Team:NYMU-Taipei/project/3c

From 2014.igem.org

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       <h>Cohesion</h>
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       <h>Care</h>
       <div class='abstract'>
       <div class='abstract'>
<ul>
<ul>
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<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>
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<li>The indicator circuit can act as an alarm clock, sending out banana odor when cavities are going to happen in our oral environment.</li>
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<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>
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<li>Aside from testing the circuit in E.coli, our ultimate plan is to use a special chassis, Streptococcus sobrinus, to express our circuit.</li>
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<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>
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<li>Because Streptococcus sobrinus is a commonly seen bacteria in human oral, we don’t need to worry much about the risk that it would trigger human immune system or damage human body.</li>
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           <div href='#3c-1'><p>purpose</p></div>
           <div href='#3c-1'><p>purpose</p></div>
           <div href='#3c-2'><p>background</p></div>
           <div href='#3c-2'><p>background</p></div>
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           <div href='#3c-3'><p>design</p></div>
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           <div href='#3c-3'><p>Chassis</p></div>
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           <div href='#3c-4'><p style="line-height: 25px;">Functional Measurement</p></div>
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           <div href='#3c-4'><p>design</p></div>
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           <div href='#3c-5'><p>result</p></div>
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          <div href='#3c-5'><p style="line-height: 25px;">Functional Measurement</p></div>
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           <div href='#3c-6'><p>result</p></div>
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       <div id='3c-1' style='height:50px;'></div>
       <h1>Purpose</h1>
       <h1>Purpose</h1>
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       <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><br>
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       <p>Exploring the situation of oral environment and send out warning signal if problems concerning tooth decay are going to emerge. With the banana fragrance getting stronger and stronger, people will be able to know that the risk of cavity in their mouth is on the rise.</p>
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      <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>
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       <div id='3c-2' style='height:50px;'></div>
       <h1>Background</h1>
       <h1>Background</h1>
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       <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>
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       <p>The special organism we adapted in this part is Streptococcus sobrinus. By using its specific biological feature, we can turn our circuit into an alarm clock, which would ring whenever humans are in danger of oral cavities. Most people don’t know about the existence of cavities in their mouths until suffering from toothaches or serious pain. However, it is difficult for people to go for dentist checks very often because of the high expenses and amount of time spent. Furthermore, for people in rural areas, there might be insufficient dental resources. Therefore, we have decided to create a system that can alert people from time to time. We aim to use banana fragrance as our indicating method instead of visual cues, avoiding the embarrassment that might result from unsightly colors appearing on our tooth.</p>
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       <h1>Design</h1>
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       <h1>Chassis</h1>
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       <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>
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       <p>For this circuit, we use a special chassis to load and express our wanting genes, which is Streptococcus sobrinus, it is an anaerobic, spherical shaped, Gram positive bacteria. The reason why Streptococcus sobrinus can serve as a good indicator is that the group scale of Streptococcus sobrinus would change dramatically when tooth decay <span style='color:blue;'>is about to happen</span>. Therefore, when there are lots of modified Streptococcus sobrinus in our oral environment, they will release strong banana odor to alert people to see the dentist.</p>
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      <p>!fig1 not yet!</p>
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      <p>!circuit fig not yet!</p>
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       <div id='3c-4' style='height:50px;'></div>
       <div id='3c-4' style='height:50px;'></div>
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       <h1>Functional Measurement</h1>
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       <h1>Design</h1>
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       <p>Working circuit:</p>
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       <p>
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      <img src=''><p>(圖1)</p>
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<b>Promoter (iGEM part BBa_J23100)</b><br>
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      <br>
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It is a commonly used constitutive promoter of iGEM part registry.<br><br>
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      <p>Testing circuit:</p>
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<b>RBS (iGEM part BBa_B0030)</b><br>
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      <img src=''><p>(圖2)</p>
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It is a commonly used ribosome binding site of iGEM part registry.<br><br>
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      <p>Method:<br>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>
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<b>The banana odor generator consists of 3 parts.</b><br>
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      <img src=''><p>(圖3)</p>
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<b>Part 1 (iGEM part BBa_J45008 [1,2,5])</b><br>
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BBa_J45008 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, BAT2 gene. The function of this part is to produce enzyme to get involved in the first step in the aging reaction of L-leucine to isoamyl alcohol. We got this part from the 2007 iGEM kit.<br><br>
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<b>Part 2 (iGEM part BBa_J45009 [1,3,6])</b><br>
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BBa_J45009 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, THI3 gene. The function of this part is to produce enzyme to get involved in the second step in the aging reaction from L-leucine to isoamyl alcohol. Because this part is not released, we cultured Saccharomyces cerevisiae S288c and extract the DNA of it.<br><br>
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<b>Part 3 (iGEM part BBa_J45014 [1,4,7])</b><br>
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BBa_J45014 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, ATF1 gene. The function of this part is to produce enzyme to get involved in the esterification reaction from isoamyl alcohol to isoamyl acetate, which brings out the banana flavor. We got this part from the 2014 iGEM kit.<br><br>
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<b>Terminator (iGEM part BBa_B0015)</b><br>
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A double terminator composed of B0010 and B0012. It has used by many iGEM teams, and have strong terminating force.<br><br>
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      </p>
       <div id='3c-5' style='height:50px;'></div>
       <div id='3c-5' style='height:50px;'></div>
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      <h1>Functional Measurement</h1>
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      <p><b>Circuits needed to be constructed</b><br>Promoter (BBa_J23100) + RBS (BBa_B0030) + Banana odor generator ( BBa_J45008+BBa_J45009 +BBa_J45014)+Terminator(BBa_B0015)
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      </p><br>
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      <p><b>Experimental steps</b></p>
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      <ol class='cont-ol'>
 +
<li>Culture modified Escheria coli in sealed agar plates overnight.</li>
 +
<li>Use syringe to extract the gas generated by Escheria coli.</li>
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<li>Use gas chromatography to analyze the sample.</li>
 +
      </ol>
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      <div id='3c-6' style='height:50px;'></div>
       <h1>Result</h1>
       <h1>Result</h1>
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       <img src=''><p>(圖4)</p>
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       <p></p>
       <h1>Reference</h1>
       <h1>Reference</h1>
       <ol>
       <ol>
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        <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>
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<li>2006 MIT iGEM team and iGEM part registry BBa_J45008 / BBa_J45009 / BBa_J45014</li>
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        <li>Quorum sensing and biofilm formation by Streptococcus mutans. Senadheera.(2008) D1, Cvitkovitch DG</li>
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<li>NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome X, complete sequence</li>
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<li>NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome IV, complete sequence</li>
 +
<li>NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome XV, complete sequence</li>
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<li>(2012)"Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis"</li>
       </ol>
       </ol>
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{{:Team:NYMU-Taipei/NYMU14_Footer}}
{{:Team:NYMU-Taipei/NYMU14_Footer}}

Revision as of 13:31, 7 October 2014

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Care
  • The indicator circuit can act as an alarm clock, sending out banana odor when cavities are going to happen in our oral environment.
  • Aside from testing the circuit in E.coli, our ultimate plan is to use a special chassis, Streptococcus sobrinus, to express our circuit.
  • Because Streptococcus sobrinus is a commonly seen bacteria in human oral, we don’t need to worry much about the risk that it would trigger human immune system or damage human body.

purpose

background

Chassis

design

Functional Measurement

result

Purpose

Exploring the situation of oral environment and send out warning signal if problems concerning tooth decay are going to emerge. With the banana fragrance getting stronger and stronger, people will be able to know that the risk of cavity in their mouth is on the rise.

Background

The special organism we adapted in this part is Streptococcus sobrinus. By using its specific biological feature, we can turn our circuit into an alarm clock, which would ring whenever humans are in danger of oral cavities. Most people don’t know about the existence of cavities in their mouths until suffering from toothaches or serious pain. However, it is difficult for people to go for dentist checks very often because of the high expenses and amount of time spent. Furthermore, for people in rural areas, there might be insufficient dental resources. Therefore, we have decided to create a system that can alert people from time to time. We aim to use banana fragrance as our indicating method instead of visual cues, avoiding the embarrassment that might result from unsightly colors appearing on our tooth.

Chassis

For this circuit, we use a special chassis to load and express our wanting genes, which is Streptococcus sobrinus, it is an anaerobic, spherical shaped, Gram positive bacteria. The reason why Streptococcus sobrinus can serve as a good indicator is that the group scale of Streptococcus sobrinus would change dramatically when tooth decay is about to happen. Therefore, when there are lots of modified Streptococcus sobrinus in our oral environment, they will release strong banana odor to alert people to see the dentist.

Design

Promoter (iGEM part BBa_J23100)
It is a commonly used constitutive promoter of iGEM part registry.

RBS (iGEM part BBa_B0030)
It is a commonly used ribosome binding site of iGEM part registry.

The banana odor generator consists of 3 parts.
Part 1 (iGEM part BBa_J45008 [1,2,5])
BBa_J45008 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, BAT2 gene. The function of this part is to produce enzyme to get involved in the first step in the aging reaction of L-leucine to isoamyl alcohol. We got this part from the 2007 iGEM kit.

Part 2 (iGEM part BBa_J45009 [1,3,6])
BBa_J45009 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, THI3 gene. The function of this part is to produce enzyme to get involved in the second step in the aging reaction from L-leucine to isoamyl alcohol. Because this part is not released, we cultured Saccharomyces cerevisiae S288c and extract the DNA of it.

Part 3 (iGEM part BBa_J45014 [1,4,7])
BBa_J45014 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, ATF1 gene. The function of this part is to produce enzyme to get involved in the esterification reaction from isoamyl alcohol to isoamyl acetate, which brings out the banana flavor. We got this part from the 2014 iGEM kit.

Terminator (iGEM part BBa_B0015)
A double terminator composed of B0010 and B0012. It has used by many iGEM teams, and have strong terminating force.

Functional Measurement

Circuits needed to be constructed
Promoter (BBa_J23100) + RBS (BBa_B0030) + Banana odor generator ( BBa_J45008+BBa_J45009 +BBa_J45014)+Terminator(BBa_B0015)


Experimental steps

  1. Culture modified Escheria coli in sealed agar plates overnight.
  2. Use syringe to extract the gas generated by Escheria coli.
  3. Use gas chromatography to analyze the sample.

Result

Reference

  1. 2006 MIT iGEM team and iGEM part registry BBa_J45008 / BBa_J45009 / BBa_J45014
  2. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome X, complete sequence
  3. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome IV, complete sequence
  4. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome XV, complete sequence
  5. (2012)"Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis"

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