Team:SYSU-China/project bacterial migration

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        <li><a href="https://2011.igem.org/Team:SYSU-China"><span></span></a></li>
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        <li><a href="https://2011.igem.org/Team:SYSU-China/main_page_news"><span>NEWS</span></a></li>
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                <li><a href="https://2011.igem.org/Team:SYSU-China/project_bacterial_migration"><span>Bacterial Migration</span></a>
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                <li><a href="https://2011.igem.org/Team:SYSU-China/project_Cesium_Absorption"><span>Cesium Absorption</span></a></li>
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                <li><a href="https://2011.igem.org/Team:SYSU-China/project_Aggregation_Recovery"><span>Aggregation & Recovery</span></a></li>
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                <li><a href="https://2011.igem.org/Team:SYSU-China/page_project_notes"><span>Notes</span></a></li>
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      <h2>Background </h2>
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          <br />
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          <p>Ionizing radiation activates the SOS repair system of bacteria through DNA damage. The single-strand DNA breaks leads to the activation of protein RecA, which leads to proteolysis of the repressor protein LexA, resulting in the increased transcription of about 20 genes, including recA and recN (Fig.1). Therefore, recAp and recNp are radiation-inducible and can be utilized to control gene expression induced by ionizing radiation. </p>
 +
          <br/>
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            <p>The rotational direction of bacteria is controlled by the flagellar motor system, of which CheY plays a pivotal role. When CheY is phosphorylated (CheY-P), it binds to the flagellar switch protein FliM and induces the flagellum to rotate clockwise, which causes the cell to tumble. Smooth swimming is restored by the phosphatase CheZ, which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise (Fig.2). E.coli lacking the cheZ gene (∆cheZ, strain RP1616, non-motile) cannot dephosphorylate CheY-P, tumble incessantly, and are thus nonmotile. So, if we controls the expression of cheZ in strain RP1616, it's possible to control the movement of bacteria.</p>
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              <li> <a href="https://static.igem.org/mediawiki/2011/1/14/BM_01_Fig_1.jpg" title="Fig.1  The SOS repair system.
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The single-strand DNA breaks leads to the activation of protein RecA, which leads to proteolysis of the repressor protein LexA, resulting in the increased transcription of about 20 genes, including recA and recN."><img src="https://static.igem.org/mediawiki/2011/f/fc/BM_01_Fig_1_small.jpg" width="150" height="144" /></a>
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                Fig.1<p>  The SOS repair system.</p>
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                </li>
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              <li> <a href="https://static.igem.org/mediawiki/2011/6/64/BM_01_Fig_2.jpg" title="Fig.2  The flagellar motor system.
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Protein CheZ is a phosphatase which interacts with various substrate including CheY. When CheY is phosphorylated, it binds to protein FliM and the flagella rotates clockwise, causing bacteria to tumble. While, when CheY is not phosphorylated, the flagella rotates counterclockwise, then bacteria moves ahead."><img src="https://static.igem.org/mediawiki/2011/d/de/BM_01_Fig_2_small.jpg" width="150" height="94" alt="Tree" /></a>
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              Fig.2 <p> The flagellar motor system. </p>
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      <h2>Modules</h2>
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      <p>Accordingly, in order to construct bacteria that move directionally towards ionizing radiation, we can place gene cheZ in the downstream of recAp. </p>
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      <br />
 +
      <p>First of all, we got recAp by PCR and constructed plasmid RecA-EGFP-pET28a (Fig.1) to verify that it functioned well (Fig.2).</p>
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      <br />
 +
      <p>Then, we tested the expression of cheZ on protein level. Within the inducement of 0.1 mM IPTG at 18℃ for 18h, we extracted the total proteins of the E.coli transformed with plasmid CheZ-pET28a (Fig.3). Western Blot results showed that the expression of protein CheZ significantly increased after the inducement, indicating that cheZ expressed well (Fig.4). Additionally, sicne a basal expression caused by lacp in pET28a, the control group also showed a low expression of CheZ. </p>
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              <li> <a href="https://static.igem.org/mediawiki/2011/2/2c/BM_02_Fig_1.jpg" title="Fig.1  The racAp testing plasmid.
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We got recAp by PCR and constructed plasmid RecA-EGFP-pET28a to test its function."><img src="https://static.igem.org/mediawiki/2011/7/77/BM_02_Fig_1_small.jpg" width="150" height="94" /></a> Fig.1  <p>The racAp testing plasmid</p> </li>
 +
              <li> <a href="https://static.igem.org/mediawiki/2011/4/4c/BM_02_Fig_3.jpg" title="Fig.3  Plasmid CheZ-pET28a."><img src="https://static.igem.org/mediawiki/2011/b/bc/BM_02_Fig_3_small.jpg" width="150" height="94" /></a> Fig.3 
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                <p>Plasmid CheZ-pET28a.</p> </li>
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              <li> <a href="https://static.igem.org/mediawiki/2011/8/8a/BM_02_Fig_4.jpg" title="Fig.4  The expression of cheZ.
 +
Within the inducement of 0.1 mM IPTG at 18℃ for 18h, the expression of protein CheZ significantly increased. Sicne a basal expression caused by lacp in pET28a, the control group also showed a low expression of CheZ. "><img src="https://static.igem.org/mediawiki/2011/f/f8/BM_02_Fig_4_small.jpg" width="150" height="98" /></a> Fig.4 
 +
                <p>The expression of cheZ.</p>
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              </li>
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              </ul>
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          </div>
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    <h2>Function</h2>
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    <br />
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        <P>Finally, we planned to test the function of gene cheZ in bacterial migration towards ionizing radiation. Because of limited conditions, we replaced ionizing radiation with UV, which has the same effect of DNA damage to activate the SOS repair system.  The ∆cheZ E.coli strain RP1616 was transformed with plasmid RecA-CheZ-pET28a (Fig.1) (RP1616(RecA-CheZ-pET28a)) whose original T7p was digested by restriction endonuclease.
 +
</P>
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    <br />
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    <p>After exposing to a certain intensity (which couldn't be measured for a lack of apposite apparatus) of UV, we observed the behavior of both wild-type (RP437, motile) and ∆cheZ (RP1616) with RecA-CheZ-pET28a. The results indicate that: </p>
 +
    <br />
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    <p>1) RP1616(RecA-CheZ-pET28a) moves directionally towards higher UV intensity (Fig.2). </p>
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            <ul>
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              <li> <a href="https://static.igem.org/mediawiki/2011/d/d7/BM_03_Fig_1.jpg" title="Fig.7  Plasmid RecA-CheZ-pET28a(without T7p)."><img src="https://static.igem.org/mediawiki/2011/e/ef/BM_03_Fig_1_small.jpg" width="150" height="94" alt="Flower" /></a>
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                Fig.7 <p> Plasmid RecA-CheZ-pET28a(without T7p).</p>
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<script type="text/javascript" src="Library/jquery.fancybox-1.3.4.pack.js"></script>
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<a id="youtube_player" href="http://www.youtube.com/watch?v=Ghxmpo7SKC0&feature=feedwll;feature=player_embedded#at=41"><img src="https://static.igem.org/mediawiki/2011/2/2e/Tour_2.jpg" width="307" height="173" /></a> <!-- #EndLibraryItem -->
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<div class="page_content_relatedBar_project_wiki2">
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  <h2>Pages we think helpful:</h2>
 +
    <h1><a href="https://2011.igem.org/Team:SYSU-China/page_project_modules_construction">see how we construct our modules</a></h1>
 +
    <h1><a href="https://2011.igem.org/Team:SYSU-China/page_project_modules_verification">see how we verify each module</a></h1>
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    <h1><a href="https://2011.igem.org/Team:SYSU-China/main_page_story">or see our whole idea in Story page</a></h1>
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    <p>&nbsp;</p>
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<div class="main_page_clearer_light"></div>
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<!--here begins the contact_bottombar!--><!-- #BeginLibraryItem "/Library/contact_bottombar.lbi" --><div class="contact_bottombar">
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      <p><a href="https://2011.igem.org/Main_Page">2011 iGEM Mainpage</a></p>
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      <p><a href="https://igem.org/Team_Wikis?year=2011">2011 iGEM Team wikis</a></p>
 +
 
 +
      <p>From the 2011 iGEM team SYSU-China (2011)</p>
 +
      <p>Sun Yat-Sen University, Guangzhou, China</p>
 +
      <p>Address: 135# Xingang Rd.(W.), Haizhu Guangzhou, P.R.China</p>
 +
      <p><a href="http://eng.sysu.edu.cn/">visit the Sun Yat-sen university website</a></p>
 +
      <p>Thanks Apycom jQuery Menus and <a href="http://apycom.com/">visit their website</a>
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Revision as of 17:33, 25 October 2011

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Modules Verification-Sun Yat-sen Univ.

Background


Ionizing radiation activates the SOS repair system of bacteria through DNA damage. The single-strand DNA breaks leads to the activation of protein RecA, which leads to proteolysis of the repressor protein LexA, resulting in the increased transcription of about 20 genes, including recA and recN (Fig.1). Therefore, recAp and recNp are radiation-inducible and can be utilized to control gene expression induced by ionizing radiation.


The rotational direction of bacteria is controlled by the flagellar motor system, of which CheY plays a pivotal role. When CheY is phosphorylated (CheY-P), it binds to the flagellar switch protein FliM and induces the flagellum to rotate clockwise, which causes the cell to tumble. Smooth swimming is restored by the phosphatase CheZ, which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise (Fig.2). E.coli lacking the cheZ gene (∆cheZ, strain RP1616, non-motile) cannot dephosphorylate CheY-P, tumble incessantly, and are thus nonmotile. So, if we controls the expression of cheZ in strain RP1616, it's possible to control the movement of bacteria.

Modules


Accordingly, in order to construct bacteria that move directionally towards ionizing radiation, we can place gene cheZ in the downstream of recAp.


First of all, we got recAp by PCR and constructed plasmid RecA-EGFP-pET28a (Fig.1) to verify that it functioned well (Fig.2).


Then, we tested the expression of cheZ on protein level. Within the inducement of 0.1 mM IPTG at 18℃ for 18h, we extracted the total proteins of the E.coli transformed with plasmid CheZ-pET28a (Fig.3). Western Blot results showed that the expression of protein CheZ significantly increased after the inducement, indicating that cheZ expressed well (Fig.4). Additionally, sicne a basal expression caused by lacp in pET28a, the control group also showed a low expression of CheZ.

Function


Finally, we planned to test the function of gene cheZ in bacterial migration towards ionizing radiation. Because of limited conditions, we replaced ionizing radiation with UV, which has the same effect of DNA damage to activate the SOS repair system. The ∆cheZ E.coli strain RP1616 was transformed with plasmid RecA-CheZ-pET28a (Fig.1) (RP1616(RecA-CheZ-pET28a)) whose original T7p was digested by restriction endonuclease.


After exposing to a certain intensity (which couldn't be measured for a lack of apposite apparatus) of UV, we observed the behavior of both wild-type (RP437, motile) and ∆cheZ (RP1616) with RecA-CheZ-pET28a. The results indicate that:


1) RP1616(RecA-CheZ-pET28a) moves directionally towards higher UV intensity (Fig.2).

2011 iGEM Mainpage

2011 iGEM Team wikis

From the 2011 iGEM team SYSU-China (2011)

Sun Yat-Sen University, Guangzhou, China

Address: 135# Xingang Rd.(W.), Haizhu Guangzhou, P.R.China

visit the Sun Yat-sen university website

Thanks Apycom jQuery Menus and visit their website