="diffchange diffchange-inline">At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. </del>Samples were <del class="diffchange diffchange-inline">then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), </del>washed <del class="diffchange diffchange-inline">once with PBS buffer and stored on ice. Once collected all samples were then dispensed in </del>PBS and diluted <del class="diffchange diffchange-inline">by a factor of </del>1/20 for <del class="diffchange diffchange-inline">flow cytometry </del>analysis.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>3. Samples were washed <ins class="diffchange diffchange-inline">into </ins>PBS<ins class="diffchange diffchange-inline">, </ins>and diluted 1/20 <ins class="diffchange diffchange-inline">in preparation </ins>for <ins class="diffchange diffchange-inline">FACS </ins>analysis.</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">FACS data </del> </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 480 nm, and a emission filter of 480 nm, </ins> </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of <del class="diffchange diffchange-inline">sensitiveity </del>to the inducing agent, with <del class="diffchange diffchange-inline">concentraitons </del>as low as 0.01% having significant inducing potential. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of <ins class="diffchange diffchange-inline">sensitivity </ins>to the inducing agent, with <ins class="diffchange diffchange-inline">concentrations </ins>as low as 0.01% having significant inducing potential. </div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138617&oldid=prevI.stansfield at 23:03, 24 October 20102010-10-24T23:03:20Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Aim</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The <del class="diffchange diffchange-inline">previous dose response </del>experiment <del class="diffchange diffchange-inline">using </del>the <del class="diffchange diffchange-inline">fluorometer (will link </del>the <del class="diffchange diffchange-inline">exp) did not show a steady increase </del>of <del class="diffchange diffchange-inline">GFP expression with increasing </del>galactose <del class="diffchange diffchange-inline">concentrations. Therefore </del>the <del class="diffchange diffchange-inline">experiment was repeated using lower concentrations </del>of <del class="diffchange diffchange-inline">this inducing agent. We have therefore tested media containing: 0.5%</del>, <del class="diffchange diffchange-inline">0</del>.<del class="diffchange diffchange-inline">1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The <ins class="diffchange diffchange-inline">aim of this </ins>experiment <ins class="diffchange diffchange-inline">was to test </ins>the <ins class="diffchange diffchange-inline">response of the GAL1 promoter in </ins>the <ins class="diffchange diffchange-inline">presence </ins>of galactose <ins class="diffchange diffchange-inline">over time, by measuring </ins>the <ins class="diffchange diffchange-inline">expression </ins>of <ins class="diffchange diffchange-inline">GFP</ins>, <ins class="diffchange diffchange-inline">the downstream gene</ins>. <ins class="diffchange diffchange-inline">Construct Gal1p-(Npep-GFP) was used in the experiments described here </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusion</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.<del class="diffchange diffchange-inline">5</del>% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.<ins class="diffchange diffchange-inline">1</ins>% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">The observed GFP induction curve suggests that the promoter behaves as an analogue switch only over a very narrow range of galactose concentrations.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
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</table>I.stansfieldhttp://2010.igem.org/wiki/index.php?title=Galactose_dose_response_of_Gal1_Promoter_in_pRS415&diff=138613&oldid=prevI.stansfield: New page: <h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1> <h3>Aim</h3> <p>The previous dose response experiment using the fluorometer (will link the ex...2010-10-24T23:02:56Z<p>New page: <h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1> <h3>Aim</h3> <p>The previous dose response experiment using the fluorometer (will link the ex...</p>
<p><b>New page</b></p><div><h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p>The previous dose response experiment using the fluorometer (will link the exp) did not show a steady increase of GFP expression with increasing galactose concentrations. Therefore the experiment was repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.5%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose. <br />
</p><br />
<br />
<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and Raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening 861 µl of this cell culture were sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.3 by 10am the following morning. <br><br />
<br />
<br><br />
3. At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. Samples were then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), washed once with PBS buffer and stored on ice. Once collected all samples were then dispensed in PBS and diluted by a factor of 1/20 for flow cytometry analysis.<br />
<br><br><br />
<br />
<h3>Results</h3><br />
<br />
FACS data </p><br />
<br />
<br><br><br />
<br />
[[Image: Gal-facs3.jpg|300 px]]<br />
<br />
<br><br><br />
<br />
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of sensitiveity to the inducing agent, with concentraitons as low as 0.01% having significant inducing potential. <br />
<br><br><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.5% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. <br />
The observed GFP induction curve suggests that the promoter behaves as an analogue switch only over a very narrow range of galactose concentrations.<br />
</p><br />
<br />
<br></div>I.stansfield