http://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&feed=atom&action=historyTeam:Hong Kong HKUST/pneumosensor/modules - Revision history2024-03-28T12:34:15ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=406808&oldid=prevJumnicole at 16:05, 1 September 20152015-09-01T16:05:36Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p class="first_letter_enhanced">To complete the story of competence regulation mechanism from <i>S. <del class="diffchange diffchange-inline">Pneumoniae</del></i>, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in <i>S. <del class="diffchange diffchange-inline">Pneumoniae</del></i>. The gene <i>comW</i> (SP0018) was found to be </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p class="first_letter_enhanced">To complete the story of competence regulation mechanism from <i>S. <ins class="diffchange diffchange-inline">pneumoniae</ins></i>, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in <i>S. <ins class="diffchange diffchange-inline">pneumoniae</ins></i>. The gene <i>comW</i> (SP0018) was found to be </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). Coexpression of ComW with &sigma;<sup>x</sup> restores the accumulation of &sigma;<sup>x</sup> and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor X against proteolysis by ClpXP and is required for full activity of &sigma;<sup>x</sup> in directing transcription of late competence genes (Piotrowski, Luo, and Morrison, 2009). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). Coexpression of ComW with &sigma;<sup>x</sup> restores the accumulation of &sigma;<sup>x</sup> and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor X against proteolysis by ClpXP and is required for full activity of &sigma;<sup>x</sup> in directing transcription of late competence genes (Piotrowski, Luo, and Morrison, 2009). </div></td></tr>
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</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=356917&oldid=prevJumnicole at 21:36, 17 October 20142014-10-17T21:36:26Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. First, we cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup><del class="diffchange diffchange-inline">, </del>and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. First, we cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup> and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br></div></td></tr>
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</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=356786&oldid=prevJumnicole at 21:35, 17 October 20142014-10-17T21:35:03Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. <del class="diffchange diffchange-inline">We firstly </del>cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup>, and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. <ins class="diffchange diffchange-inline">First, we </ins>cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup>, and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br></div></td></tr>
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</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=345775&oldid=prevJordyevan at 19:37, 17 October 20142014-10-17T19:37:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h7>&sigma;<sup>x</sup> and ComW protein are both produced by a constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, which continuously expresses &sigma;<sup>x</sup> required for P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters induction, and ComW protein is required for &sigma;<sup>x</sup> stabilization. ComW protein acts as a barrier that protects &sigma;<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increases the production of &sigma;<sup>x</sup>. The increase in &sigma;<sup>x</sup> production will increase the expression of green fluorescence protein by P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters.</h7></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h7>&sigma;<sup>x</sup> and ComW protein are both produced by a constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, which continuously expresses &sigma;<sup>x</sup> required for P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters induction, and ComW protein is required for &sigma;<sup>x</sup> stabilization. ComW protein acts as a barrier that protects &sigma;<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increases the production of &sigma;<sup>x</sup>. The increase in &sigma;<sup>x</sup> production will increase the expression of green fluorescence protein by P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters.</h7></div></td></tr>
</table>Jordyevanhttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=345710&oldid=prevJumnicole at 19:37, 17 October 20142014-10-17T19:37:08Z<p></p>
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</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=343255&oldid=prevGtong at 19:02, 17 October 20142014-10-17T19:02:07Z<p></p>
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</table>Gtonghttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=325999&oldid=prevJumnicole at 14:59, 17 October 20142014-10-17T14:59:08Z<p></p>
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</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=323319&oldid=prevJumnicole at 14:11, 17 October 20142014-10-17T14:11:22Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> cognate response regulator (RR) ComE<a href="http://parts.igem.org/Part:BBa_K1379051"> <del class="diffchange diffchange-inline">(</del>BBa_K1379051<del class="diffchange diffchange-inline">)</del></a>. When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> cognate response regulator (RR) ComE <ins class="diffchange diffchange-inline">(</ins><a href="http://parts.igem.org/Part:BBa_K1379051">BBa_K1379051</a><ins class="diffchange diffchange-inline">)</ins>. When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> expressed in our Pneumosensor.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> expressed in our Pneumosensor.</div></td></tr>
</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=323266&oldid=prevJumnicole at 14:10, 17 October 20142014-10-17T14:10:23Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> cognate response regulator (RR) ComE. When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> cognate response regulator (RR) ComE<ins class="diffchange diffchange-inline"><a href="http://parts.igem.org/Part:BBa_K1379051"> (BBa_K1379051)</a></ins>. When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> expressed in our Pneumosensor.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> expressed in our Pneumosensor.</div></td></tr>
</table>Jumnicolehttp://2014.igem.org/wiki/index.php?title=Team:Hong_Kong_HKUST/pneumosensor/modules&diff=321660&oldid=prevJumnicole at 13:38, 17 October 20142014-10-17T13:38:28Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p class="first_letter_enhanced">In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of <i>S. pneumoniae</i>. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in <i>S. pneumoniae</i> which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative &sigma;<sup>x</sup> (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the <del class="diffchange diffchange-inline">Competence Signal Peptide </del>(CSP). Upon stimulation by CSP, &sigma;<sup>x</sup> will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>x</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by &sigma;<sup>x</sup> were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the <i>S. pneumoniae</i> sensing device of high specificity by incorporating this system into <i>E. coli</i>. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p class="first_letter_enhanced">In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of <i>S. pneumoniae</i>. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in <i>S. pneumoniae</i> which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative &sigma;<sup>x</sup> (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the <ins class="diffchange diffchange-inline">competence-stimulating peptide </ins>(CSP). Upon stimulation by CSP, &sigma;<sup>x</sup> will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>x</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by &sigma;<sup>x</sup> were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the <i>S. pneumoniae</i> sensing device of high specificity by incorporating this system into <i>E. coli</i>. <br></div></td></tr>
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</table>Jumnicole