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| <div id="description_area"> | | <div id="description_area"> |
| <h2>Pneumosensor Future Work</h2> | | <h2>Pneumosensor Future Work</h2> |
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| <td class= "content_cell" colspan= "2"> | | <td class= "content_cell" colspan= "2"> |
| <div class= "content_area_two_row"> | | <div class= "content_area_two_row"> |
- | <p>
| + | <p> |
| Our team designed but was unable to test this module due to the limitation of not being able to work directly with <i>Streptococcus | | Our team designed but was unable to test this module due to the limitation of not being able to work directly with <i>Streptococcus |
| Pneumoniae</i> (Biosafety level 2) in our lab. This module proposes to kill <i>Streptococcus Pneumoniae</i> upon detection when coupled | | Pneumoniae</i> (Biosafety level 2) in our lab. This module proposes to kill <i>Streptococcus Pneumoniae</i> upon detection when coupled |
- | with the detection and regulation modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal. | + | with the detection and modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal. |
| </p> | | </p> |
| <p> | | <p> |
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- | <p> The enzymes are tagged with osmY (Washington 2012) via a linker to be exported out of <i>Escherichia coli</i>. Both enzymes have | + | <p> The enzymes are tagged with osmY (<a href="http://parts.igem.org/Part:BBa_K892008?title=Part:BBa_K892008">BBa_K892008</a>, Washington 2012) via a linker to be exported out of <i>Escherichia coli</i>. Both enzymes have |
| very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases. | | very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases. |
| Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic | | Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic |
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| </div> | | </div> |
| </table> | | </table> |
| </div> | | </div> |
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- | | + | <div class='content_1'><h3>P<sub>comCDE</sub></h3> |
| + | <table class="content_table" align= "center" > |
| + | <tr class= "content_row"> |
| + | <td class= "content_cell"> |
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| + | <p>P<sub>comCDE</sub> is a promoter induced by phosphorylated ComE, which can be replaced by a phosphorylmimetic mutant (ComE<sup>D58E</sup>) in experiment. We could not |
| + | manage to verify the sequence of our ligated product, P<sub>comCDE</sub>- BBa_E0240. Therefore, in the future, we hope to continue the verification process, and complete our |
| + | construct.</p> |
| + | |
| + | </td> |
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| + | </div> |
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| <!-- one row of content , two column one picture left--> | | <!-- one row of content , two column one picture left--> |
- | <div class='content_1'><h3><i>comX</i>, P<sub>celA</sub>, P<sub>helicase</sub> </h3> | + | <div class='content_1'><h3>σ<sup>x</sup>, P<sub>celA</sub>, P<sub>comFA</sub> </h3> |
| <table class="content_table" align= "center" > | | <table class="content_table" align= "center" > |
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- | <p> 1. Put inducible promoter upstream of RBS (BBa_B0034), comX gene, and terminator (BBa_B0015). An example of inducible promoter is BBa_I0500. Hence, by putting an inducible promoter, we can tune the level of comX protein expression and characterize combox promoters (PcelA and Phelicase) on different level of comX concentration. | + | <p> P<sub>celA</sub> and P<sub>comFA</sub> promoters are promoter that is regulated by σ<sup>x</sup>. We have proven that in the presence of σ<sup>x</sup>, P<sub>celA</sub> and P<sub>comFA</sub> could express GFP. However, we did not manage to characterize the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> in different concentration of σ<sup>x</sup>. So, a possible future work is to put inducible promoter upstream of RBS (BBa_B0034), σ<sup>x</sup> gene, and terminator (BBa_B0015). An example of inducible promoter is BBa_I0500. Hence, by putting an inducible promoter, we can tune the level of σ<sup>x</sup> expression and characterize Com-Box promoters (P<sub>celA</sub> and P<sub>comFA</sub>) on different level of σ<sup>x</sup> concentration. |
- | <br>
| + | |
- | <br>
| + | |
- | 2. Further characterization of combox promoters (PcelA and Phelicase) by making a 3-D Graph for time, comX concentration, and Fluorescence expression.
| + | |
- | <br> | + | |
- | <br>
| + | |
- | 3. Characterizing combox promoters (PcelA and Phelicase) specificity by introducing other proteins with similar structure as comX protein.
| + | |
| <br> | | <br> |
| <br> | | <br> |
- | 4. Continue comW construction, ligate with terminator (BBa_B0015), and introduce it to E.coli DH10B strain. | + | Moreover,ComW is a protein that function to protect σ<sup>x</sup> from degradation. It is necessary to have ComW protein as it could increase the amount of σ<sup>x</sup> to regulate P<sub>celA</sub> and P<sub>comFA</sub> promoters. However, due to the time constrains, we were unable to finish the construct of ComW generator. Hence, possible future work would be continuing ComW generator construct by ligating BBa_K880005-<i>comW</i> to a double terminator (BBa_B0015), and introduce it to <i>E.coli</i> DH10B strain. Then, characterization of <i>comW</i> could be performed by measuring the amount of σ<sup>x</sup> with and without ComW protein. |
| <br> | | <br> |
- | <br>
| + | |
- | 5. Characterization of comW by measuring the amount of comX protein with and without comW protein. (The function of comW protein is to protect comX protein from being degraded by ClpXP degradation enzyme)
| + | |
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| </p> | | </p> |
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| <p><br> | | <p><br> |
| <u>References</u> | | <u>References</u> |
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| and -Resistant <i>Streptococcus pneumoniae</i> Strains" 2003 | | and -Resistant <i>Streptococcus pneumoniae</i> Strains" 2003 |
| </p> | | </p> |
- | </td> | + | </div></td> |
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- | <td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor">Pneumosensor</a></h4>
| + | <div style= "text-align:center; background-color:#00CAAA; font-size: 12px;" class="scrollToTop"> |
- | <ul class= 'site_list'> | + | <br> |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_one">Detection module</a></li>
| + | Back to top |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_two"><i>S. pneumoniae</i> sigma X promoters module</a></li>
| + | </div> |
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| + | }} |
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- | </ul>
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- | </td> | + | |
- | <td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator">Riboregulator</a></h4>
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- | <ul class= 'site_list'>
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- | </ul>
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- | </td> | + | |
- | <td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice">Human Practice</a></h4> | + | |
- | <ul class= 'site_list'>
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- | </ul>
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- | </ul>
| + | |
- | </td> | + | |
- | <td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team">Team</a></h4> | + | |
- | <ul class= 'site_list'>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/members">Members</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/advisers">Advisers</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/instructors">Instructors</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/attribution">Attributions</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/acknowledgement">Acknowledgement</a></li>
| + | |
- | <li class='site_link'><a href="https://igem.org/Team.cgi?year=2014">Official Team Profile</a></li>
| + | |
- | </ul>
| + | |
- | </td> | + | |
- | <td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab">WetLab</a></h4> | + | |
- | <ul class= 'site_list'>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/notebook">Notebook</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/safety">Safety</a></li>
| + | |
- | </ul>
| + | |
- | </td> | + | |
- | <td class= 'site_map_column' style="float: none;"><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/Achievements">Achievement</a></h4>
| + | |
- | <ul class= 'site_list'>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/medal_requirement">Medal Requirements</a></li>
| + | |
- | <li class='site_link'><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/deliverable">Deliverable</a></li>
| + | |
- | </ul>
| + | |
- | </td>
| + | |
- | | + | |
- | </tr>
| + | |
- |
| + | |
- | </table>
| + | |
- | | + | |
- |
| + | |
- |
| + | |
- | </body>
| + | |
- | </html> | + | |