http://2014.igem.org/wiki/index.php?title=Special:Contributions/GraceWang&feed=atom&limit=50&target=GraceWang&year=&month=2014.igem.org - User contributions [en]2024-03-29T11:00:35ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:NYMU-Taipei/project/hoperfusionTeam:NYMU-Taipei/project/hoperfusion2014-10-17T22:04:19Z<p>GraceWang: </p>
<hr />
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<h>HOPErfusion</h><br />
<div class='article indent'><br />
<p>HOPErfusion is a handmade device which combines HOPE box and perfusion machine into a cool apparatus. Its mainly function is to simulate the oral environment for facilitating the anti-biofilm experiment. <a href='/Team:NYMU-Taipei/project/2c'>[Cleanse-Antibiofilm]</a>.</p><br />
<p>In HOPE box, we put modified slide glass by coating by saliva(37。C,1 hr) to simulate the surface of oral cavity. Closed space of HOPE box is anoxic atmosphere like oral cavity . Perfusion machine is to drop BHI and saliva on slide glass to mimic the flowing atmosphere(28.3 mm/min). Finally, all the material we used is germ-free to prevent other germs to influence our experiment.</p> <br />
<p>HOPErfusion is user-friendly,costly and most importantly,effective. HOPErfusion has successfully helped us with easily mimicking the atmosphere of oral cavity in our experiments.So we have confidence that HOPErfusion can be implemented to future testing and research related to oral biology.</p> <br />
<br />
<video muted controls autoplay poster="/wiki/images/e/e9/NYMU14_video_waiting.gif" style="<br />
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<br />
<h3>saliva collection step</h3><br />
<img src='/wiki/images/b/bc/NYMU14_hoperfusion_fig1.png' style='width:730px;'><br />
<h3>HOPErfusion assembly</h3><br />
<img src='/wiki/images/d/db/NYMU14_hoperfusion_fig2.png' style='width:730px;'><br />
<img src='/wiki/images/b/b4/NYMU14_hoperfusion_fig3.png' style='width:730px;'><br />
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{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/hoperfusionTeam:NYMU-Taipei/project/hoperfusion2014-10-17T21:58:04Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>HOPErfusion</h><br />
<div class='article indent'><br />
<p>HOPErfusion is a handmade device which combines HOPE box and perfusion machine into a cool apparatus. Its mainly function is to simulate the oral environment for facilitating the anti-biofilm experiment. <a href='/Team:NYMU-Taipei/project/2c'>[Cleanse-Antibiofilm]</a>.</p><br />
<p>In HOPE box, we put modified slide glass by coating by saliva(37。C,1 hr) to simulate the surface of oral cavity. Closed space of HOPE box is anoxic atmosphere like oral cavity . Perfusion machine is to drop BHI and saliva on slide glass to mimic the flowing atmosphere(28.3 mm/min). Finally, all the material we used is germ-free to prevent other germs to influence our experiment.</p> <br />
<p>HOPErfusion is user-friendly,costly and most importantly,effective. HOPErfusion has successfully helped us with easily mimicking the atmosphere of oral cavity in our experiments.So we have confidence that HOPErfusion can be implemented to future testing and research related to oral biology.</p> <br />
<br />
<video muted controls autoplay poster="/wiki/images/e/e9/NYMU14_video_waiting.gif" style="<br />
width: 513px;<br />
height: 306px;<br />
margin-left: 200px;<br />
margin-bottom: 20px;<br />
z-index: 100;"><br />
<source src="/wiki/images/5/50/NYMU-Taipei_HOPErfusion_assemble.mov" type="video/mp4"><br />
Your browser does not support HTML5 video. You can <a href="/wiki/images/a/a0/NYMU14_hp15_movie.ogg">Download</a> it.<br />
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<br />
<h3>saliva collection step</h3><br />
<img src='/wiki/images/b/bc/NYMU14_hoperfusion_fig1.png' style='width:700px;'><br />
<h3>HOPErfusion assembly</h3><br />
<img src='/wiki/images/d/db/NYMU14_hoperfusion_fig2.png' style='width:700px;'><br />
<img src='/wiki/images/b/b4/NYMU14_hoperfusion_fig3.png' style='width:700px;'><br />
</div> <br />
</div><br />
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{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/notebook/labnotesTeam:NYMU-Taipei/notebook/labnotes2014-10-17T21:28:53Z<p>GraceWang: </p>
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<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
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<link href="https://2014.igem.org/Team:NYMU-Taipei/note.css?action=raw&ctype=text/css" type="text/css" rel="stylesheet"><br />
<h>Lab notes</h><br />
<div id='labnote'><br />
<div class='note-tab'><p>labnote 1</p></div><br />
<div class='note-tab'><p>labnote 2</p></div><br />
<div class='note-tab'><p>labnote 3</p></div><br />
<div class='note-tab'><p>labnote 4</p></div><br />
</div><br />
<embed src='/wiki/images/8/87/NYMU14_Labnote_Target1.pdf' class='note-pdf' style='display:block;'><br />
<embed src='/wiki/images/c/c6/NYMU14_Labnote_Target2.pdf' class='note-pdf' style='display:none;'><br />
<embed src='/wiki/images/e/eb/NYMU14_Labnote_Target3.pdf' class='note-pdf' style='display:none;'><br />
<embed src='/wiki/images/9/93/NYMU14_labnote_inhibitor1.pdf' class='note-pdf' style='display:none;'><br />
<embed src='' class='note-pdf' style='display:none;'><br />
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{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/File:NYMU14_labnote_inhibitor1.pdfFile:NYMU14 labnote inhibitor1.pdf2014-10-17T21:21:36Z<p>GraceWang: </p>
<hr />
<div></div>GraceWanghttp://2014.igem.org/File:Labnote4.pdfFile:Labnote4.pdf2014-10-17T20:59:57Z<p>GraceWang: </p>
<hr />
<div></div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1cTeam:NYMU-Taipei/project/1c2014-10-16T17:01:49Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Target</h><br />
<h1>Here’s the Gist…</h1><br />
<div class='abstract'><br />
<ul><br />
<li>Utilize the <i>Streptococcus phage M102</i> to infect <i>S. mutans</i> and act as a sensor.</li><br />
<li>When <i>S. mutans</i> population exceeds a threshold, <i>M102</i> sends out a quorum-sensing signal to our probiotic.<br />
<li>This signal then turns on the probiotic’s killing module and brings down the <i>S. mutans</i> population.<br />
</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c-1'><p>Get started.</p></div><br />
<div href='#1c-2'><p>How to do it?</p></div><br />
<div href='#1c-3'><p>Test it!</p></div><br />
<div href='#1c-4'><p>Our result~</p></div><br />
<div href='#1c-5'><p>Reference</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px; visibility: hidden;"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c-1'>Before we get started:</h1><br />
<p>"Control" is so-called because it is used to control the number of <i>S. mutans</i>, instead of killing it completely off, which wouldn't help the matter as indicated in [<a href='/Team:NYMU-Taipei/modeling/m2'>Modeling: Competition</a>]. When the number of <i>S. mutans</i> exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess <i>S. mutans</i>.</p><br />
<p>In order to find and confirm the threshold that causes cavities, we designed <b>different kinds and numbers of terminators</b> (each with different leakage rates) to create the threshold. Moreover, we combined both wet lab results and modelling to decide which design is more suitable.</p><br />
<p>Over the years, it has been found that the <b>competence stimulating peptide, CSP</b>, a quorum sensing chemical, is released in every competence <i>S. mutan</i>s. Thus, we can detect the number of <i>S. mutans</i> by marking the amount of CSP.</p><br />
<img src='/wiki/images/d/dd/NYMU14_target-sensor.gif'><br />
<p>In <i>S. mutans</i> the CSP bind to the membrane receptor, "comD", thereby phosphorylating the response regulator, "comE". The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and the release of mutacin, an antibiotic peptide. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene "nlmC"( non-lantibiotic mutacin C) in S. mutans has the best performance under the stimulation of CSP.</p><br />
<p>At this point, the two main organisms we adapted for our project are our probiotics and <i>M102</i> bacteriophage. We used one of the most commonly seen <b>quorum sensing chemicals, AHL</b>, N-Acyl-homoserine-lactone, <b>as our indicator between phage and E.coli</b>. Due to the idea of phage threshold control of the number of <i>S. mutans</i> in prevention of the second-dominant strain in the oral cavity, we envision a possibility to trigger another killing system to reach synergistic effect on diminishing <i>S. mutans</i>. If the threshold terminators were passed, the downstream signaling pathway would be switched on and contact with the engineered probiotics.</p><br />
<p>When the amount of the <i>S. mutans</i> is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the <b>luxR-luxI system</b>. LuxI acts as a gene which would generate AHL-synthase that yielding the quorum-sensing signal AHL right after the threshold level of <i>S. mutans</i> is exceeded. LuxR is a protein which forms a complex with AHL, and then turns into an activated form which will afterwards bind with the promoter pLuxR and trigger it.<br />
The signal sequence and endolysin downstream would be secreted and eliminate the excess <i>S. mutans</i>.</p><br />
<div style='float:right'><img src='/wiki/images/2/2c/NYMU14_target_cellwall.png' style='width: 450px;padding-left: 20px;'><p style='width: 450px;font-size: 14px;'>Figure 1. Gram positive bacteria have only one layer of cell wall outside of cell membrane, composing of peptidoglycan, while the cell wall of gram negative bacteria has additional protection outside peptidoglycan cell wall.[1]</p></div><br />
<p>Endolysin is an enzyme expressed by phage-infected bacteria. The C-terminus bind to the host cell wall, while the N-terminus is enzymatic domain. Working with holin, which lyses cell membrane, endolysin could break the cell wall of bacteria. Usually serve as peptidase, endolysin tend to work on gram positive species, which have layers of peptidoglycan cell wall. Though originally intended to be utilized inside the cell wall, endolysin can also be applied outside cell according to previous research. Furthermore, it is even less harmful to gram negative bacteria this way because of the lipid layer of those bacteria composes cell wall.</p><br />
<br />
<h1 id='1c-2' style='float:left;'>So how did we do it?</h1><br />
<img src='/wiki/images/f/fa/NYMU14_target_sensor_ter.png' style='width:500px;'><br />
<p>In our circuit, we incorporate nlmC promoter and <b>different combination of terminators to create the threshold</b>.</p><br />
<p>When the amount of <i>S. mutans</i> is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the <b>luxR-luxI system</b>. LuxI in <i>S. mutans</i> will be triggered by nlmC and activate AHL which will combine with LuxR produced by probiotics continuously. When LuxR combine with AHL to form AHL-LuxR complex, it will induce pLuxR promoter and trigger the Killer Module.</p><br />
<img src='/wiki/images/3/31/NYMU14_targetall.png' style='width:760px;'><br />
<h3>Killer Module</h3><br />
<img src='/wiki/images/a/a7/NYMU_killing_module.png' style='width:580px;'><br />
<p>YebF is a protein that is naturally secreted by <i>E.coli</i>. It would transport proteins attached to it to the periplasm (space between cell membrane and cell wall), and later secrete it through porin on the cell wall. We chose YebF for its two advantages: one, it can be secreted in experimental strains which usually do not secrete proteins, along with other diverse passenger characters. Two, YebF can prevent endolysin from toxicating <i>E. coli</i>.</p><br />
<br />
<h1 id='1c-3'>Putting it to the test!</h1><br />
<h3>Stage one: How well does nlmC function?</h3><br />
<p>In order to test if nlmC functions as we expected it to, we constructed two circuits. The first is constructed with a constitutive promoter and GFP, and will serve as our control:</p><br />
<img src='/wiki/images/d/da/NYMU14_Target_T1.png' style='width: 300px;'><br />
<p>The other is constructed instead with nlmC and GFP, and will serve as the testing circuit for comparison between the efficacy of a constitutive promoter and the nlmC promoter:</p><br />
<img src='/wiki/images/b/b5/NYMU14_Target_T1-2.png' style='width: 300px;'><br />
<p>To compare these circuits, we applied different amounts of synthetic CSP in different time lapses to our experimental group. If everything works as planned, the experimental group should display fluorescence in a wide array of intensity depending on CSP concentration, while the control group displays a uniform bright green.</p><br />
<br />
<h3>Stage two: Which threshold combination should we use?</h3><br />
<p>After confirming that the nlmC promoter works and responds to different levels of CSP, we need to design a test to determine which terminator combination we can best use as an effective threshold. We designed two circuits with different terminator thresholds, B0012 and double B0012, respectively, and GFP after them as a reporter.</p><br />
<img src='/wiki/images/f/fa/NYMU14_target_sensor_ter.png' style='width:500px;'><br />
<p>The testing is similar to Stage 1, and we simply recorded the GFP production after induction with commercial CSP.</p><br />
<p>In the future, this protocol can be used to test many other combinations to find the "perfect" threshold for HOPE. After obtaining some experimental data, we employed extensive modeling to incorporate these numbers into other combinations of terminators, and we’ll be looking to validate these results after the Jamboree is over to further strengthen HOPE. Please see our <a href='Team:NYMU-Taipei/modeling/m3'>[Model: Threshold]</a> section for more information.</p><br />
<br />
<h3>Stage three: Mechanisms of the Signal Receptor in our Probiotic</h3><br />
<p>After all the fragments of the circuit is linked, we need a test to tell us that the fragments are well-connected, and that the mechanisms linking them has not lost its function. We created a circuit which is identical to our final circuit, except with RFP in place of the killing module:</p><br />
<img src='/wiki/images/8/8c/NYMU14_Target_T3.png'><br />
<p>We then transformed our circuit into <i>E. coli</i>, and added in commercial AHL. If the mechanisms remain robust after splicing, we should see a strong display of red from RFP.</p><br />
<img src='/wiki/images/2/2a/NYMU14_Target_Construction_Check_2.png' style='width: 300px;margin-left: 40px;'><br />
<br />
<h3>Stage four: Mechanisms of the Signal Producer in <i>S. mutans</i></h3><br />
<img src='/wiki/images/b/b2/NYMU14_Target_Test2.png' style='float: right;width: 340px;padding-left: 20px;padding-bottom: 20px;padding-top: 20px;'><br />
<p>One main concern with our mechanism is that AHL is a Gram-negative quorum sensing-signal… and yet we are implanting it into the Gram-positive <i>S.mutans</i> via <i>M102</i>. We need to test if the AHL production still works in <i>S. mutans</i>.</p><br />
<p>In order to confirm that whether the engineered circuit could successfully yield AHL, we adopt the purification and structural examination processes referred to related references.The LuxI gene we embedded inside our circuit coded BBa_C0061 in IGEM registry and it synthesizes N-3-(Oxohexanoyl)-L-homoserine lactone. We uses preparative-TLC to purify the chemical and conduct 1HNMR to check the structure.</p><br />
<p>According to paper research, we prepared our sample through the protocol described below:</p><br />
<ol><br />
<li>S.mutans liquid cultured till its stationary phase</li><br />
<li>Add lysozyme to 5ml cultures</li><br />
<li>Sonication</li><br />
<li>Centrifugation</li><br />
<li>Extract the supernatant with equal volume of ethyl acetate X2</li><br />
<li>Dry with anhydrous magnesium sulfate</li><br />
<li>Filtering</li><br />
<li>Evaporate to dry</li><br />
<li>Dissolve with 50-100uL HPLC-grade ethyl acetate</li><br />
<li>Run TLC</li><br />
</ol><br />
<br />
<h3>Stage five: Does YebF work?</h3><br />
<p>Before we check the functionality of the entire construct, we need to test the YebF protein for its ability to transport attached parts out of the membrane. We began by constructing two circuits:</p><br />
<img src='/wiki/images/d/d3/NYMU14_target_YebF_t.png' style='width: 550px;'><br />
<p>These circuits are then transformed into <i>E. coli</i>, cultivated in liquid culture, and centrifuged. Afterwards, the supernatants are analyzed for their OD value. If YebF works as we expected, we should see the first circuit successfully secrete RFP into the supernatant :</p><br />
<img src='/wiki/images/a/a6/NYMU14_target_YebF_rfp.png' style='width: 220px;margin-left: 40px;'><br />
<p>While the control should only contain RFP which is not exported:</p><br />
<img src='/wiki/images/8/87/NYMU14_target_YebF_norfp.png' style='width: 190px;margin-left: 40px;'><br />
<br />
<h3>Stage six: Does Endolysin work?</h3><br />
<p>To test the antimicrobial activity of endolysin, we also created two circuits:</p><br />
<img src='/wiki/images/d/d1/NYMU14_target_endolysin_t.png' style='width: 550px;'><br />
<p>The first circuit contains YebF and endolysin, which serves as the experimental circuit, and the second contains just YebF, and is the control. These are transformed into <i>E. coli</i>, and after culturing them overnight and extracting the supernatant, we spread the supernatant on plates of <i>S. mutans</i> to test for their antimicrobial activity.</p><br />
<br />
<h3>Stage seven: Does AHL stimulate the killing program as anticipated?</h3><br />
<p>In this stage, we put the final probiotic circuit into <i>E. coli</i> to see how well it performs.</p><br />
<img src='/wiki/images/3/39/NYMU14_Target_T7.png' style='width: 700px;'><br />
<p>The two different germs lines are involved in this test, the <i>E.coli</i> constructed circuit (<b>pSB1C3 + luxR + pluxR + YebF + Endolysin</b>) and <i>S.mutans</i> constructed circuit (luxI).<br />
<br />
The culturing of constructed <i>E.coli</i> with the supernatant of constructed <i>S.mutans</i> (containing AHL) supposed to be secretion of endolysin. Then we cultured the wild type <i>S.mutans</i> with the supernatant of previous culture.</p><br />
<img src='/wiki/images/b/ba/NYMU14_Target_Test5.png' style='width: 500px;margin-left: 40px;'><br />
<br />
<h3>Stage eight: Finally, let’s check <i>Everything</i>!</h3><br />
<p>In this last stage we put all the constructed circuits intended for our probiotic and <i>M102</i> into <i>E. coli</i> and <i>S. mutans</i>, respectively. Then we co-cultured these two transformed strains together to test our full circuit.</p><br />
<br />
<h1 id='1c-4'>Our result</h1><br />
<br />
<h1 id='1c-5'>Reference</h1><br />
<ol><br />
<li>http://www.expertsmind.com/topic/microbiology/bacterial-cell-wall-92313.aspx</li><br />
</ol><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1cTeam:NYMU-Taipei/project/1c2014-10-16T16:56:48Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Target</h><br />
<h1>Here’s the Gist…</h1><br />
<div class='abstract'><br />
<ul><br />
<li>Utilize the <i>Streptococcus phage M102</i> to infect <i>S. mutans</i> and act as a sensor.</li><br />
<li>When <i>S. mutans</i> population exceeds a threshold, <i>M102</i> sends out a quorum-sensing signal to our probiotic.<br />
<li>This signal then turns on the probiotic’s killing module and brings down the <i>S. mutans</i> population.<br />
</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c-1'><p>Get started.</p></div><br />
<div href='#1c-2'><p>How to do it?</p></div><br />
<div href='#1c-3'><p>Test it!</p></div><br />
<div href='#1c-4'><p>Our result~</p></div><br />
<div href='#1c-5'><p>Reference</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px; visibility: hidden;"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c-1'>Before we get started:</h1><br />
<p>"Control" is so-called because it is used to control the number of <i>S. mutans</i>, instead of killing it completely off, which wouldn't help the matter as indicated in [<a href='/Team:NYMU-Taipei/modeling/m2'>Modeling: Competition</a>]. When the number of <i>S. mutans</i> exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess <i>S. mutans</i>.</p><br />
<p>In order to find and confirm the threshold that causes cavities, we designed <b>different kinds and numbers of terminators</b> (each with different leakage rates) to create the threshold. Moreover, we combined both wet lab results and modelling to decide which design is more suitable.</p><br />
<p>Over the years, it has been found that the <b>competence stimulating peptide, CSP</b>, a quorum sensing chemical, is released in every competence <i>S. mutan</i>s. Thus, we can detect the number of <i>S. mutans</i> by marking the amount of CSP.</p><br />
<img src='/wiki/images/d/dd/NYMU14_target-sensor.gif'><br />
<p>In <i>S. mutans</i> the CSP bind to the membrane receptor, "comD", thereby phosphorylating the response regulator, "comE". The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and the release of mutacin, an antibiotic peptide. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene "nlmC"( non-lantibiotic mutacin C) in S. mutans has the best performance under the stimulation of CSP.</p><br />
<p>At this point, the two main organisms we adapted for our project are our probiotics and <i>M102</i> bacteriophage. We used one of the most commonly seen <b>quorum sensing chemicals, AHL</b>, N-Acyl-homoserine-lactone, <b>as our indicator between phage and E.coli</b>. Due to the idea of phage threshold control of the number of <i>S. mutans</i> in prevention of the second-dominant strain in the oral cavity, we envision a possibility to trigger another killing system to reach synergistic effect on diminishing <i>S. mutans</i>. If the threshold terminators were passed, the downstream signaling pathway would be switched on and contact with the engineered probiotics.</p><br />
<p>When the amount of the <i>S. mutans</i> is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the <b>luxR-luxI system</b>. LuxI acts as a gene which would generate AHL-synthase that yielding the quorum-sensing signal AHL right after the threshold level of <i>S. mutans</i> is exceeded. LuxR is a protein which forms a complex with AHL, and then turns into an activated form which will afterwards bind with the promoter pLuxR and trigger it.<br />
The signal sequence and endolysin downstream would be secreted and eliminate the excess <i>S. mutans</i>.</p><br />
<div style='float:right'><img src='/wiki/images/2/2c/NYMU14_target_cellwall.png' style='width: 450px;padding-left: 20px;'><p style='width: 450px;font-size: 14px;'>Figure 1. Gram positive bacteria have only one layer of cell wall outside of cell membrane, composing of peptidoglycan, while the cell wall of gram negative bacteria has additional protection outside peptidoglycan cell wall.[1]</p></div><br />
<p>Endolysin is an enzyme expressed by phage-infected bacteria. The C-terminus bind to the host cell wall, while the N-terminus is enzymatic domain. Working with holin, which lyses cell membrane, endolysin could break the cell wall of bacteria. Usually serve as peptidase, endolysin tend to work on gram positive species, which have layers of peptidoglycan cell wall. Though originally intended to be utilized inside the cell wall, endolysin can also be applied outside cell according to previous research. Furthermore, it is even less harmful to gram negative bacteria this way because of the lipid layer of those bacteria composes cell wall.</p><br />
<br />
<h1 id='1c-2' style='float:left;'>So how did we do it?</h1><br />
<img src='/wiki/images/f/fa/NYMU14_target_sensor_ter.png' style='width:500px;'><br />
<p>In our circuit, we incorporate nlmC promoter and <b>different combination of terminators to create the threshold</b>.</p><br />
<p>When the amount of <i>S. mutans</i> is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the <b>luxR-luxI system</b>. LuxI in <i>S. mutans</i> will be triggered by nlmC and activate AHL which will combine with LuxR produced by probiotics continuously. When LuxR combine with AHL to form AHL-LuxR complex, it will induce pLuxR promoter and trigger the Killer Module.</p><br />
<img src='/wiki/images/3/31/NYMU14_targetall.png' style='width:760px;'><br />
<h3>Killer Module</h3><br />
<img src='/wiki/images/a/a7/NYMU_killing_module.png' style='width:580px;'><br />
<p>YebF is a protein that is naturally secreted by <i>E.coli</i>. It would transport proteins attached to it to the periplasm (space between cell membrane and cell wall), and later secrete it through porin on the cell wall. We chose YebF for its two advantages: one, it can be secreted in experimental strains which usually do not secrete proteins, along with other diverse passenger characters. Two, YebF can prevent endolysin from toxicating <i>E. coli</i>.</p><br />
<br />
<h1 id='1c-3'>Putting it to the test!</h1><br />
<h3>Stage one: How well does nlmC function?</h3><br />
<p>In order to test if nlmC functions as we expected it to, we constructed two circuits. The first is constructed with a constitutive promoter and GFP, and will serve as our control:</p><br />
<img src='/wiki/images/d/da/NYMU14_Target_T1.png' style='width: 300px;'><br />
<p>The other is constructed instead with nlmC and GFP, and will serve as the testing circuit for comparison between the efficacy of a constitutive promoter and the nlmC promoter:</p><br />
<img src='/wiki/images/b/b5/NYMU14_Target_T1-2.png' style='width: 300px;'><br />
<p>To compare these circuits, we applied different amounts of synthetic CSP in different time lapses to our experimental group. If everything works as planned, the experimental group should display fluorescence in a wide array of intensity depending on CSP concentration, while the control group displays a uniform bright green.</p><br />
<br />
<h3>Stage two: Which threshold combination should we use?</h3><br />
<p>After confirming that the nlmC promoter works and responds to different levels of CSP, we need to design a test to determine which terminator combination we can best use as an effective threshold. We designed two circuits with different terminator thresholds, B0012 and double B0012, respectively, and GFP after them as a reporter.</p><br />
<img src='/wiki/images/f/fa/NYMU14_target_sensor_ter.png' style='width:500px;'><br />
<p>The testing is similar to Stage 1, and we simply recorded the GFP production after induction with commercial CSP.</p><br />
<p>In the future, this protocol can be used to test many other combinations to find the "perfect" threshold for HOPE. After obtaining some experimental data, we employed extensive modeling to incorporate these numbers into other combinations of terminators, and we’ll be looking to validate these results after the Jamboree is over to further strengthen HOPE. Please see our <a href='Team:NYMU-Taipei/modeling/m3'>[Model: Threshold]</a> section for more information.</p><br />
<br />
<h3>Stage three: Mechanisms of the Signal Receptor in our Probiotic</h3><br />
<p>After all the fragments of the circuit is linked, we need a test to tell us that the fragments are well-connected, and that the mechanisms linking them has not lost its function. We created a circuit which is identical to our final circuit, except with RFP in place of the killing module:</p><br />
<img src='/wiki/images/8/8c/NYMU14_Target_T3.png'><br />
<p>We then transformed our circuit into <i>E. coli</i>, and added in commercial AHL. If the mechanisms remain robust after splicing, we should see a strong display of red from RFP.</p><br />
<img src='/wiki/images/2/2a/NYMU14_Target_Construction_Check_2.png' style='width: 300px;margin-left: 40px;'><br />
<br />
<h3>Stage four: Mechanisms of the Signal Producer in <i>S. mutans</i></h3><br />
<img src='/wiki/images/b/b2/NYMU14_Target_Test2.png' style='float: right;width: 340px;padding-left: 20px;padding-bottom: 20px;padding-top: 20px;'><br />
<p>One main concern with our mechanism is that AHL is a Gram-negative quorum sensing-signal… and yet we are implanting it into the Gram-positive <i>S.mutans</i> via <i>M102</i>. We need to test if the AHL production still works in <i>S. mutans</i>.</p><br />
<p>In order to confirm that whether the engineered circuit could successfully yield AHL, we adopt the purification and structural examination processes referred to related references.The LuxI gene we embedded inside our circuit coded BBa_C0061 in IGEM registry and it synthesizes N-3-(Oxohexanoyl)-L-homoserine lactone. We uses preparative-TLC to purify the chemical and conduct 1HNMR to check the structure.</p><br />
<p>According to paper research, we prepared our sample through the protocol described below:<br />
1.S.mutans liquid cultured till its stationary phase<br />
2.Add lysozyme to 5ml cultures <br />
3.Sonication<br />
4.Centrifugation<br />
5.Extract the supernatant with equal volume of ethyl acetate X2<br />
6.Dry with anhydrous magnesium sulfate<br />
7.Filtering<br />
8.Evaporate to dry<br />
9.Dissolve with 50-100uL HPLC-grade ethyl acetate<br />
10.Run TLC<br />
</p><br />
<br />
<h3>Stage five: Does YebF work?</h3><br />
<p>Before we check the functionality of the entire construct, we need to test the YebF protein for its ability to transport attached parts out of the membrane. We began by constructing two circuits:</p><br />
<img src='/wiki/images/d/d3/NYMU14_target_YebF_t.png' style='width: 550px;'><br />
<p>These circuits are then transformed into <i>E. coli</i>, cultivated in liquid culture, and centrifuged. Afterwards, the supernatants are analyzed for their OD value. If YebF works as we expected, we should see the first circuit successfully secrete RFP into the supernatant :</p><br />
<img src='/wiki/images/a/a6/NYMU14_target_YebF_rfp.png' style='width: 220px;margin-left: 40px;'><br />
<p>While the control should only contain RFP which is not exported:</p><br />
<img src='/wiki/images/8/87/NYMU14_target_YebF_norfp.png' style='width: 190px;margin-left: 40px;'><br />
<br />
<h3>Stage six: Does Endolysin work?</h3><br />
<p>To test the antimicrobial activity of endolysin, we also created two circuits:</p><br />
<img src='/wiki/images/d/d1/NYMU14_target_endolysin_t.png' style='width: 550px;'><br />
<p>The first circuit contains YebF and endolysin, which serves as the experimental circuit, and the second contains just YebF, and is the control. These are transformed into <i>E. coli</i>, and after culturing them overnight and extracting the supernatant, we spread the supernatant on plates of <i>S. mutans</i> to test for their antimicrobial activity.</p><br />
<br />
<h3>Stage seven: Does AHL stimulate the killing program as anticipated?</h3><br />
<p>In this stage, we put the final probiotic circuit into <i>E. coli</i> to see how well it performs.</p><br />
<img src='/wiki/images/3/39/NYMU14_Target_T7.png' style='width: 700px;'><br />
<p>The two different germs lines are involved in this test, the <i>E.coli</i> constructed circuit (<b>pSB1C3+luxR+pluxR+yebF+Endolysin</b>) and <i>S.mutans</i> constructed circuit (luxI).<br />
<br />
The culturing of constructed <i>E.coli</i> with the supernatant of constructed <i>S.mutans</i> (containing AHL) supposed to be secretion of endolysin. Then we cultured the wild type <i>S.mutans</i> with the supernatant of previous culture.</p><br />
<img src='/wiki/images/b/ba/NYMU14_Target_Test5.png' style='width: 500px;margin-left: 40px;'><br />
<br />
<h3>Stage eight: Finally, let’s check <i>Everything</i>!</h3><br />
<p>In this last stage we put all the constructed circuits intended for our probiotic and <i>M102</i> into <i>E. coli</i> and <i>S. mutans</i>, respectively. Then we co-cultured these two transformed strains together to test our full circuit.</p><br />
<br />
<h1 id='1c-4'>Our result</h1><br />
<br />
<h1 id='1c-5'>Reference</h1><br />
<ol><br />
<li>http://www.expertsmind.com/topic/microbiology/bacterial-cell-wall-92313.aspx</li><br />
</ol><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1cTeam:NYMU-Taipei/project/1c2014-10-16T16:51:13Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Target</h><br />
<h1>Here’s the Gist…</h1><br />
<div class='abstract'><br />
<ul><br />
<li>Utilize the <i>Streptococcus phage M102</i> to infect <i>S. mutans</i> and act as a sensor.</li><br />
<li>When <i>S. mutans</i> population exceeds a threshold, <i>M102</i> sends out a quorum-sensing signal to our probiotic.<br />
<li>This signal then turns on the probiotic’s killing module and brings down the <i>S. mutans</i> population.<br />
</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c-1'><p>Get started.</p></div><br />
<div href='#1c-2'><p>How to do it?</p></div><br />
<div href='#1c-3'><p>Test it!</p></div><br />
<div href='#1c-4'><p>Our result~</p></div><br />
<div href='#1c-5'><p>Reference</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px; visibility: hidden;"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c-1'>Before we get started:</h1><br />
<p>"Control" is so-called because it is used to control the number of <i>S. mutans</i>, instead of killing it completely off, which wouldn't help the matter as indicated in [<a href='/Team:NYMU-Taipei/modeling/m2'>Modeling: Competition</a>]. When the number of <i>S. mutans</i> exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess <i>S. mutans</i>.</p><br />
<p>In order to find and confirm the threshold that causes cavities, we designed <b>different kinds and numbers of terminators</b> (each with different leakage rates) to create the threshold. Moreover, we combined both wet lab results and modelling to decide which design is more suitable.</p><br />
<p>Over the years, it has been found that the <b>competence stimulating peptide, CSP</b>, a quorum sensing chemical, is released in every competence <i>S. mutan</i>s. Thus, we can detect the number of <i>S. mutans</i> by marking the amount of CSP.</p><br />
<img src='/wiki/images/d/dd/NYMU14_target-sensor.gif'><br />
<p>In <i>S. mutans</i> the CSP bind to the membrane receptor, "comD", thereby phosphorylating the response regulator, "comE". The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and the release of mutacin, an antibiotic peptide. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene "nlmC"( non-lantibiotic mutacin C) in S. mutans has the best performance under the stimulation of CSP.</p><br />
<p>At this point, the two main organisms we adapted for our project are our probiotics and <i>M102</i> bacteriophage. We used one of the most commonly seen <b>quorum sensing chemicals, AHL</b>, N-Acyl-homoserine-lactone, <b>as our indicator between phage and E.coli</b>. Due to the idea of phage threshold control of the number of <i>S. mutans</i> in prevention of the second-dominant strain in the oral cavity, we envision a possibility to trigger another killing system to reach synergistic effect on diminishing <i>S. mutans</i>. If the threshold terminators were passed, the downstream signaling pathway would be switched on and contact with the engineered probiotics.</p><br />
<p>When the amount of the <i>S. mutans</i> is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the <b>luxR-luxI system</b>. LuxI acts as a gene which would generate AHL-synthase that yielding the quorum-sensing signal AHL right after the threshold level of <i>S. mutans</i> is exceeded. LuxR is a protein which forms a complex with AHL, and then turns into an activated form which will afterwards bind with the promoter pLuxR and trigger it.<br />
The signal sequence and endolysin downstream would be secreted and eliminate the excess <i>S. mutans</i>.</p><br />
<div style='float:right'><img src='/wiki/images/2/2c/NYMU14_target_cellwall.png' style='width: 450px;padding-left: 20px;'><p style='width: 450px;font-size: 14px;'>Figure 1. Gram positive bacteria have only one layer of cell wall outside of cell membrane, composing of peptidoglycan, while the cell wall of gram negative bacteria has additional protection outside peptidoglycan cell wall.[1]</p></div><br />
<p>Endolysin is an enzyme expressed by phage-infected bacteria. The C-terminus bind to the host cell wall, while the N-terminus is enzymatic domain. Working with holin, which lyses cell membrane, endolysin could break the cell wall of bacteria. Usually serve as peptidase, endolysin tend to work on gram positive species, which have layers of peptidoglycan cell wall. Though originally intended to be utilized inside the cell wall, endolysin can also be applied outside cell according to previous research. Furthermore, it is even less harmful to gram negative bacteria this way because of the lipid layer of those bacteria composes cell wall.</p><br />
<br />
<h1 id='1c-2' style='float:left;'>So how did we do it?</h1><br />
<img src='/wiki/images/f/fa/NYMU14_target_sensor_ter.png' style='width:500px;'><br />
<p>In our circuit, we incorporate nlmC promoter and <b>different combination of terminators to create the threshold</b>.</p><br />
<p>When the amount of <i>S. mutans</i> is so large that the phages couldn't afford the loading, CSP would pass the threshold value and turn on the <b>luxR-luxI system</b>. LuxI in <i>S. mutans</i> will be triggered by nlmC and activate AHL which will combine with LuxR produced by probiotics continuously. When LuxR combine with AHL to form AHL-LuxR complex, it will induce pLuxR promoter and trigger the Killer Module.</p><br />
<img src='/wiki/images/3/31/NYMU14_targetall.png' style='width:760px;'><br />
<h3>Killer Module</h3><br />
<img src='/wiki/images/a/a7/NYMU_killing_module.png' style='width:580px;'><br />
<p>YebF is a protein that is naturally secreted by <i>E.coli</i>. It would transport proteins attached to it to the periplasm (space between cell membrane and cell wall), and later secrete it through porin on the cell wall. We chose YebF for its two advantages: one, it can be secreted in experimental strains which usually do not secrete proteins, along with other diverse passenger characters. Two, YebF can prevent endolysin from toxicating <i>E. coli</i>.</p><br />
<br />
<h1 id='1c-3'>Putting it to the test!</h1><br />
<h3>Stage one: How well does nlmC function?</h3><br />
<p>In order to test if nlmC functions as we expected it to, we constructed two circuits. The first is constructed with a constitutive promoter and GFP, and will serve as our control:</p><br />
<img src='/wiki/images/d/da/NYMU14_Target_T1.png' style='width: 300px;'><br />
<p>The other is constructed instead with nlmC and GFP, and will serve as the testing circuit for comparison between the efficacy of a constitutive promoter and the nlmC promoter:</p><br />
<img src='/wiki/images/b/b5/NYMU14_Target_T1-2.png' style='width: 300px;'><br />
<p>To compare these circuits, we applied different amounts of synthetic CSP in different time lapses to our experimental group. If everything works as planned, the experimental group should display fluorescence in a wide array of intensity depending on CSP concentration, while the control group displays a uniform bright green.</p><br />
<br />
<h3>Stage two: Which threshold combination should we use?</h3><br />
<p>After confirming that the nlmC promoter works and responds to different levels of CSP, we need to design a test to determine which terminator combination we can best use as an effective threshold. We designed two circuits with different terminator thresholds, B0012 and double B0012, respectively, and GFP after them as a reporter.</p><br />
<img src='/wiki/images/f/fa/NYMU14_target_sensor_ter.png' style='width:500px;'><br />
<p>The testing is similar to Stage 1, and we simply recorded the GFP production after induction with commercial CSP.</p><br />
<p>In the future, this protocol can be used to test many other combinations to find the "perfect" threshold for HOPE. After obtaining some experimental data, we employed extensive modeling to incorporate these numbers into other combinations of terminators, and we’ll be looking to validate these results after the Jamboree is over to further strengthen HOPE. Please see our <a href='Team:NYMU-Taipei/modeling/m3'>[Model: Threshold]</a> section for more information.</p><br />
<br />
<h3>Stage three: Mechanisms of the Signal Receptor in our Probiotic</h3><br />
<p>After all the fragments of the circuit is linked, we need a test to tell us that the fragments are well-connected, and that the mechanisms linking them has not lost its function. We created a circuit which is identical to our final circuit, except with RFP in place of the killing module:</p><br />
<img src='/wiki/images/8/8c/NYMU14_Target_T3.png'><br />
<p>We then transformed our circuit into <i>E. coli</i>, and added in commercial AHL. If the mechanisms remain robust after splicing, we should see a strong display of red from RFP.</p><br />
<img src='/wiki/images/2/2a/NYMU14_Target_Construction_Check_2.png' style='width: 300px;margin-left: 40px;'><br />
<br />
<h3>Stage four: Mechanisms of the Signal Producer in <i>S. mutans</i></h3><br />
<img src='/wiki/images/b/b2/NYMU14_Target_Test2.png' style='float: right;width: 340px;padding-left: 20px;padding-bottom: 20px;padding-top: 20px;'><br />
<p>One main concern with our mechanism is that AHL is a Gram-negative quorum sensing-signal… and yet we are implanting it into the Gram-positive <i>S.mutans</i> via <i>M102</i>. We need to test if the AHL production still works in <i>S. mutans</i>.</p><br />
<p>In this experiment, we first made a standard curve of the concentration of commercialized AHL versus absorption value. After that, we transformed the circuit containing LuxI into <i>S. mutans</i>. By extracting the supernatant of the culturing medium of the engineered <i>S.mutans</i>, and using a spectrometer to measure the concentration of AHL compared to the standard curve made previously, we can determine whether or not AHL is being expressed successfully in <i>S. mutans</i>.</p><br />
<br />
<h3>Stage five: Does YebF work?</h3><br />
<p>Before we check the functionality of the entire construct, we need to test the YebF protein for its ability to transport attached parts out of the membrane. We began by constructing two circuits:</p><br />
<img src='/wiki/images/d/d3/NYMU14_target_YebF_t.png' style='width: 550px;'><br />
<p>These circuits are then transformed into <i>E. coli</i>, cultivated in liquid culture, and centrifuged. Afterwards, the supernatants are analyzed for their OD value. If YebF works as we expected, we should see the first circuit successfully secrete RFP into the supernatant :</p><br />
<img src='/wiki/images/a/a6/NYMU14_target_YebF_rfp.png' style='width: 220px;margin-left: 40px;'><br />
<p>While the control should only contain RFP which is not exported:</p><br />
<img src='/wiki/images/8/87/NYMU14_target_YebF_norfp.png' style='width: 190px;margin-left: 40px;'><br />
<br />
<h3>Stage six: Does Endolysin work?</h3><br />
<p>To test the antimicrobial activity of endolysin, we also created two circuits:</p><br />
<img src='/wiki/images/d/d1/NYMU14_target_endolysin_t.png' style='width: 550px;'><br />
<p>The first circuit contains YebF and endolysin, which serves as the experimental circuit, and the second contains just YebF, and is the control. These are transformed into <i>E. coli</i>, and after culturing them overnight and extracting the supernatant, we spread the supernatant on plates of <i>S. mutans</i> to test for their antimicrobial activity.</p><br />
<br />
<h3>Stage seven: Does AHL stimulate the killing program as anticipated?</h3><br />
<p>In this stage, we put the final probiotic circuit into <i>E. coli</i> to see how well it performs.</p><br />
<img src='/wiki/images/3/39/NYMU14_Target_T7.png' style='width: 700px;'><br />
<p>The two different germs lines are involved in this test, the <i>E.coli</i> constructed circuit (<b>pSB1C3+luxR+pluxR+yebF+Endolysin</b>) and <i>S.mutans</i> constructed circuit (luxI).<br />
<br />
The culturing of constructed <i>E.coli</i> with the supernatant of constructed <i>S.mutans</i> (containing AHL) supposed to be secretion of endolysin. Then we cultured the wild type <i>S.mutans</i> with the supernatant of previous culture.</p><br />
<img src='/wiki/images/b/ba/NYMU14_Target_Test5.png' style='width: 500px;margin-left: 40px;'><br />
<br />
<h3>Stage eight: Finally, let’s check <i>Everything</i>!</h3><br />
<p>In this last stage we put all the constructed circuits intended for our probiotic and <i>M102</i> into <i>E. coli</i> and <i>S. mutans</i>, respectively. Then we co-cultured these two transformed strains together to test our full circuit.</p><br />
<br />
<h1 id='1c-4'>Our result</h1><br />
<br />
<h1 id='1c-5'>Reference</h1><br />
<ol><br />
<li>http://www.expertsmind.com/topic/microbiology/bacterial-cell-wall-92313.aspx</li><br />
</ol><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/File:%E5%9C%96%E7%89%871.pngFile:圖片1.png2014-10-14T21:25:30Z<p>GraceWang: </p>
<hr />
<div></div>GraceWanghttp://2014.igem.org/File:Inhibitor_circuit.pngFile:Inhibitor circuit.png2014-10-14T18:02:49Z<p>GraceWang: </p>
<hr />
<div></div>GraceWanghttp://2014.igem.org/File:NYMU_killing_module.pngFile:NYMU killing module.png2014-10-14T16:26:49Z<p>GraceWang: </p>
<hr />
<div></div>GraceWanghttp://2014.igem.org/File:NYMU14_targetall.pngFile:NYMU14 targetall.png2014-10-14T15:50:08Z<p>GraceWang: </p>
<hr />
<div></div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1c1Team:NYMU-Taipei/project/1c12014-10-10T18:36:17Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Inhibitor</h><br />
<div class='abstract'><br />
<ul><br />
<li>'''Interrupt biofilm formation through artificial genetic interference within S. mutans.'''</li><br />
<li>We synthesized a 24 bp non-coding DNA and transcribed into sRNA. This small RNA will bind to translation initiation region of biofilm formation-related mRNA and inhibit the translation.</li><br />
<li>Two target biofilm formation-related mRNA: Histidine kinase & G protein.</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c1-1'><p>Before we get started:</p></div><br />
<div href='#1c1-2'><p>So how did we do it?</p></div><br />
<div href='#1c1-3'><p>Putting it to the test!</p></div><br />
<div href='#1c1-4'><p>Our result</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c1-1'>Before we get started:</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1 id='1c1-2'>So how did we do it?</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1 id='1c1-3'>Putting it to the test!</h1><br />
<h1 id='1c1-4'>Our result</h1><br />
<h1>Reference</h1><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1c1Team:NYMU-Taipei/project/1c12014-10-10T18:05:00Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Inhibitor</h><br />
<div class='abstract'><br />
<ul><br />
<li>Our aim is to decrease the biofilm formation from the inside of S.mutans.</li><br />
<li>We synthesized a 24 bp non-coding DNA and transcribed into sRNA. This small RNA will bind to translation initiation region of biofilm formation-related mRNA and inhibit the translation.</li><br />
<li>Two target biofilm formation-related mRNA: Histidine kinase & G protein.</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c1-1'><p>Before we get started:</p></div><br />
<div href='#1c1-2'><p>So how did we do it?</p></div><br />
<div href='#1c1-3'><p>Putting it to the test!</p></div><br />
<div href='#1c1-4'><p>Our result</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c1-1'>Before we get started:</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1 id='1c1-2'>So how did we do it?</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1 id='1c1-3'>Putting it to the test!</h1><br />
<h1 id='1c1-4'>Our result</h1><br />
<h1>Reference</h1><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1c1Team:NYMU-Taipei/project/1c12014-10-10T17:58:30Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Inhibitor</h><br />
<div class='abstract'><br />
<ul><br />
<li>Our aim is to decrease the biofilm formation from the inside of S.mutans.</li><br />
<li>We synthesized a 24 bp non-coding DNA and transcribed into sRNA. This small RNA will bind to translation initiation region of biofilm formation-related mRNA and inhibit the translation.</li><br />
<li>Two target biofilm formation-related mRNA: Histidine kinase & G protein.</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c1-1'><p>purpose</p></div><br />
<div href='#1c1-2'><p>background</p></div><br />
<div href='#1c1-3'><p>design</p></div><br />
<div href='#1c1-4'><p>result</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c1-1'>Before we get started:</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1 id='1c1-3'>So how did we do it?</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1 id='1c1-4'>Putting ti to the test!</h1><br />
<h1 id='1c1-5'>Result</h1><br />
<h1>Reference</h1><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1c1Team:NYMU-Taipei/project/1c12014-10-10T17:56:52Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Inhibitor</h><br />
<div class='abstract'><br />
<ul><br />
<li>Our aim is to decrease the biofilm formation from the inside of S.mutans.</li><br />
<li>We synthesized a 24 bp non-coding DNA and transcribed into sRNA. This small RNA will bind to translation initiation region of biofilm formation-related mRNA and inhibit the translation.</li><br />
<li>Two target biofilm formation-related mRNA: Histidine kinase & G protein.</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c1-1'><p>purpose</p></div><br />
<div href='#1c1-2'><p>background</p></div><br />
<div href='#1c1-3'><p>design</p></div><br />
<div href='#1c1-4'><p>result</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c1-1'>Before we get started:</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1 id='1c1-3'>So how did we do it?</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1 id='1c1-4'>Putting ti to the test!</h1><br />
<h1>Reference</h1><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1c1Team:NYMU-Taipei/project/1c12014-10-10T17:49:09Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Inhibitor</h><br />
<div class='abstract'><br />
<ul><br />
<li>Our aim is to decrease the biofilm formation from the inside of S.mutans.</li><br />
<li>We synthesized a 24 bp non-coding DNA and transcribed into sRNA. This small RNA will bind to translation initiation region of biofilm formation-related mRNA and inhibit the translation.</li><br />
<li>Two target biofilm formation-related mRNA: Histidine kinase & G protein.</li><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#1c1-1'><p>purpose</p></div><br />
<div href='#1c1-2'><p>background</p></div><br />
<div href='#1c1-3'><p>design</p></div><br />
<div href='#1c1-4'><p>result</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='1c1-1'>Before we get started</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1 id='1c1-3'>So how did we do it?</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1 id='1c1-4'>Result</h1><br />
<h1>Reference</h1><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/3cTeam:NYMU-Taipei/project/3c2014-10-10T17:42:46Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Care</h><br />
<div class='abstract'><br />
<ul><br />
<li>This circuit can act as an alarm clock, sending out banana odor when cavities are going to happen in our oral environment.</li><br />
<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><br />
<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><br />
</ul><br />
</div><br />
<div class='cont-panel'><br />
<div href='#3c-1'><p>purpose</p></div><br />
<div href='#3c-2'><p>background</p></div><br />
<div href='#3c-3'><p>Chassis</p></div><br />
<div href='#3c-4'><p>design</p></div><br />
<div href='#3c-5'><p style="line-height: 25px;">Functional Measurement</p></div><br />
<div href='#3c-6'><p>result</p></div><br />
<div style="display:inline-block; width: 640px; height: 0.1px; border: none; margin: 0px"></div><br />
</div><br />
<div class='article indent'><br />
<h1 id='3c-1'>Purpose</h1><br />
<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><br />
<h1 id='3c-2'>Background</h1><br />
<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><br />
<h1 id='3c-3'>Chassis</h1><br />
<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><br />
<h1 id='3c-4'>Design</h1><br />
<br />
<h3>Promoter (iGEM part BBa_J23100)</h3><br />
<p>It is a commonly used constitutive promoter of iGEM part registry.</p><br />
<h3>RBS (iGEM part BBa_B0030)</h3><br />
<p>It is a commonly used ribosome binding site of iGEM part registry.</p><br />
<h3>The banana odor generator consists of 3 parts.</h3><br />
<h3>Part 1 (iGEM part BBa_J45008 [1,2,5])</h3><br />
<p>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.</p><br />
<h3>Part 2 (iGEM part BBa_J45009 [1,3,6])</h3> <br />
<p>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.</p><br />
<h3>Part 3 (iGEM part BBa_J45014 [1,4,7])</h3><br />
<p>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.</p><br />
<h3>Terminator (iGEM part BBa_B0015)</h3><br />
<p>A double terminator composed of B0010 and B0012. It has used by many iGEM teams, and have strong terminating force.</p><br />
<br />
<h1 id='3c-5'>Functional Measurement</h1><br />
<h3>Circuits needed to be constructed</h3><p class='noindent'>Promoter (BBa_J23100) + RBS (BBa_B0030) + Banana odor generator ( BBa_J45008+BBa_J45009 +BBa_J45014)+Terminator(BBa_B0015)<br />
</p><br><br />
<h3>Experimental steps</h3><br />
<ol class='cont-ol'><br />
<li>Culture modified E. coli in sealed agar plates overnight.</li><br />
<li>Use syringe to extract the gas generated by E. coli.</li><br />
<li>Use gas chromatography to analyze the sample.</li><br />
</ol><br />
<h1 id='3c-6'>Result</h1><br />
<p></p><br />
<h1>Reference</h1><br />
<ol><br />
<li>2006 MIT iGEM team and iGEM part registry BBa_J45008 / BBa_J45009 / BBa_J45014</li><br />
<li>NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome X, complete sequence</li><br />
<li>NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome IV, complete sequence</li><br />
<li>NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome XV, complete sequence</li><br />
<li>(2012)"Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis"</li><br />
</ol><br />
</div> <!-- article --><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1cTeam:NYMU-Taipei/project/1c2014-09-13T16:47:43Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Sensor</h><br />
<div class='abstract'><br />
<p><br />
▶We incorporate our circuit into Streptococcus phage M102, using the lysis gene of phage to kill S.mutans.<br><br />
▶In order to control the number of Streptococcus mutans, we use constitutive promoter to sense S.mutans by using a quorum sensing chemical compound which will be released in every competence S. mutans .<br><br />
▶To control the number of S.mutans more accurately, we design the threshold composed of different terminators.<br><br />
▶When the number of S.mutans reach the threshold, the lysis gene of Streptococcus phage will be triggered, and killing the S.mutans.<br />
</p><br />
</div><br />
<h1>Purpose</h1><br />
<p>We are going to use the “control part” to control the number of Streptococcus mutans (S. mutans). When the number of S. mutans exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess S.mutans.</p><br />
<h1>Background</h1><br />
<p>It is said that the number of S. mutans is highly related to the outbreak of cavities. Therefore, controlling the number of S. mutans has been our first priority. To reach our purpose, we need to detect the number of S. mutans, determine the threshold that causes cavities, and finally, kill the S. mutans. These three main designs of our first C, “control”, will be explained in the following paragraphs.</p><br><br />
<p>First of all, we need to detect the number of S. mutans. Over the years, it has been found that the <b>competence stimulating peptide, CSP</b>, a quorum sensing chemical, will be released in every competence S. mutans. Thus, we can detect the number of S.mutans by marking the amount of CSP.</p><br><br />
<p>In S.mutans, the CSP will bind to the membrane receptor, “comD”, thereby phosphorylating the response regulator, “comE”. The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and mutacin release. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene “nlmC”( non-lantibiotic mutacin C) in S.mutans has the best performance under the stimulation of CSP. As a result, we have decided to use this promoter in our design.</p><br><br />
<p>Secondly, we need to find and confirm the threshold that causes cavities. In this part, we will use different kinds and numbers of terminators (each with different leakage rates) to create the threshold. Moreover, we will combine both wet lab results and modelling to decide which design is more suitable.</p><br><br />
<p>Lastly, we need to kill the S. mutans when the amount of S. mutans exceeds the threshold. To fulfill our goal, we decided to incorporate our circuit into the Streptococcus phage M102, which is highly specific to S. mutans, in order to precisely control the lysis genes in phage M102.</p><br />
<h1>Design</h1><br />
<p>!figure not yet!</p><br />
<p>In our circuit, we incorporate nlmC promoter and different combination of terminators. When the number of S. mutans exceed the threshold, the first “C” , control mechanism will activate the lysis genes of phage M102. Thus, control the number of S. mutans.</p><br><br />
<p>In order to test the function of the “control” part of this project, we incorporate two stages of functional tests.<br><br />
<br><br />
<b>The first stage</b><br><br />
We will construct two circuits. One is served as a positive control, constructed with a constitutive promoter and the GFP gene from the iGEM kit, E0040. The other is served as an experimental group, constructed with a nlmC promoter and E0040. When doing the functional measurement, we will apply different amount of synthetic CSP in different time lapse into our experimental group after the circuit being transformed into S. mutans.<br> <br />
In the first stage of functional test, it is hoped to see the high amount of green fluorescent in the positive control, while different amount of green fluorescent being observed in our experimental group.<br><br />
<br><br />
<b>The second stage</b><br><br />
We will test the circuit constructed with nlmC promoter and different kinds of terminator, also using E0040 to measure the threshold we create with terminators. In this stage, we will test three different terminators, B0012, B0015, and one derived from S.mutans in different combination, namely, we will construct nine circuits:</p><br />
<ul type='circle'><br />
<li>nlmC promoter + B0012 + E0040</li><br />
<li>nlmC promoter + B0015 + E0040</li><br />
<li>nlmC promoter + terminator from S.mutans + E0040</li><br />
<li>nlmC promoter + B0012 + B0012 + E0040</li><br />
<li>nlmC promoter + B0012 + B0015 + E0040</li><br />
<li>nlmC promoter + B0012 + terminator from S.mutans + E0040</li><br />
<li>nlmC promoter + B0015 + B0015 + E0040</li><br />
<li>nlmC promoter + B0015 + terminator from S.mutans + E0040</li><br />
<li>nlmC promoter + terminator from S.mutans+ terminator from S.mutans +E0040</li><br />
</ul><br />
<p>In the second stage of our functional test, we are looking forward to see different amount of green fluorescent after applying different amount of synthetic CSP in different time lapse into these nine circuits.<br><br />
After the second stage of the function test, we will hand over our data to our modeling group, to see which threshold suit best in killing S.mutans.</p><br />
<h1>Result</h1><br />
<h1>Reference</h1><br />
<ol><br />
<li>Kreth, J., Hung, D. C. I., Merritt, J., Perry, J., Zhu, L., Goodman, S. D., Cvitkovitch, D. G., ... Qi, F. (January 01, 2007). The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis. Microbiology Reading-, 153, 1799-1807.</li><br />
<li>Hung, D. C. I., Downey, J. S., Ayala, E. A., Kreth, J., Mair, R., Senadheera, D. B., Qi, F., ... Goodman, S. D. (June 28, 2011). Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans. Journal of Bacteriology, 193, 14, 3642-3652.</li><br />
<li>Liu, T., Xue, S., Cai, W., Liu, X., Liu, X., Zheng, R., Luo, H., ... Qi, W. (March 22, 2014). ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans. Annals of Microbiology, 64, 1, 31-38.</li><br />
<li>van der Ploeg, J R. (2007). Genome sequence of Streptococcus mutans bacteriophage M102. Wiley-Blackwell.</li><br />
</ol><br />
<!-----------------------------------><br />
<h>Control-Inhibitor</h><br />
<h1>Purpose</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1>Background</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<h1>Design</h1><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1>Result</h1><br />
<h1>Reference</h1><br />
</div><br />
</html><br />
{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWanghttp://2014.igem.org/Team:NYMU-Taipei/project/1cTeam:NYMU-Taipei/project/1c2014-09-13T16:47:04Z<p>GraceWang: </p>
<hr />
<div>{{:Team:NYMU-Taipei/NYMU14_Menu}}<br />
<html><br />
<div id="main-content"><br />
<h>Control-Sensor</h><br />
<div class='abstract'><br />
<p><br />
▶We incorporate our circuit into Streptococcus phage M102, using the lysis gene of phage to kill S.mutans.<br><br />
▶In order to control the number of Streptococcus mutans, we use constitutive promoter to sense S.mutans by using a quorum sensing chemical which will be released in every competence S. mutans .<br><br />
▶To control the number of S.mutans more accurately, we design the threshold composed of different terminators.<br><br />
▶When the number of S.mutans reach the threshold, the lysis gene of Streptococcus phage will be triggered, and killing the S.mutans.<br />
</p><br />
</div><br />
<h1>Purpose</h1><br />
<p>We are going to use the “control part” to control the number of Streptococcus mutans (S. mutans). When the number of S. mutans exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess S.mutans.</p><br />
<h1>Background</h1><br />
<p>It is said that the number of S. mutans is highly related to the outbreak of cavities. Therefore, controlling the number of S. mutans has been our first priority. To reach our purpose, we need to detect the number of S. mutans, determine the threshold that causes cavities, and finally, kill the S. mutans. These three main designs of our first C, “control”, will be explained in the following paragraphs.</p><br><br />
<p>First of all, we need to detect the number of S. mutans. Over the years, it has been found that the <b>competence stimulating peptide, CSP</b>, a quorum sensing chemical, will be released in every competence S. mutans. Thus, we can detect the number of S.mutans by marking the amount of CSP.</p><br><br />
<p>In S.mutans, the CSP will bind to the membrane receptor, “comD”, thereby phosphorylating the response regulator, “comE”. The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and mutacin release. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene “nlmC”( non-lantibiotic mutacin C) in S.mutans has the best performance under the stimulation of CSP. As a result, we have decided to use this promoter in our design.</p><br><br />
<p>Secondly, we need to find and confirm the threshold that causes cavities. In this part, we will use different kinds and numbers of terminators (each with different leakage rates) to create the threshold. Moreover, we will combine both wet lab results and modelling to decide which design is more suitable.</p><br><br />
<p>Lastly, we need to kill the S. mutans when the amount of S. mutans exceeds the threshold. To fulfill our goal, we decided to incorporate our circuit into the Streptococcus phage M102, which is highly specific to S. mutans, in order to precisely control the lysis genes in phage M102.</p><br />
<h1>Design</h1><br />
<p>!figure not yet!</p><br />
<p>In our circuit, we incorporate nlmC promoter and different combination of terminators. When the number of S. mutans exceed the threshold, the first “C” , control mechanism will activate the lysis genes of phage M102. Thus, control the number of S. mutans.</p><br><br />
<p>In order to test the function of the “control” part of this project, we incorporate two stages of functional tests.<br><br />
<br><br />
<b>The first stage</b><br><br />
We will construct two circuits. One is served as a positive control, constructed with a constitutive promoter and the GFP gene from the iGEM kit, E0040. The other is served as an experimental group, constructed with a nlmC promoter and E0040. When doing the functional measurement, we will apply different amount of synthetic CSP in different time lapse into our experimental group after the circuit being transformed into S. mutans.<br> <br />
In the first stage of functional test, it is hoped to see the high amount of green fluorescent in the positive control, while different amount of green fluorescent being observed in our experimental group.<br><br />
<br><br />
<b>The second stage</b><br><br />
We will test the circuit constructed with nlmC promoter and different kinds of terminator, also using E0040 to measure the threshold we create with terminators. In this stage, we will test three different terminators, B0012, B0015, and one derived from S.mutans in different combination, namely, we will construct nine circuits:</p><br />
<ul type='circle'><br />
<li>nlmC promoter + B0012 + E0040</li><br />
<li>nlmC promoter + B0015 + E0040</li><br />
<li>nlmC promoter + terminator from S.mutans + E0040</li><br />
<li>nlmC promoter + B0012 + B0012 + E0040</li><br />
<li>nlmC promoter + B0012 + B0015 + E0040</li><br />
<li>nlmC promoter + B0012 + terminator from S.mutans + E0040</li><br />
<li>nlmC promoter + B0015 + B0015 + E0040</li><br />
<li>nlmC promoter + B0015 + terminator from S.mutans + E0040</li><br />
<li>nlmC promoter + terminator from S.mutans+ terminator from S.mutans +E0040</li><br />
</ul><br />
<p>In the second stage of our functional test, we are looking forward to see different amount of green fluorescent after applying different amount of synthetic CSP in different time lapse into these nine circuits.<br><br />
After the second stage of the function test, we will hand over our data to our modeling group, to see which threshold suit best in killing S.mutans.</p><br />
<h1>Result</h1><br />
<h1>Reference</h1><br />
<ol><br />
<li>Kreth, J., Hung, D. C. I., Merritt, J., Perry, J., Zhu, L., Goodman, S. D., Cvitkovitch, D. G., ... Qi, F. (January 01, 2007). The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis. Microbiology Reading-, 153, 1799-1807.</li><br />
<li>Hung, D. C. I., Downey, J. S., Ayala, E. A., Kreth, J., Mair, R., Senadheera, D. B., Qi, F., ... Goodman, S. D. (June 28, 2011). Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans. Journal of Bacteriology, 193, 14, 3642-3652.</li><br />
<li>Liu, T., Xue, S., Cai, W., Liu, X., Liu, X., Zheng, R., Luo, H., ... Qi, W. (March 22, 2014). ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans. Annals of Microbiology, 64, 1, 31-38.</li><br />
<li>van der Ploeg, J R. (2007). Genome sequence of Streptococcus mutans bacteriophage M102. Wiley-Blackwell.</li><br />
</ol><br />
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<h>Control-Inhibitor</h><br />
<h1>Purpose</h1><br />
<p>Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.</p><br />
<h1>Background</h1><br />
<p>Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.</p><br><br />
<p>G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.</p><br />
<h1>Design</h1><br />
<p>We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.</p><br />
<h1>Result</h1><br />
<h1>Reference</h1><br />
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{{:Team:NYMU-Taipei/NYMU14_Footer}}</div>GraceWang