http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&feed=atom&action=historyTeam:Buenos Aires/Results/Strains - Revision history2024-03-29T06:49:15ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298728&oldid=prevAbush84: /* At different initial OD and proportions */2012-10-27T04:04:47Z<p><span class="autocomment">At different initial OD and proportions</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>As shown in <del class="diffchange diffchange-inline">graph </del>and table there is a basal growth that does not depend on the initial OD or strain proportion<del class="diffchange diffchange-inline">, of </del>a growth factor of 6 approximately.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>As shown in <ins class="diffchange diffchange-inline">the figure </ins>and table there is a basal growth that does not depend on the initial OD or strain proportion<ins class="diffchange diffchange-inline">. This residual growth produces </ins>a growth factor of 6 approximately.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>However we observed a much higher growth at the proportion 1:1 when the initial OD 0.25 and 0.1. <del class="diffchange diffchange-inline">Therefore we can assume </del>that at these proportions there is a natural cooperation between the strains <del class="diffchange diffchange-inline">and that should be the level </del>of <del class="diffchange diffchange-inline">growth that we would like to assess through our bioengineering. Besides we would like to be able in </del>the <del class="diffchange diffchange-inline">future </del>to <del class="diffchange diffchange-inline">tune the strains in order </del>to <del class="diffchange diffchange-inline">be able to obtain in the </del>proportions <del class="diffchange diffchange-inline">9:1 and 1:9 similar results to those obtained in the 1:1, at our own will</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>However we observed a much higher growth at the proportion 1:1 when the initial OD 0.25 and 0.1. <ins class="diffchange diffchange-inline">This suggests </ins>that at these proportions there is a natural cooperation between the strains<ins class="diffchange diffchange-inline">. The objective </ins>of the <ins class="diffchange diffchange-inline">project is </ins>to <ins class="diffchange diffchange-inline">build upon this natural cooperation and </ins>to <ins class="diffchange diffchange-inline">allow for tunable </ins>proportions.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== At the same initial OD: 0.2, followed over time ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== At the same initial OD: 0.2, followed over time ====</div></td></tr>
</table>Abush84http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298527&oldid=prevAbush84: /* Strain proportion measurement */2012-10-27T03:59:21Z<p><span class="autocomment">Strain proportion measurement</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).</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">Strain </del>proportion measurement ==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== <ins class="diffchange diffchange-inline">Strains </ins>proportion measurement ==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.</div></td></tr>
</table>Abush84http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298504&oldid=prevAbush84: /* Screening of strain proportion */2012-10-27T03:58:42Z<p><span class="autocomment">Screening of strain proportion</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).</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">Screening of strain </del>proportion ==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== <ins class="diffchange diffchange-inline">Strain </ins>proportion <ins class="diffchange diffchange-inline">measurement </ins>==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.</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>We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We mixed strains <ins class="diffchange diffchange-inline">TCY-</ins>3281 (<ins class="diffchange diffchange-inline">that </ins>expresses YFP) and <ins class="diffchange diffchange-inline">TCY-</ins>3265 (<ins class="diffchange diffchange-inline">that </ins>expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; 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>'''Description of Mixtures'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Description of Mixtures'''</div></td></tr>
</table>Abush84http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298451&oldid=prevAbush84: /* Measurement of strains fluorescence */2012-10-27T03:57:00Z<p><span class="autocomment">Measurement of strains fluorescence</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Discussion'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Discussion'''</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase<ins class="diffchange diffchange-inline">. Therefore we decided to use epifluorescence microscopy (see below)</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Screening of strain proportion ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Screening of strain proportion ==</div></td></tr>
</table>Abush84http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298359&oldid=prevAbush84: /* Description of strains */2012-10-27T03:54:53Z<p><span class="autocomment">Description of strains</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|CFP +(induced YFP)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|CFP +(induced YFP)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|Positive Control</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|Positive Control</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|TCY 3128</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|TCY 3128</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| style="text-align: center;" |(-H-T)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| style="text-align: center;" |(-H-T)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|CFP </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|CFP </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|His device testing</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|His device testing</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">|-</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|TCY 3081</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|TCY 3081</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| style="text-align: center;" |(-H-T)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| style="text-align: center;" |(-H-T)</div></td></tr>
</table>Abush84http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298345&oldid=prevAbush84: /* Description of strains */2012-10-27T03:54:25Z<p><span class="autocomment">Description of strains</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|Positive Control</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|Positive Control</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;">|TCY 3128</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;">| style="text-align: center;" |(-H-T)</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;">|CFP </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;">|His device testing</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;">|TCY 3081</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;">| style="text-align: center;" |(-H-T)</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;">|YFP </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;">|Trp device testing</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; 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>| rowspan="2" style="text-align: center;" |</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| rowspan="2" style="text-align: center;" |</div></td></tr>
</table>Abush84http://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=298070&oldid=prevVparasco: /* Result */2012-10-27T03:44:47Z<p><span class="autocomment">Result</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Table:''' Number of colonies counted per plate.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Table:''' Number of colonies counted per plate.</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>We <del class="diffchange diffchange-inline">expect </del>to see a decrease in the number of colonies <del class="diffchange diffchange-inline">- </del>because of cell death. We found that this was not the case<del class="diffchange diffchange-inline">, </del> in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We <ins class="diffchange diffchange-inline">expected </ins>to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.</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>[[File:Bsas2012kdeathcells.png| 500px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Bsas2012kdeathcells.png| 500px]]</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;"></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>We can infer from this data that though they have not died, they may have enter into a <del class="diffchange diffchange-inline">'''...Alan </del>state<del class="diffchange diffchange-inline">'''</del>. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We can infer from this data that though they have not died, they may have enter into a <ins class="diffchange diffchange-inline">persistant </ins>state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.</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> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">These results are consistent with the chosen parameters</ins>. <ins class="diffchange diffchange-inline">Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">{|</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">|[[File:BsAs2012_celldeath</del>.<del class="diffchange diffchange-inline">png | 100px]]</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">|[[File:BsAs2012_celldeath2.png| 100px]]</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">|</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">|}</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> FigureX</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
</table>Vparascohttp://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=297753&oldid=prevLucho.Moro: /* Measurement of Trp in medium and Basal Production */2012-10-27T03:34:50Z<p><span class="autocomment">Measurement of Trp in medium and Basal Production</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Growth in coculture was puzzling, as it resulted in more colonies than the expected. If cooperation was effective, we expected to see as many colonies as "seed" cells, not more. Revertion of cells from the "lawn" doesn't explain the number of colonies either. Probably a combination of both these effects are taking place.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Growth in coculture was puzzling, as it resulted in more colonies than the expected. If cooperation was effective, we expected to see as many colonies as "seed" cells, not more. Revertion of cells from the "lawn" doesn't explain the number of colonies either. Probably a combination of both these effects are taking place.</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>== Measurement of Trp in medium <del class="diffchange diffchange-inline">and Basal Production </del>==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== Measurement of Trp in medium ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; 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>To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted by natural strains to the medium, so we can compare. With that end in mind, we designed a protocol for measurement of tryptophan in medium, based in its fluorescense at 350nm, when excited with 295nm light.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted by natural strains to the medium, so we can compare. With that end in mind, we designed a protocol for measurement of tryptophan in medium, based in its fluorescense at 350nm, when excited with 295nm light.</div></td></tr>
</table>Lucho.Morohttp://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=297739&oldid=prevLucho.Moro: /* Measurement of Trp in medium and Basal Production */2012-10-27T03:34:27Z<p><span class="autocomment">Measurement of Trp in medium and Basal Production</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To determine Trp concentration, we must first have a way to transform our readings (intensity) to a more useful output, so we made a calibration curve, through serialized 1:2 dilutions of our medium, which Trp's concentration is 20μg/ml, until approximately constant intensity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To determine Trp concentration, we must first have a way to transform our readings (intensity) to a more useful output, so we made a calibration curve, through serialized 1:2 dilutions of our medium, which Trp's concentration is 20μg/ml, until approximately constant intensity.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The procedure to measure secretion rates will be growing the strain from a known OD in exponential growth phase in -T medium and plotting it's OD over time, spin-drying at time=t, retrieving the supernatant's Trp concentration and dividing it by the integral of OD vs. time between time=0 and time=t, so we get to a rate which will be proportional to the number of cells in the culture, which means we can actually compare between different strains. Since our medium is free from Trp, all of it should come from within the cells, and if the culture is growing at exponential rates, lysis should be negligible, so the only explanation would be cells exporting their own Trp.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The procedure to measure secretion rates will be growing the strain from a known OD in exponential growth phase in -T medium and plotting it's OD over time, spin-drying at time=t, retrieving the supernatant's Trp concentration and dividing it by the integral of OD vs. time between time=0 and time=t, so we get to a rate which will be proportional to the number of cells in the culture, which means we can actually compare between different strains. Since our medium is free from Trp, all of it should come from within the cells, and if the culture is growing at exponential rates, lysis should be negligible, so the only explanation would be cells exporting <ins class="diffchange diffchange-inline">and diffusing </ins>their own Trp.</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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Through this experiment we can be sure that we would be able to measure increase of Trp in medium as it is exported from the cells, within the biological range of export.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Through this experiment we can be sure that we would be able to measure increase of Trp in medium as it is exported from the cells, within the biological range of export.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/ml, we assume that's the saturation point of the curve. If we get bigger intensities than the one corresponding to it, we will dilute the sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/ml, we assume that's the saturation point of the curve. If we get bigger intensities than the one corresponding to it, we will dilute the sample.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<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;">Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.</del></div></td><td colspan="2"> </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>== Growth dependence on the Trp and His concentrations ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Growth dependence on the Trp and His concentrations ==</div></td></tr>
</table>Lucho.Morohttp://2012.igem.org/wiki/index.php?title=Team:Buenos_Aires/Results/Strains&diff=297702&oldid=prevLucho.Moro: /* Measurement of Trp in medium and Basal Production */2012-10-27T03:33:04Z<p><span class="autocomment">Measurement of Trp in medium and Basal Production</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As a previous step, we checked that none of the other aminoacids used in the medium interferes, by graphically comparing the spectres for uncomplemented medium and medium complemented with leucine, uracile and histidine, at an appropiate range.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As a previous step, we checked that none of the other aminoacids used in the medium interferes, by graphically comparing the spectres for uncomplemented medium and medium complemented with leucine, uracile and histidine, at an appropiate range.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To determine Trp concentration, we must first have a way to transform our readings (intensity) to a more useful output, so we made a calibration curve, through serialized 1:2 dilutions of our medium, which Trp's concentration is <del class="diffchange diffchange-inline">50mg</del>/<del class="diffchange diffchange-inline">mL</del>, until approximately constant intensity.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To determine Trp concentration, we must first have a way to transform our readings (intensity) to a more useful output, so we made a calibration curve, through serialized 1:2 dilutions of our medium, which Trp's concentration is <ins class="diffchange diffchange-inline">20μg</ins>/<ins class="diffchange diffchange-inline">ml</ins>, until approximately constant intensity.</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>The procedure to measure secretion rates will be growing the strain from a known OD in exponential growth phase in -T medium and plotting it's OD over time, spin-drying at time=t, retrieving the supernatant's Trp concentration and dividing it by the integral of OD vs. time between time=0 and time=t, so we get to a rate which will be proportional to the number of cells in the culture, which means we can actually compare between different strains. Since our medium is free from Trp, all of it should come from within the cells, and if the culture is growing at exponential rates, lysis should be negligible, so the only explanation would be cells exporting their own Trp.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The procedure to measure secretion rates will be growing the strain from a known OD in exponential growth phase in -T medium and plotting it's OD over time, spin-drying at time=t, retrieving the supernatant's Trp concentration and dividing it by the integral of OD vs. time between time=0 and time=t, so we get to a rate which will be proportional to the number of cells in the culture, which means we can actually compare between different strains. Since our medium is free from Trp, all of it should come from within the cells, and if the culture is growing at exponential rates, lysis should be negligible, so the only explanation would be cells exporting their own Trp.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| class="wikitable" border="1"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| class="wikitable" border="1"</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> |-</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> |-</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> |<!--column1-->[[File:<del class="diffchange diffchange-inline">Trp-bsas2012</del>.png | 250px]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> |<!--column1-->[[File:<ins class="diffchange diffchange-inline">Curva</ins>.png | 250px]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> |-</div></td><td class='diff-marker'> </td><td style="background: #eee; 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> |Graph:Tryptophan calibration curve</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> |Graph:Tryptophan calibration curve</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Results ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Results ====</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">As can be seen from the graph the screening of the concentration of the Trp in medium describes an almost lineal function. </del>Through this experiment we can be sure that we would be able to measure increase of Trp in medium as it is exported from the cells, within the biological range of export.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Through this experiment we can be sure that we would be able to measure increase of Trp in medium as it is exported from the cells, within the biological range of export.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The sensitivity of this method seems to be enough to detect concentrations as low as ~0.<del class="diffchange diffchange-inline">02mg</del>/<del class="diffchange diffchange-inline">mL</del>, and as high as <del class="diffchange diffchange-inline">50mg</del>/<del class="diffchange diffchange-inline">mL</del>, maybe more. Since our medium is <del class="diffchange diffchange-inline">50mg</del>/<del class="diffchange diffchange-inline">mL</del>, we assume that's the saturation point of the curve. If we get bigger intensities than the one corresponding to it, we will dilute the sample.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The sensitivity of this method seems to be enough to detect concentrations as low as ~0.<ins class="diffchange diffchange-inline">01μg</ins>/<ins class="diffchange diffchange-inline">ml</ins>, and as high as <ins class="diffchange diffchange-inline">20μg</ins>/<ins class="diffchange diffchange-inline">ml</ins>, maybe more. Since our medium is <ins class="diffchange diffchange-inline">20μg</ins>/<ins class="diffchange diffchange-inline">ml</ins>, we assume that's the saturation point of the curve. If we get bigger intensities than the one corresponding to it, we will dilute the sample.</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>Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.</div></td></tr>
</table>Lucho.Moro