http://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&feed=atom&action=historyTeam:Bologna/Project - Revision history2024-03-28T17:51:31ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=106043&oldid=prevMavi.amaduzzi: /* Collaboration with other iGEM2008 Teams */2008-11-20T16:52:25Z<p><span class="autocomment">Collaboration with other iGEM2008 Teams</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2008.igem.org/Team:Bologna/Project ''Up'']</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2008.igem.org/Team:Bologna/Project ''Up'']</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=Collaboration with other <del class="diffchange diffchange-inline">iGEM2008 </del>Teams =</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Collaboration with other <ins class="diffchange diffchange-inline">iGEM 2008 </ins>Teams =</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After the last year competition, at the beginning of the 2008, we decided to get a new team started for iGEM2008 competition. In April, we got in contact with Prof. Paolo Magni, who wanted to start a new team in [[Team:UNIPV-Pavia|Pavia]] for iGEM2008. So, in order to share experiences and ideas about iGEM, and to show him what kind of wet lab resources are necessary to develop a Synthetic Biology project, we met at the Cellular and Molecular Engineering Laboratory of the University of Bologna- Cesena Campus. After this first meeting, there have been other chances to meet during the summer. In particular, several conference calls were organized and two meetings were scheduled in Pisa and Bressanone (Italy). It was fundamental to compare lab protocols and techniques to help each other avoiding mistakes and speeding up project progress. The main topics of our discussion were the optimization of plasmid resuspension and ligation reaction steps as well as how to measure fluorescence. Finally, before DNA Repository quality control publication on the Registry web site, we cross-checked some parts that showed problems after DNA transformation. Problems had been confirmed by quality control results (parts' sequences classified as "inconsistent"). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After the last year competition, at the beginning of the 2008, we decided to get a new team started for iGEM2008 competition. In April, we got in contact with Prof. Paolo Magni, who wanted to start a new team in [[Team:UNIPV-Pavia|Pavia]] for iGEM2008. So, in order to share experiences and ideas about iGEM, and to show him what kind of wet lab resources are necessary to develop a Synthetic Biology project, we met at the Cellular and Molecular Engineering Laboratory of the University of Bologna- Cesena Campus. After this first meeting, there have been other chances to meet during the summer. In particular, several conference calls were organized and two meetings were scheduled in Pisa and Bressanone (Italy). It was fundamental to compare lab protocols and techniques to help each other avoiding mistakes and speeding up project progress. The main topics of our discussion were the optimization of plasmid resuspension and ligation reaction steps as well as how to measure fluorescence. Finally, before DNA Repository quality control publication on the Registry web site, we cross-checked some parts that showed problems after DNA transformation. Problems had been confirmed by quality control results (parts' sequences classified as "inconsistent"). </div></td></tr>
</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=106042&oldid=prevMavi.amaduzzi at 16:51, 20 November 20082008-11-20T16:51:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>= Ecoli.PROM: an Erasable and Programmable Genetic Memory in ''E. coli''=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>= Ecoli.PROM: an Erasable and Programmable Genetic Memory in ''E. coli''=</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">__FORCETOC__</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Circuito2.jpg|510px|right|thumbnail|Figure 1: Genetic circuit]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Circuito2.jpg|510px|right|thumbnail|Figure 1: Genetic circuit]]</div></td></tr>
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</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103265&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:30:03Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</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 Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to <del class="diffchange diffchange-inline">evoid </del>random memory switching but it is possible to set (or to reset) the memory with proper [[Team:Bologna/Modeling#Numerical_simulations|UV and IPTG stimulations]]. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to <ins class="diffchange diffchange-inline">avoid </ins>random memory switching but it is possible to set (or to reset) the memory with proper [[Team:Bologna/Modeling#Numerical_simulations|UV and IPTG stimulations]]. </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>Since the Registry lacks promoters with the K-parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K-parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Thus, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences each with a different repressor [[Team:Bologna/Wetlab#At_last..._Operator_sites_as_BioBricks.21|binding affinity]] to the repressor protein. We isolated each operator with the intention to clone them into BioBrick standard assembly plasmids.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Thus, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences each with a different repressor [[Team:Bologna/Wetlab#At_last..._Operator_sites_as_BioBricks.21|binding affinity]] to the repressor protein. We isolated each operator with the intention to clone them into BioBrick standard assembly plasmids.</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">assess </del>the functionality of independent operators by assembling one circuit (Figure 3, closed-loop configuration) where the constitutive promoter BBa_J23118 and LacI operator 2 were auto-regulated by the LacI repressor. GFP was used as the reporter. An identical construct without the LacI operator site was used as the negative control (Figure 3, open-loop configuration).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We <ins class="diffchange diffchange-inline">assessed </ins>the functionality of independent operators by assembling one circuit (Figure 3, closed-loop configuration) where the constitutive promoter BBa_J23118 and LacI operator 2 were auto-regulated by the LacI repressor. GFP was used as the reporter. An identical construct without the LacI operator site was used as the negative control (Figure 3, open-loop configuration).</div></td></tr>
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</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103206&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:28:34Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In the genetic Flip-Flop, the amount of LacI to set ON the memory is induced by an <del class="diffchange diffchange-inline">[https://2008.igem.org/Team:Bologna/Modeling#The_genetic_Flip-Flop </del>UV-sensitive trigger <del class="diffchange diffchange-inline">]</del>. To test the lexA operator functionality, we assembled the BBa_K079050 construct (Figure 6).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In the genetic Flip-Flop, the amount of LacI to set ON the memory is induced by an UV-sensitive trigger. To test the lexA operator functionality, we assembled the BBa_K079050 construct (Figure 6).</div></td></tr>
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</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103157&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:26:59Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K-parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K-parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Thus, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences each with a different repressor [[binding affinity]] to the repressor protein. We isolated each operator with the intention to clone them into BioBrick standard assembly plasmids.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Thus, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences each with a different repressor [[<ins class="diffchange diffchange-inline">Team:Bologna/Wetlab#At_last..._Operator_sites_as_BioBricks.21|</ins>binding affinity]] to the repressor protein. We isolated each operator with the intention to clone them into BioBrick standard assembly plasmids.</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>We assess the functionality of independent operators by assembling one circuit (Figure 3, closed-loop configuration) where the constitutive promoter BBa_J23118 and LacI operator 2 were auto-regulated by the LacI repressor. GFP was used as the reporter. An identical construct without the LacI operator site was used as the negative control (Figure 3, open-loop configuration).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We assess the functionality of independent operators by assembling one circuit (Figure 3, closed-loop configuration) where the constitutive promoter BBa_J23118 and LacI operator 2 were auto-regulated by the LacI repressor. GFP was used as the reporter. An identical construct without the LacI operator site was used as the negative control (Figure 3, open-loop configuration).</div></td></tr>
</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103136&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:25:55Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</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 Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with proper [[UV and IPTG stimulations]]. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with proper [[<ins class="diffchange diffchange-inline">Team:Bologna/Modeling#Numerical_simulations|</ins>UV and IPTG stimulations]]. </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>Since the Registry lacks promoters with the K-parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K-parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td></tr>
</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103124&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:25:10Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with proper [[UV and IPTG stimulations]]. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with proper [[UV and IPTG stimulations]]. </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>Since the Registry lacks promoters with the K parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K<ins class="diffchange diffchange-inline">-</ins>parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Thus, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences each with a different repressor [[binding affinity]] to the repressor protein. We isolated each operator with the intention to clone them into BioBrick standard assembly plasmids.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Thus, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences each with a different repressor [[binding affinity]] to the repressor protein. We isolated each operator with the intention to clone them into BioBrick standard assembly plasmids.</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>
</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103113&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:24:42Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</span></p>
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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>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with [[<del class="diffchange diffchange-inline">proper </del>UV and IPTG stimulations]]. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with <ins class="diffchange diffchange-inline">proper </ins>[[UV and IPTG stimulations]]. </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>Since the Registry lacks promoters with the K parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td></tr>
</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103104&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:24:23Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</span></p>
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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>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with proper UV and IPTG <del class="diffchange diffchange-inline">stimulation</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>As shown in Figure 2, the coexistence of two stable equilibria (LacI-ON and TerR-ON) is guaranteed for[[image:ki.jpg]]and [[image:kr.jpg]]model parameters greater than 3 in a large range of values (bistability range). To have a good robustness we fixed [[image:ki.jpg]] and [[image:kr.jpg]] equal to 10. For this value the circuit is sufficently distant from the bifurcation lines to evoid random memory switching but it is possible to set (or to reset) the memory with <ins class="diffchange diffchange-inline">[[</ins>proper UV and IPTG <ins class="diffchange diffchange-inline">stimulations]]</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>Since the Registry lacks promoters with the K parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since the Registry lacks promoters with the K parameter equal to 10, we decided to introduce operator site parts in order to build regulated promoter with the fixed transcriptional strenght and repression sensitivity.</div></td></tr>
</table>Mavi.amaduzzihttp://2008.igem.org/wiki/index.php?title=Team:Bologna/Project&diff=103083&oldid=prevMavi.amaduzzi: /* Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli */2008-10-30T03:23:37Z<p><span class="autocomment">Ecoli.PROM: an Erasable and Programmable Genetic Memory in E. coli</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The molecular circuit can switch between two different stable states (LacI-ON and TetR-ON), driven by the external stimuli UVc and IPTG. LacI-ON represents the stable state in which LacI gene is active and LacI protein represses the TetR gene expression, with a positive feedback. Therefore, the LacI-ON state coincides with the TetR-OFF condition. On the contrary, the TetR-ON represents the state with the TetR gene active and the LacI gene silenced (LacI-OFF). Owing to the coexistence of two stable states (bistability), this circuit is capable of serving as a binary memory. We denominated it genetic Flip-Flop since it works as a SR Latch: LacI state is the [[Image:q.jpg]] output and TetR state is the [[Image:qneg.jpg]] output. Uvc is the set signal and IPTG is the reset signal. Indeed, IPTG stimulation inhibits LacI repressor, thus can cause the transition from the LacI-ON state to the TetR-ON. UVc radiation, inactivating LexA repressor through the [[Team:Bologna/Modeling#UV_Radiation:_SOS_system|SOS response]] [Friedberg et al. 1995] can cause the opposite transition from LacI-ON to TetR-ON.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The molecular circuit can switch between two different stable states (LacI-ON and TetR-ON), driven by the external stimuli UVc and IPTG. LacI-ON represents the stable state in which LacI gene is active and LacI protein represses the TetR gene expression, with a positive feedback. Therefore, the LacI-ON state coincides with the TetR-OFF condition. On the contrary, the TetR-ON represents the state with the TetR gene active and the LacI gene silenced (LacI-OFF). Owing to the coexistence of two stable states (bistability), this circuit is capable of serving as a binary memory. We denominated it genetic Flip-Flop since it works as a SR Latch: LacI state is the [[Image:q.jpg]] output and TetR state is the [[Image:qneg.jpg]] output. Uvc is the set signal and IPTG is the reset signal. Indeed, IPTG stimulation inhibits LacI repressor, thus can cause the transition from the LacI-ON state to the TetR-ON. UVc radiation, inactivating LexA repressor through the [[Team:Bologna/Modeling#UV_Radiation:_SOS_system|SOS response]] [Friedberg et al. 1995] can cause the opposite transition from LacI-ON to TetR-ON.</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 core elements of this epigenetic memory are the two mutually regulated promoters (see Figure 1), each designed as a constitutive promoter flanking an indipendent operator site. In this way, the promoter transcriptional strength and the repressor binding affinity can be independently fixed. [[Team:Bologna/Modeling#Mathematical_Model|Mathematical model analysis and computer simulations]] were used to obtain a rational design of the regulated promoters. By the model we found the analytic relationships to quote the regulated promoter in terms of transcriptional strength and sensitivity to the repressor and we established such a relevant circuit properties as the bistability and the [[dynamical response to inputs]].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The core elements of this epigenetic memory are the two mutually regulated promoters (see Figure 1), each designed as a constitutive promoter flanking an indipendent operator site. In this way, the promoter transcriptional strength and the repressor binding affinity can be independently fixed. [[Team:Bologna/Modeling#Mathematical_Model|Mathematical model analysis and computer simulations]] were used to obtain a rational design of the regulated promoters. By the model we found the analytic relationships to quote the regulated promoter in terms of transcriptional strength and sensitivity to the repressor and we established such a relevant circuit properties as the bistability and the [[<ins class="diffchange diffchange-inline">Team:Bologna/Modeling#Numerical_simulations|</ins>dynamical response to inputs]].</div></td></tr>
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</table>Mavi.amaduzzi