http://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&feed=atom&action=historySynchronization of Cell Cycles - Revision history2024-03-28T17:42:16ZRevision history for this page on the wikiMediaWiki 1.16.5http://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=20182&oldid=prevSugat: /* Constructs */2006-10-30T18:57:13Z<p><span class="autocomment">Constructs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cell cycle modulation: DnaA sequestration</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cell cycle modulation: DnaA sequestration</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>DnaA binding sites <del class="diffchange diffchange-inline">are put </del>downstream to pLux and pLac promoters.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">DnaA is cell cycle regulator in ''E. coli''. We have used </ins>DnaA binding sites downstream to pLux and pLac promoters <ins class="diffchange diffchange-inline">to regulate free DnaA concentration</ins>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td></tr>
</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19973&oldid=prevSugat: /* Constructs */2006-10-30T13:23:00Z<p><span class="autocomment">Constructs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Sync circuit.png|thumb|left|Synchronization circuit]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Sync circuit.png|thumb|left|Synchronization circuit]]</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">In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core </del>oscillator.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Core </ins>oscillator<ins class="diffchange diffchange-inline">: The E</ins>.<ins class="diffchange diffchange-inline">coli cell cycle</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Pcya regulate expression of LuxI-cfp(LVA) fusion protein. LuxI is fused to cfp(LVA) in order to monitor its expression and presence of LVA tag on the fusion protein would reduce the half life. This would increse peak to trough ratio. </ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Oscillator coupling: Vibrio quorum sensing machinery</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Oscillators are coupled by feeding the oscillatory signal through the quorum sensing circuit. AiiA an intracellular protein that drgrades AI is expresed from an inducible promoter in order to increase peak to trough ratio for AI.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Cell cycle modulation: DnaA sequestration</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">DnaA binding sites are put downstream to pLux and pLac promoters.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td></tr>
</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19963&oldid=prevSugat at 13:17, 30 October 20062006-10-30T13:17:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Constructs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Constructs===</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;">[[Image:Sync circuit.png|thumb|left|Synchronization circuit]]</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.</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>
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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:Sync circuit.png|thumb|left|Synchronization circuit]]</del></div></td><td colspan="2"> </td></tr>
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</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19962&oldid=prevSugat at 13:16, 30 October 20062006-10-30T13:16:37Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Sync circuit.png|thumb|left|Synchronization circuit]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Sync circuit.png|thumb|left|Synchronization circuit]]</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td></tr>
</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19961&oldid=prevSugat: /* Constructs */2006-10-30T13:15:58Z<p><span class="autocomment">Constructs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Constructs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Constructs===</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>In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.</div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[Image:Sync circuit.png|thumb|left|Synchronization circuit]]</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Testing oscillations===</div></td></tr>
</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19954&oldid=prevSugat at 13:09, 30 October 20062006-10-30T13:09:38Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Aim===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Aim===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our aim is to develop a synthetic gene network to synchronize ''E. coli'' cell cycles<del class="diffchange diffchange-inline">. Cell cycle is a robust biological oscillator. We plan to couple these oscillator using LuxI-LuxR based quorum sensing signaling. The cell cycle phase will be modulated using DnaA sequestration</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our aim is to develop a synthetic gene network to synchronize ''E. coli'' cell cycles. </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>===Approach===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Approach===</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 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;">Cell cycle is a robust biological oscillator. Theoretical work predicts that weak coupling of cellular oscillators should result in their synchronization [Garcia-Ojalvo et al., 2005]. Our core oscilltor is a cell cycle phase dependant promoter- Pcya.</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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We plan to couple these oscillators using LuxI-LuxR based quorum sensing signaling machinery.The core oscilltor will regulate expression luxI(an enzyme that produces an autoinducer acyl-homoserine-lactone).Oscilltory expression of luxI would result in oscilltory production of the autoinducer(AI). This in turn would cause oscillatory expression from a luxR-AI dependant promoter. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">The cell cycle phase will be modulated using DnaA sequestration. DnaA is a cell cycle regulator in ''E.coli''. Cell cycle progression is affected by decrease in free and active DnaA molecules is a cell. DnaA temperature sensitive mutants are used for cell cycle synchronization. We have used DnaA binding sites on plasmid to 'sequester' free DnaA molecules. We expect transcription from upstream promoter would change concentration of free DnaA inside a cell. We have put luxR dependant promoter upstream to DnaA binding sites. Thus, concentration of free DnaA concentration is dependant on AI concentation. This would lead to regulation of local cell cycle phase based on global phase.</ins></div></td></tr>
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</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19915&oldid=prevSugat: /* Aim */2006-10-30T12:24:15Z<p><span class="autocomment">Aim</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Aim===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Aim===</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">Our aim is to develop a synthetic gene network to synchronize ''E. coli'' </ins>cell cycles<ins class="diffchange diffchange-inline">. Cell cycle is a robust biological oscillator. We plan to couple these oscillator using LuxI-LuxR based quorum sensing signaling. The cell cycle phase will be modulated using DnaA sequestration</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">We are interested in synthetically synchronizing </del>cell cycles. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Approach===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Approach===</div></td></tr>
</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19857&oldid=prevSugat: /* Synchronization of Cell Cycles */2006-10-30T11:11:10Z<p><span class="autocomment">Synchronization of Cell Cycles</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[NCBS_Bangalore_2006#The_Living_Networks_Wiki|IGEM Bangalore HOME]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[NCBS_Bangalore_2006#The_Living_Networks_Wiki|IGEM Bangalore HOME]]</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">==Synchronization of Cell Cycles==</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Group II==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Group II==</div></td></tr>
</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=19856&oldid=prevSugat at 11:10, 30 October 20062006-10-30T11:10:50Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Synchronization of Cell Cycles==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Synchronization of Cell Cycles==</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">I am documenting the data that I presented in the Living Netwok Jamboree on June 3rd.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">==Group II==</ins></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We are interested in synthetically synchronizing cell cycles. In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.LuxI tagged with cfp(+LVA) is expressed from this promoter.LuxI oscillations will result in AI(autoinducer) oscillations and this would result in oscillations of AI dependant promoter. YFP is expressed from such a promoter.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Aim===</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We are interested in synthetically synchronizing cell cycles. </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Approach===</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </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">===Constructs===</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Testing oscillations===</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LuxI tagged with cfp(+LVA) is expressed from this promoter.LuxI oscillations will result in AI(autoinducer) oscillations and this would result in oscillations of AI dependant promoter. YFP is expressed from such a promoter.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>CFP expression is readout for the core oscillator-Pcya and YFP is readout of the expression of the quorum sensing promoter-Plux.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>CFP expression is readout for the core oscillator-Pcya and YFP is readout of the expression of the quorum sensing promoter-Plux.</div></td></tr>
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</table>Sugathttp://2006.igem.org/wiki/index.php?title=Synchronization_of_Cell_Cycles&diff=18511&oldid=prevDhanya at 09:07, 28 October 20062006-10-28T09:07:24Z<p></p>
<p><b>New page</b></p><div>[[NCBS_Bangalore_2006#The_Living_Networks_Wiki|IGEM Bangalore HOME]]<br />
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==Synchronization of Cell Cycles==<br />
<br />
I am documenting the data that I presented in the Living Netwok Jamboree on June 3rd.<br />
<br />
We are interested in synthetically synchronizing cell cycles. In our genetic construct, we have used Pcya (adenylate cyclase promoter-that is known to oscillate in cell cycle dependant manner) as a core oscillator.LuxI tagged with cfp(+LVA) is expressed from this promoter.LuxI oscillations will result in AI(autoinducer) oscillations and this would result in oscillations of AI dependant promoter. YFP is expressed from such a promoter.<br />
CFP expression is readout for the core oscillator-Pcya and YFP is readout of the expression of the quorum sensing promoter-Plux.<br />
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[[Image:cfp.jpg|thumb|left|160 px|Testing the core oscillator Pcya]]<br />
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Y axis is the mean cfp fluorescence of a cell. X-axis is a correlate of cell cycle phase.Here, we have used cell length as a crude correlate for cell cycle phase.The same data is platted twice for visual representation of oscillation. <br />
RFP expression is control.<br />
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[[Image:yfp.jpg|thumb|left|160 px|Testing oscillations at Plux ]]<br />
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Y axis is the mean yfp fluorescence of a cell. X-axis is major axis length of a cell which is a correlate of cell cycle phase.<br />
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--[[User:Sugat|Sugat]] 06:49, 8 July 2006 (EDT)</div>Dhanya