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| | <div id="main-content"> | | <div id="main-content"> |
| | <h>Cleanse-Attachment</h> | | <h>Cleanse-Attachment</h> |
| | + | <h1>Here’s the Gist…</h1> |
| | <div class='abstract'> | | <div class='abstract'> |
| | <ul> | | <ul> |
| - | <li>To evaluate the efficiency of killing module and antibiofilm module, we develop cohesion part to shorten the geographical distance between S. mutans and E. coli.</li> | + | <li>Help anchor E. coli to the surface of <i>S. mutans</i> for efficient killing and biofilm degradation.</li> |
| - | <li>We use C16, a trunked quorum sensing pheromone as an anchor to attach e-coli to the surface of S. mutans. C16 is displayed on E. coli by INPNC, an E. coli surface display protein.</li>
| + | |
| - | <li>We measure the function of our circuit by three stages, including the function test of INPNC, the display pattern of C16,and the interaction between S. mutans and our modified e-coli.</li>
| + | |
| | </ul> | | </ul> |
| | </div> | | </div> |
| | <div class='cont-panel'> | | <div class='cont-panel'> |
| - | <div href='#2c1-1'><p>purpose</p></div> | + | <div href='#2c1-1'><p>Get started.</p></div> |
| - | <div href='#2c1-2'><p>background</p></div> | + | <div href='#2c1-2'><p>How to do it?</p></div> |
| - | <div href='#2c1-3'><p>design</p></div> | + | <div href='#2c1-3'><p>Test it!</p></div> |
| - | <div href='#2c1-4'><p style="line-height: 25px;">Functional Measurement</p></div> | + | <div href='#2c1-4'><p>Our result~</p></div> |
| - | <div href='#2c1-5'><p>result</p></div> | + | <div href='#2c1-5'><p>Reference</p></div> |
| | <div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div> | | <div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div> |
| | </div> | | </div> |
| | <div class='article indent'> | | <div class='article indent'> |
| - | <h1 id='2c1-1'>Purpose</h1> | + | <h1 id='2c1-1'>Before we get started:</h1> |
| - | <p> To facilitate the efficiency of our killing and anti-biofilm parts, we designed a cohesion module. The cohesion module will attach our modified E. coli to the surface of the S. mutans, so we can shorten the distance between our helper E.coli and our targeted enemy, S. mutans . Then, we can evaluate the efficiency of the other mechanism, the “Completion” part in our project.</ p> | + | <p>Although there are a plethora of oral bacteria, only a handful cause tooth decay, and chief among those is <b><i>S. mutans</i> </b>(see <a href='overview'>[Project Overview]</a> for more information). For our modified <i>E. coli</i> to deal with this threat, we need to find a way for them to <b>get close to <i>S. mutans</i></b> colonies in the oral environment.</p> |
| - | <p>For the Completion-killing part, when the helper E. coli received the signal sent by modified phage, the antibiotic secreted E. coli can kill the S. mutans faster if they are closer to each other. Additionally, E. coli can sense the increase of biofilm formation in a shorter period of time and thus activate the antibioflim module.In conclusion, the “Cohesion” part allows the E. coli to serve as a supervisor around S. mutans and reduces the time and distance of the attacks.</p>
| + | |
| - | <h1 id='2c1-2'>Background</h1>
| + | <p>Competence Stimulating Peptide, or <b>CSP</b>, is an important <b>quorum sensing (QS) pheromone specific to <i>S. mutans</i></b>, playing a huge role in their genetic response to population density. The system works since <i>S. mutans</i> continuously release CSP, which <b>bind to the surface receptors</b> of other <i>S. mutans</i> nearby. Check out <a href='1c'>[Control: Target]</a> for more information on CSP. </p> |
| - | <p>In order to make E. coli a supervisor of the population of S. mutans, we have to find an anchor to attach E. coli to S. mutans. Thus, we take advantage of the CSP. CSP is a kind of quorum sensing pheromone, which enables Streptococcus mutans to alter their gene expression when the critical density of cell population is reached. Recent studies showed that density-dependent quorum sensing (QS) system is primarily comprised of the Competence Stimulating Peptide (CSP) and the ComD/ComE two-component signal transduction system. In addition to biofilm formation, the CSP-mediated QS system in S. mutans also affects its acidogenicity, aciduricity, genetic transformation and bacteriocin production. Most importantly,CSP is specificly secreted by S. mutans, which is the reason why we chose it as anchor targeting S. mutans.</p>
| + | |
| - | <h1 id='2c1-3'>Design</h1> | + | <p>The exclusivity of CSP and the ubiquity of its receptors in <i>S. mutans</i> makes it a great candidate for creating an anchoring module! </p> |
| - | <p> We use competence stimulating peptide (CSP ) as a targeting material to attach E. coli to the surface of Streptococcus mutans. To do so, we utilize Surface display protein INPNC (designed by < a href="https://2011.igem.org/Team:Edinburgh" target="_blank"> 2011 iGEM Edinburgh team</a> ) to display CSP on the membrane of E. coli , which act as the “anchor”. CSP binds to both the receptors on the surface of Streptococcus mutans and our engineered helper E. coli .</ p> | + | |
| - | <p>!fig1 not yet!</p>
| + | <h1 id='2c1-2'>So how did we do it?</h1> |
| - | <p> !circuit fig not yet!</ p> | + | <p>Because of size constraints, we did not want to use the actual CSP molecule itself. Instead, we found <b>C16</b>, a functionally active <b>fragment of CSP</b> which still binds to CSP receptors. Compared to CSP, C16 is smaller and more suitable for attachment on the <i>E. coli</i> surface. </p> |
| - | <h1 id='2c1-4'>Functional Measurement</h1>
| + | |
| - | <h3>Working circuit:</h3>
| + | <p>Now that the attachment candidate is found, what’s left is to express it on the surface of <i>E. coli</i>, so it can interact with <i>S. mutans</i>.</p> |
| - | <img src=''><p>(圖1)</p>
| + | |
| - | <br>
| + | <p>For that purpose, we found <b>INPNC</b>, a <b>surface display protein</b> in <i>E. coli</i> biobricked by the 2011 Edinburgh iGEM team. Using INPNC as basis, we created the INPNC-C16 fusion protein, which displays C16 on the surface of <i>E. coli</i> to achieve the results we want. </p> |
| - | <h3>Testing circuit:</h3>
| + | |
| - | | + | <p>At last, our circuit is complete! </p> |
| - | <h3>Method:</h3>
| + | <img src=”/wiki/images/7/7d/NYMU14_attach_Attachment.png”> |
| - | <p>Use 5cc LB culture and 5 ul chloramphenicol antibiotic to incubate bacteria transformed by function test circuit( K523013 + CSP16 + E1010 and B0015)and control circuit (K523013 + CSP16 + and B0015 )12~16 hour. Then centrifuge 5cc bacteria culture at 13000 rps for 2 minute.</p> | + | |
| - | <img src=''><p>(圖3)</p> | + | <h1 id='2c1-3'>Putting it to the test!</h1> |
| - | <h1 id='2c1-5'>Result</h1> | + | <h1 id='2c1-4'>Our result</h1> |
| - | <img src=''><p>(圖4)</p>
| + | <h1 id='2c1-5'>Reference</h1> |
| - | <h1>Reference</h1>
| + | <ol class="cont-ol"> |
| - | <ol>
| + | <li>Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (January 01, 2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology, 31, 2, 170-4.</li> |
| - | <li>Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide(2006)Randal Eckert1, Jian He2, Daniel K. Yarbrough2, Fengxia Qi2,Maxwell H. Anderson3 and Wenyuan Shi</li>
| + | <li>Li, Y.-H., Lau, P. C. Y., Tang, N., Svensater, G., Ellen, R. P., & Cvitkovitch, D. G. (November 15, 2002). Novel Two-Component Regulatory System Involved in Biofilm Formation and Acid Resistance in Streptococcus mutans. Journal of Bacteriology, 184, 22, 6333-6342.</li> |
| - | <li>Quorum sensing and biofilm formation by Streptococcus mutans. Senadheera.(2008) D1, Cvitkovitch DG</li>
| + | <li>Baev, D., England, R., & Kuramitsu, H. K. (January 01, 1999). Stress-induced membrane association of the Streptococcus mutans GTP-binding protein, an essential G protein, and investigation of its physiological role by utilizing an antisense RNA strategy. Infection and Immunity, 67, 9, 4510-6.</li> |
| - | </ol>
| + | <li>Yoshida, A., & Kuramitsu, H. K. (December 01, 2002). Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation. Applied and Environmental Microbiology, 68, 12, 6283-6291.</li> |
| - | </div> <!-- article--> | + | <li>Biswas, I., Jha, J. K., & Fromm, N. (August 01, 2008). Shuttle expression plasmids for genetic studies in Streptococcus mutans. Microbiology, 154, 8, 2275-2282.</li> |
| | + | <li>Li, Yung-Hua, Tang, Nan, Aspiras, Marcelo B., Lau, Peter C. Y., Lee, Janet H., Ellen, Richard P., & Cvitkovitch, Dennis G. (n.d.). A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation. American Society for Microbiology.</li> |
| | + | <li>Wu, J., Cho, M. I., & Kuramitsu, H. K. (January 01, 1995). Expression, purification, and characterization of a novel G protein, SGP, from Streptococcus mutans. Infection and Immunity, 63, 7, 2516-21.</li> |
| | + | </ol> |
| | + | </div> <!-- article --> |
| | </div> | | </div> |
| | </html> | | </html> |
| | {{:Team:NYMU-Taipei/NYMU14_Footer}} | | {{:Team:NYMU-Taipei/NYMU14_Footer}} |
Cleanse-Attachment
Here’s the Gist…
- Help anchor E. coli to the surface of S. mutans for efficient killing and biofilm degradation.
Before we get started:
Although there are a plethora of oral bacteria, only a handful cause tooth decay, and chief among those is S. mutans (see [Project Overview] for more information). For our modified E. coli to deal with this threat, we need to find a way for them to get close to S. mutans colonies in the oral environment.
Competence Stimulating Peptide, or CSP, is an important quorum sensing (QS) pheromone specific to S. mutans, playing a huge role in their genetic response to population density. The system works since S. mutans continuously release CSP, which bind to the surface receptors of other S. mutans nearby. Check out [Control: Target] for more information on CSP.
The exclusivity of CSP and the ubiquity of its receptors in S. mutans makes it a great candidate for creating an anchoring module!
So how did we do it?
Because of size constraints, we did not want to use the actual CSP molecule itself. Instead, we found C16, a functionally active fragment of CSP which still binds to CSP receptors. Compared to CSP, C16 is smaller and more suitable for attachment on the E. coli surface.
Now that the attachment candidate is found, what’s left is to express it on the surface of E. coli, so it can interact with S. mutans.
For that purpose, we found INPNC, a surface display protein in E. coli biobricked by the 2011 Edinburgh iGEM team. Using INPNC as basis, we created the INPNC-C16 fusion protein, which displays C16 on the surface of E. coli to achieve the results we want.
At last, our circuit is complete!
Putting it to the test!
Our result
Reference
- Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (January 01, 2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology, 31, 2, 170-4.
- Li, Y.-H., Lau, P. C. Y., Tang, N., Svensater, G., Ellen, R. P., & Cvitkovitch, D. G. (November 15, 2002). Novel Two-Component Regulatory System Involved in Biofilm Formation and Acid Resistance in Streptococcus mutans. Journal of Bacteriology, 184, 22, 6333-6342.
- Baev, D., England, R., & Kuramitsu, H. K. (January 01, 1999). Stress-induced membrane association of the Streptococcus mutans GTP-binding protein, an essential G protein, and investigation of its physiological role by utilizing an antisense RNA strategy. Infection and Immunity, 67, 9, 4510-6.
- Yoshida, A., & Kuramitsu, H. K. (December 01, 2002). Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation. Applied and Environmental Microbiology, 68, 12, 6283-6291.
- Biswas, I., Jha, J. K., & Fromm, N. (August 01, 2008). Shuttle expression plasmids for genetic studies in Streptococcus mutans. Microbiology, 154, 8, 2275-2282.
- Li, Yung-Hua, Tang, Nan, Aspiras, Marcelo B., Lau, Peter C. Y., Lee, Janet H., Ellen, Richard P., & Cvitkovitch, Dennis G. (n.d.). A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation. American Society for Microbiology.
- Wu, J., Cho, M. I., & Kuramitsu, H. K. (January 01, 1995). Expression, purification, and characterization of a novel G protein, SGP, from Streptococcus mutans. Infection and Immunity, 63, 7, 2516-21.
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