Team:NYMU-Taipei/project/1c1

From 2014.igem.org

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       <h>Control-Inhibitor</h>
       <h>Control-Inhibitor</h>
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      <h1>Here’s the gist…</h1>
       <div class='abstract'>
       <div class='abstract'>
<ul>
<ul>
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<li>Interrupt biofilm formation through artificial genetic interference within S. mutans.</li>
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<li>Interrupt biofilm formation through artificial genetic interference within <i>S. mutans.</i></li>
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<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>
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<li>Two target biofilm formation-related mRNA: Histidine kinase & G protein.</li>
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</ul>
</ul>
       </div>
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           <div href='#1c1-3'><p>Test it!</p></div>
           <div href='#1c1-3'><p>Test it!</p></div>
           <div href='#1c1-4'><p>Our result~</p></div>
           <div href='#1c1-4'><p>Our result~</p></div>
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          <div href='#1c1-5'><p>Reference</p></div>
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       <div class='article indent'>
       <h1 id='1c1-1'>Before we get started:</h1>
       <h1 id='1c1-1'>Before we get started:</h1>
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       <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>
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       <p><b>Biofilm</b> formation is part of the reason <i>S. mutans</i> is so devastating to oral health (see Project Overview for more information). For adequate oral protection, biofilm formation is a problem we must solve, and this means we need a way to silence the appropriate proteins.</p>
 +
      <p>So how are proteins silenced? As we know, proteins are translated from single-stranded mRNAs. Just like eukaryotic cells, bacteria can <b>selectively silence</b> mRNA by transcribing sequences of <b>sRNA, or bacterial small RNA</b>.</p>
 +
      <p>These small RNA molecules are usually less than 50bp long, and function by <b>binding to the specified, complementary mRNA strand to prevent ribosome attachment</b>. We decided that targeted sRNA would give us a good shot at silencing HK11 and SGP in <i>S. mutans.</i></p>
 +
      <p>Next up, we need to find some proteins to silence!</p>
       <h1 id='1c1-2'>So how did we do it?</h1>
       <h1 id='1c1-2'>So how did we do it?</h1>
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       <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>
+
       <p>After careful literature search, we found two candidates:</p>
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       <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>
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      <p><b>Histidine kinase 11 (HK11)</b> is a sensor kinase from a two-component signal transduction system. Literature indicates that a knock-out of HK11 resulted in dramatically decreased biofilm formation.</p>
-
       <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>
+
       <p><b>G protein in <i>S. mutans</i> (SGP)</b> is involved in the regulation of intracellular GTP/GDP ratio, response to stress, and other diverse cellular functions. As with HK11, a knock-out of SGP resulted in dramatically decreased biofilm density.</p>
 +
       <p>In order to silence these two proteins with sRNA, we synthesized a 24bp strand of non-coding DNA, and transcribed it into the corresponding RNA. This small RNA sequence is our artificial sRNA, and will <b>bind to the TIR (translation initiation region)</b> of our target mRNAs. This prevents the target mRNA from undergoing translation.</p>
 +
      <p>The final thing we need is something to help smooth out the whole process. The <b>MicC scaffold</b> is a specially constructed sequence which recruits the <b>Hfq protein</b>. The Hfq protein then <b>helps our sRNA hybridize with its target mRNA</b>, while <b>stabilizing the sRNA-mRNA complex</b>.</p>
 +
      <p>At last, our circuit is complete!</p>
       <h1 id='1c1-3'>Putting it to the test!</h1>
       <h1 id='1c1-3'>Putting it to the test!</h1>
       <h1 id='1c1-4'>Our result</h1>
       <h1 id='1c1-4'>Our result</h1>
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       <h1>Reference</h1>
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       <h1 id='1c1-5'>Reference</h1>
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       </div> <!-- article -->
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{{:Team:NYMU-Taipei/NYMU14_Footer}}

Revision as of 17:38, 14 October 2014

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Control-Inhibitor

Here’s the gist…

  • Interrupt biofilm formation through artificial genetic interference within S. mutans.

Get started.

How to do it?

Test it!

Our result~

Reference

Before we get started:

Biofilm formation is part of the reason S. mutans is so devastating to oral health (see Project Overview for more information). For adequate oral protection, biofilm formation is a problem we must solve, and this means we need a way to silence the appropriate proteins.

So how are proteins silenced? As we know, proteins are translated from single-stranded mRNAs. Just like eukaryotic cells, bacteria can selectively silence mRNA by transcribing sequences of sRNA, or bacterial small RNA.

These small RNA molecules are usually less than 50bp long, and function by binding to the specified, complementary mRNA strand to prevent ribosome attachment. We decided that targeted sRNA would give us a good shot at silencing HK11 and SGP in S. mutans.

Next up, we need to find some proteins to silence!

So how did we do it?

After careful literature search, we found two candidates:

Histidine kinase 11 (HK11) is a sensor kinase from a two-component signal transduction system. Literature indicates that a knock-out of HK11 resulted in dramatically decreased biofilm formation.

G protein in S. mutans (SGP) is involved in the regulation of intracellular GTP/GDP ratio, response to stress, and other diverse cellular functions. As with HK11, a knock-out of SGP resulted in dramatically decreased biofilm density.

In order to silence these two proteins with sRNA, we synthesized a 24bp strand of non-coding DNA, and transcribed it into the corresponding RNA. This small RNA sequence is our artificial sRNA, and will bind to the TIR (translation initiation region) of our target mRNAs. This prevents the target mRNA from undergoing translation.

The final thing we need is something to help smooth out the whole process. The MicC scaffold is a specially constructed sequence which recruits the Hfq protein. The Hfq protein then helps our sRNA hybridize with its target mRNA, while stabilizing the sRNA-mRNA complex.

At last, our circuit is complete!

Putting it to the test!

Our result

Reference