Project

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     <td height="54" colspan="2" valign="bottom" nowrap="nowrap" bgcolor="#03438A" id="logo"><img src="https://static.igem.org/mediawiki/2008/f/fc/Logonew1.jpg" width="340" height="120" /><a href="https://2008.igem.org/Team:Tianjin" class="STYLE1"> <span class="STYLE2">aaa</span><span class="STYLE3">Home</span> <span class="STYLE2">aa</span></a></td>
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     <td width="442" height="54" valign="bottom" nowrap="nowrap" bgcolor="#03438A" id="logo"><img src="https://static.igem.org/mediawiki/2008/f/fc/Logonew1.jpg" width="340" height="120" /><a href="https://2008.igem.org/Team:Tianjin" class="STYLE1"><span class="STYLE3">Home</span></a></td>
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     <td width="8" valign="top" bgcolor="#03438A">&nbsp;</td>
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         <td height="124" colspan="2" bgcolor="#03438A" class="subHeader"><div align="center" class="STYLE16" style="margin-bottom: 0">
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           <p>&nbsp;</p>
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           <p><strong>PROJECT 1 </strong></p>
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          <p>A Synthetic Plasmid Self-Assembly system</p>
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            <p class="STYLE26">Foolproof Plasmid Self-Assembly system</p>
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        </div>          </td>
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         <td colspan="2" bgcolor="#03438A" class="subHeader STYLE4"><p class="STYLE29"><strong>Objectives</strong>: </p>
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           <p class="STYLE7">Bacterial assembly is aimed to be achieved based on the mechanism of site-specific recombination systems, So that the expensive reagent as well as the laboring tasks could be saved in gene cloning experiments.</p></td>
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            <td height="39" colspan="2" bgcolor="#03438A"><span class="STYLE1" style="margin-bottom: 0"><strong>Background</strong></span></td>
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            <td height="88" colspan="2" bgcolor="#03438A" class="STYLE9"><p><strong>Site-specific recombination</strong><br />
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Site-specific  recombination differs from general recombination in that short specific  sequences which are required for the recombination, are the only sites at which  recombination occurs. These reactions invariably require specialized proteins  to recognize these sites and to catalyze the recombination reaction at these  sites.</p></td>
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            <td width="513" bgcolor="#03438A" class="STYLE9"><p><span class="STYLE10"><strong>Inverted  repeats</strong> <br />
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                If the two sites  at which recombination will take place are oriented oppositely to one another  in the same DNA molecule then the following illustrates the sequence of events  that will take place:</span></p></td>
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            <td width="480" bgcolor="#03438A" class="STYLE9"><p><span class="STYLE10"><strong>Direct repeats</strong> <br />
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                If the two sites at which recombination  will take place are oriented in the same direction in the same DNA molecule  then the following illustrates the sequence of events:</span></p></td>
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            <td bgcolor="#03438A"><a href="http://www.mun.ca/biochem/courses/3107/images/rec_invert.GIF" target="_blank"><img src="https://static.igem.org/mediawiki/2008/a/a0/567.jpg" width="509" height="100" /></a></td>
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            <td bgcolor="#03438A"><a href="http://www.mun.ca/biochem/courses/3107/images/rec_direct.GIF" target="_blank"><img src="https://static.igem.org/mediawiki/2008/6/60/I8uy.jpg" width="476" height="100" /></a></td>
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            <td height="118" bgcolor="#03438A" class="STYLE9"><p class="STYLE10">The net result is that <strong>the segment of  DNA between the two recombinogenic sites has inverted</strong> with respect to the  rest of the DNA molecule.<br />
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              In other words, <strong><u>recombination at  inverted repeats causes an inversion</u></strong></p></td>
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            <td bgcolor="#03438A" class="STYLE9"><p class="STYLE10">The net result is that <strong>the segment of  DNA between the two recombinogenic sites has been deleted</strong> from the rest of  the DNA molecule and appears as a circular molecule.<br />
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              In other words, <strong><u>recombination at  direct repeats causes a deletion</u></strong>.</p></td>
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            <td height="54" colspan="2" bgcolor="#03438A" class="STYLE9"><p><span class="STYLE10"><strong>Note</strong> that the reverse reaction -- the recombination of a  circular molecule with another DNA molecule (either circular or linear), brings  about a fusion of both molecules or the integration of one molecule into the  other. The integrated segment will be flanked by directly repeating sequences which can, of course, be used to excise the integrated segment again.</span></p></td>
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            <td height="53" colspan="2" bgcolor="#03438A"><span class="STYLE7"><strong>Integration of bacteriophage lambda</strong></span></td>
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            <td height="54" colspan="2" bgcolor="#03438A"><p class="STYLE10">              <span class="STYLE9">In order for the lambda prophage to exist  in a host <em>E.&nbsp;coli</em> cell, it must integrate into the host chromosome  which it does by means of a <strong>site-specific recombination reaction</strong>. </span><br />
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            <td height="55" bgcolor="#03438A"><img src="https://static.igem.org/mediawiki/2008/3/30/消费税.jpg" width="510" height="320" /></td>
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            <td height="55" bgcolor="#03438A"><p class="STYLE9">The <em>E.&nbsp;coli</em> chromosome contains  one <strong>attachment site</strong> which is designated <strong><em>attB</em></strong>. The site is  only 30 bp in size and contains a conserved central 15 bp region where the  recombination reaction will take place. The structure of the recombination site  is usually represented as <strong>BOB'</strong>.</p>
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              <p class="STYLE9">The bacteriophage recombination site - <strong><em>attP</em></strong> - contains the identical central 15 bp region as <strong><em>attB</em></strong>. The overall structure can  be represented as <strong>POP'</strong>.</p>
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              <p class="STYLE9">Integration of bacteriophage lambda  requires one phage-encoded protein - <strong>Int</strong>, which is the <strong>integrase</strong> - and one bacterial protein - <strong>IHF</strong>, which is <strong>Integration</strong> <strong>Host</strong> <strong>Factor</strong>. Both of these proteins bind to sites on the <strong>P</strong> and <strong>P'</strong> arms of <strong><em>attP</em></strong> to form a complex in which the central conserved 15  bp elements of <strong><em>attP</em></strong> and <strong><em>attB</em></strong> are properly aligned. </p></td>
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                <td width="42%"><p align="center"><a href="http://www.mun.ca/biochem/courses/3107/images/POP_BOB.jpg"></a><img src="https://static.igem.org/mediawiki/2008/d/d9/Qweerw.jpg" width="500" height="110" /></p></td>
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                <td width="58%"><p align="center"><a href="http://www.mun.ca/biochem/courses/3107/images/BOP_POB.jpg"></a><img src="https://static.igem.org/mediawiki/2008/6/63/Y.jpg" width="491" height="110" /></p></td>
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         <td height="23" colspan="3" bgcolor="#03438A" class="STYLE11"><p class="STYLE11 STYLE4">The result of recombination is that the integrated prophage is flanked by two attachment sites but now they are slightly different: <em>attL</em> has the structure BOP' and <em>attR</em> has the structure POB'. </p></td>
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         <td width="536" height="46" bgcolor="#03438A" class=" STYLE13"><p class="STYLE7">Our design</p>
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          <p class="STYLE7">We have innovatively utilized the site-specific systems to build a foolproof bacterial assembly  system to future reduce the labor and cost involved in gene cloning experiments. We have designed three standardized vectors which perform as the  donors, receptor vector respectively.</p>
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              <td width="954" height="39" bgcolor="#03438A"><span class="STYLE16" style="margin-bottom: 0"><strong><a href="https://2008.igem.org/B11111">Background</a></strong></span></td>
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          <p align="left"><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Tianjin">Parts contributed to the registry</a><br>
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          </p></td>
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        <td height="46" bgcolor="#03438A" class=" STYLE13"><img name="" src="https://static.igem.org/mediawiki/2008/4/45/Zong.gif" width="350" height="350" alt=""></td>
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         <td height="23" colspan="3" bgcolor="#03438A" class=" STYLE10"><p class="STYLE10 STYLE4">Cre-Lox  recombination is a special  type of site-specific recombination, which is often applied as a gene knockout  tool. <br />
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         <td colspan="2" align="left" bgcolor="#03438A" class="subHeader"><p align="center" class="STYLE15"><span class="STYLE31">How do they work? </span></p>
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          Cre is a site-specific  DNA recombinase, which can catalyse the recombination of DNA between specific  sites, e.g. loxP in a DNA molecule. When cells that have loxP sites in  their genome express Cre, a reciprocal recombination event will occur between  the loxP sites. The double stranded DNA is cut at both loxP sites  by the Cre protein. The strands are then rejoined with <a href="http://en.wikipedia.org/wiki/DNA_ligase" title="DNA ligase">DNA ligase</a>. The efficiency of recombination  depends on the orientation of the loxP sites. For two lox sites on the  same chromosome arm, inverted loxP sites will cause an inversion, while  a direct repeat of loxP sites will cause a deletion event.</p>
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          <p class="STYLE10 STYLE4">           <span class="STYLE11">Lox P site</span><br />
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            <span class="STYLE1">First, we define the  Receptor as the vector that has already existed in the cell (E.coli.), and the  Donor as the vector containing the desired gene that we intend to integrate into the Receptor. The gene circuits for these plasmids are illustrated below.</span></p>
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           Lox P (locus of X-over P1) is  a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric  8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking  it. The detailed structure is given below.</p></td>
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           <p class="STYLE1">When the Donor vector carrying the gene of interest GENE1 was introduced to the E Coli which contains the Receptor vector, the site-specific recombination will occur between the <em>attB1</em> site and the <em>attP1</em> site, so that the two sequences will be integrated into one circular DNA.</p>
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          <p class="STYLE1">The recombinant DNA then could be selected in the liquid culture containing both ampicillin and  kanamycin. Then, under inducible conditions, Cre will be expressed and the  recombined sequence will be divided into two separate plasmids; one will retain  the desired gene 1, while the other will preserve the killer gene ccdB, which is under the control of another inducible promoter. Because the two plasmids  have shared origin site, plasmid incompatibility will occur thus the two kinds of plasmids will be separated into different cells. </p>
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          <p class="STYLE1">When induced, the expressed CCDB will lead to the Programed Cell Death (PCD). </p>
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        <td height="23" colspan="3" bgcolor="#03438A" class=" STYLE13"><span class="STYLE2">aaaaaa</span><img src="https://static.igem.org/mediawiki/2008/6/6e/09875.jpg" width="803" height="104" align="middle" /></td>
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          <p class="STYLE1">In order to realize the linkage of GENE 1 with GENE 2, we will introduce the new plasmid containing the desired GENE2 to the survival cells, in which the plasmids containing GENE 1 will behave as the new Receptor plasmid. Very similarly recombination between the <em>attB2 </em>and<em> attP2 </em>and the cleavage between the two <em>loxp </em> sites will be  performed, and plasmids containing the linked GENE1 and GENE2 will be selected  when the promoter expresses CCDB is induced. </p>        
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          <p class="STYLE1">The reason for us  to use two sets of<em> attB/attP</em> specific  sites is to avoid the combination within one module.</p>          </td>
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        <td height="23" colspan="3" bgcolor="#03438A" class="STYLE1"><strong>Objectives</strong>: Bacterial assembly is aimed to be achieved based on  the mechanism of site-specific recombination systems, So that the expensive reagent as well as the laboring tasks could be saved in gene cloning experiments.</td>
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        <td height="46" colspan="3" bgcolor="#03438A" class=" STYLE13"><p class="STYLE1">Our design</p>
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          <p class="STYLE9">We have innovatively utilized the  site-specific systems mentioned above to build a foolproof bacterial assembly system to future reduce the labor and cost involved in gene cloning  experiments. We have designed three standardized vectors which perform as the  donors, receptor vector respectively.</p></td>
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        <td width="8" bgcolor="#03438A">&nbsp;</td>
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        <td width="468" bgcolor="#03438A" class="STYLE9"><p class="STYLE15">&nbsp;</p>          <p class="STYLE1">&nbsp;</p>          <p>&nbsp;</p>          <p class="STYLE9">How do they work? <br />
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          When the donor vector carrying the gene of interest GENE1 was introduced to the E Coli which contains the Receptor vector, the site-specific recombination will occur between the <em>attB1</em> site and the <em>attP1</em> site, so that the two sequences will be integraded into one circular DNA, and then, under inducible conditions, Cre will be expressed and the recombined sequence will be divided into two separate plasmids; one will retain the desired gene 1, while the other preserves the killer gene ccdB, which is under the control of another inducible promoter. When induced, the promoter will  express CcdB so that cells containing CcdB will be killed. In order to link GENE 1 with GENE 2, we will introduce the new plasmid containing the desired GENE2 to the survival cells, in which the plasmids containing GENE 1 will behave as the new Receptor plasmid. Very similarly recombination between the <em>attB2 </em>and<em> attP2 </em>and the cleavage between the two <em>loxp </em> sites will be  performed, and plasmids containing the linked GENE1 and GENE2 will be selected  when the promoter expresses CcdB is induced. </p></td>
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        <td colspan="4"><p class="STYLE1">Where  have we been?</p></td>
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          <td colspan="2" bgcolor="#03438A"><p align="center" class="STYLE16">The whole process</p>         </td>
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          <td width="557" bgcolor="#FFFFFF"><p class="subHeader"><span class="STYLE1"><img src="https://static.igem.org/mediawiki/2008/2/21/%EF%BC%91%EF%BC%92%EF%BC%93%EF%BC%91%EF%BC%91%EF%BC%92.jpg" width="520" height="137"></span></p>
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          <td width="402" bgcolor="#B9E5ED"><span class="subHeader"><span class="STYLE1"><span class="STYLE30">a</span><img src="https://static.igem.org/mediawiki/2008/4/4c/Where_have_we_been.gif" width="381" height="286" align="absmiddle"></span></span></td>
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                    <param name="quality" value="high" />
 +
                    <param name="BGCOLOR" value="#03438A">
 +
                    <embed src="https://static.igem.org/mediawiki/2008/d/d6/1111.swf" width="529" height="447" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" bgcolor="#03438A"></embed>
 +
                  </object>
 +
                </span></td>
 +
              </tr>
 +
            </table></td>
         </tr>
         </tr>
-
      <tr>
+
        <tr>
-
        <td bgcolor="#0000FF"><span class="STYLE4"></span></td>
+
          <td colspan="2" bgcolor="#0000FF"><span class="STYLE36">a</span></td>
-
        <td bgcolor="#0000FF"><span class="STYLE4"></span></td>
+
        </tr>
-
        <td width="235" bgcolor="#0000FF"><span class="STYLE4"></span></td>
+
      </table>
-
        <td width="62" bgcolor="#0000FF"><span class="STYLE4"></span></td>
+
      <table width="967" height="1108" border="0" align="center" cellpadding="2" cellspacing="0">
-
      </tr>
+
        <tr>
-
    </table>
+
          <td height="59" colspan="2" bgcolor="#03438A" class="subHeader"><p align="center" class="STYLE3" style="margin-bottom: 0">PROJECT 2 </p>
-
    <table width="1029" height="1232" border="0" cellpadding="2" cellspacing="0">
+
            <p align="center" class="STYLE3"> Synthetic convertible  ecosystem</p></td>
-
      <tr>
+
        </tr>
-
        <td height="59" colspan="3" bgcolor="#03438A" class="subHeader"><p align="center" class="STYLE3" style="margin-bottom: 0">The synthetic convertible  ecosystem</p>         </td>
+
        <tr>
-
      </tr>
+
          <td height="141" colspan="2" bgcolor="#03438A" class="subHeader STYLE5"><p class="STYLE25"><br />
-
      <tr>
+
              <span class="STYLE20">There is no mono-culture in nature! And in industry, coculture of species/strains are widely used to either improve productivity or lower the cost. The manufacturing of Vitamin C in China, which has contributed to 60  percent of its world production, could serve as an excellent example to  validate the significance of coculture in industry. Thus to understand the interactions between coexistent ecosystems will not only contribute to human’s perception of nature but also to human practices in engineering.</span></p>             </td>
-
        <td height="23" colspan="3" bgcolor="#03438A" class="subHeader STYLE5"><p class="STYLE4"><span class="STYLE12">Background</span><br />
+
        </tr>
-
          There is no mono-culture in nature! And in industry, coculture of species/strains are widely used to either improve productivity or lower the cost, thus to understand the interactions between coexistent ecosystems will not only contribute to human’s perception of nature but also to human practices in engineering.</p>
+
        <tr>
-
            <p class="STYLE4">Most of the existent ecosystem could not be  simply defined as symbiosis or competition, for instance. A huge amount species  living in a symbiosis ecosystem will somehow compete with each other for food  and space or other, such as the bacteria living in human intestine. The  interweaving and intricate relationships in natural coexistent ecosystems  shadow the human endeavor to deeply understand the dynamics of symbiosis or  competition ecosystems. Thus a lot of effort has been made to fabricate a  simplified ecosystem to emulate natural ecosystems. Approaches like auxotroph  have been applied to achieve this goal.  However, most of the natural coexistent systems are based on  cell-to-cell communication mechanisms, among which, quorum sensing plays a  large role, which is one of basis for our built. </p>
+
          <td width="419" height="250" rowspan="2" align="center" bgcolor="#03438A" class="subHeader"><table width="418" border="0">
-
          <p class="STYLE4">We aim to build an ecosystem, the  relationship within which could be regulated by culture conditions. </p></td>
+
            <tr>
-
      </tr>
+
              <td><img src="https://static.igem.org/mediawiki/2008/b/b9/Hounan.jpg" width="410" height="321"></td>
-
      <tr>
+
            </tr>
-
        <td height="30" colspan="3" bgcolor="#03438A" class="subHeader"><span class="STYLE7">The Tools</span></td>
+
            <tr>
-
      </tr>
+
              <td><img src="https://static.igem.org/mediawiki/2008/9/9e/Hounasna.jpg" width="410" height="336"></td>
-
      <tr>
+
            </tr>
-
        <td height="30" colspan="3" bgcolor="#03438A" class="subHeader"><table width="937" height="384" border="0" align="center">
+
            <tr>
-
            <tr>
+
              <td height="80" bgcolor="#03438A"><p class="STYLE25"><span class="STYLE28"><a href="https://2008.igem.org/B22222">Background</a></span></p>
-
              <td width="284" bordercolor="#03438A" bgcolor="#03438A"><p class="STYLE8"><span class="STYLE10"><strong>Toggle switch</strong>-----toggle switch is a switch on the basis of two mutually-repressive promoters, the product of each represses the express  of that of the other, and both the repressors could be deactivated in certain conditions. And the state of the cell could be regulated by the change of the  culture variations.</span></p></td>
+
                </td>
-
              <td width="261" bordercolor="#03438A" bgcolor="#03438A"><p class="STYLE11"><span class="STYLE4"><strong>Quoru
+
            </tr>
-
               
+
          </table></td>
-
                m  sensing</strong>-----Th
+
          <td width="528" height="41" bgcolor="#03438A" class="sidebarHeader STYLE5 STYLE4 STYLE12">Our Design </td>
-
               
+
        </tr>
-
                at is the way how various bacteria “talk”  to each other. It is the mechanism ensures that certain genes will keep silent  before the cell density of the species pass a threshold. </span></p></td>
+
        <tr>
-
              <td width="336" bordercolor="#03438A" bgcolor="#03438A"><p class="STYLE5"><span class="STYLE9"><strong>Prisoners’ Dilemma----</strong>It is the dilemma in which the two suspects could either choose to cooperate with or betray each other. In  conditions when they could communicate freely with each other, they will cooperate,  which maximizes their benefits as a whole; while when they are inquisited separately, they will both choose to betray one another to lower the risks of long sentence. </span></p></td>
+
          <td height="821" valign="top" bgcolor="#03438A" class="bodyText STYLE5 STYLE4 STYLE10"><p>The design for the cell 1: The genetic circuit can be  divided into three different functional sections. <br>
-
            </tr>
+
The first one in the graph is the detecting Section. By using this section you can detect the cell density according to the  intensity of the red fluorescence. The detecting section is especially useful when  you incubate two different kinds of E.coli in a coculture.</p>
-
            <tr>
+
            <p>The second one is the Helper section. We call it helper  section because the LuxR protein is the prerequisite for the activation of  PLux. Here we used a constitutive promoter to regulate the expression of the LuxR protein. </p>
-
              <td height="196" bordercolor="#03438A" bgcolor="#03438A"><img src="https://static.igem.org/mediawiki/2008/2/2d/Qwe.jpg" width="318" height="195" /></td>
+
            <p>The core section is the convertible switch. Convertible switch  is a genetic device that can switch between two convertible states, which,  here, represents a different survival strategy for the cells each. </p>
-
              <td bordercolor="#03438A" bgcolor="#03438A"><img src="https://static.igem.org/mediawiki/2008/6/6e/%E6%9C%AA%E5%91%BD%E5%90%8D%C2%B7.jpg" width="319" height="196" /></td>
+
            <p>When adding Arabinose/AHL different genes will get expressed behind the two mutually-repressive promoters. That means when added  into the culture, AHL will diffuse into the cell bind the LuxR protein and form  a complex which can activate the LuxPr promoter and then the genes of rhII capR  and araC will express. Then the AraC protein will bind to the PBad/araC promoter and repress the expression of the aiiA and another capR gene. However,  you can turn the switch to the other side by adding Arobinose. When adding  Arobinose into the culture, the repression functional molecular AraC protein will get released from the PBad/AraC promoter. With the expression of the aiiA  gene the signal molecular will get digested and therefore decrease to a proper  level which is not high enough to activate the LuxPr promoter.<br>
-
              <td bordercolor="#03438A" bgcolor="#03438A"><img src="https://static.igem.org/mediawiki/2008/0/0f/Prison.jpg" width="339" height="194" /></td>
+
              The most important thing in this section is the  capacity of the two different promoters luxPr and PBad/araC are quite different. When the luxRr promoter is activated, its higher capacity will express more chloromycetin  resistant protein and another important thing is by sensing the AHL which is  sent out by cell 2 it can produce another kind of signal molecular BHL.<br>
-
            </tr>
+
            Cell 2 is similarly designed as Cell 1</p>
-
        </table></td>
+
          </td>
-
      </tr>
+
        </tr>
-
      <tr>
+
      </table>     
-
        <td width="535" height="250" rowspan="2" align="center" bgcolor="#03438A" class="subHeader"><span class="STYLE11"><span class="STYLE4"><strong>
+
          <table width="971" height="168" border="0">
-
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+
        <tr>
-
            <param name="movie" value="https://static.igem.org/mediawiki/igem.org/c/c7/2.swf" />
+
          <td colspan="2" bgcolor="#03438A"><p><strong><span class="STYLE7">Mutualism and </span></strong><span class="STYLE1">Competition</span></p></td>
-
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+
        </tr>
-
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+
        <tr>
-
          </object>
+
          <td width="556" height="50" bgcolor="#03438A"><span class="STYLE7"><strong>Competition</strong></span></td>
-
        </strong></span></span></td>
+
          <td width="408" rowspan="2" bgcolor="#03438A"><p class="STYLE15">In the culture that both ampicillin and chloromycetin are available, it requires the expression of the both resistant genes for both antibiotics for a strain’s survival. Without  adding any signal molecular as the initially inducing factor into the culture,  the two kinds of E.coli can not communicate with each other so they will keep  on the competitive stage. In this stage each kind of cell must survive all by it self in some method as assimilating the nutrition in the culture. As a result  the two different kinds of E.coli fight with each other for the space and nutrient  ingredient.  </p>
-
        <td width="1" rowspan="2" bgcolor="#03438A">&nbsp;</td>
+
          <p class="STYLE15">By adding some  AHL into the culture, the luxPr promoter will get activated by the AHL and LuxR  complex. And then the expression product of the rhlI gene, BHL will diffuse into the cell 2 which can sense BHL-RhIR complex by binding to the PrhI promoter and turning on the expression of luxI kanR and lacI genes. The LacI protein will bind to the PBad/araC promoter and therefore stop the digestion of the signal  molecular by the expression of the aiiA gene. At the same time the expression  of the luxI gene will send out AHL. By using a very similar mechanism the  cell 1 can sense the AHL molecular.</p></td>
-
        <td width="481" height="41" bgcolor="#03438A" class="sidebarHeader STYLE5 STYLE4 STYLE12">Our Design </td>
+
        </tr>
-
      </tr>
+
        <tr>
-
      <tr>
+
          <td bgcolor="#03438A"><object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=7,0,19,0" width="607" height="507">
-
        <td height="450" valign="top" bgcolor="#03438A" class="bodyText STYLE5 STYLE4 STYLE10"><p>We aim to build an ecosystem, the  relationship within which could be regulated by culture conditions. To realize  this goal, toggle switches have been built to regulate the interactions between  the two strains. As is shown in the illustration above, we used kanamycin and&nbsp;chloramphenicol&nbsp;as  the selective forces. </p>
+
            <param name="movie" value="https://static.igem.org/mediawiki/2008/8/8a/Arobinosq3e2e.swf">
-
            <p>When  auto-inducers, either AHL or BHL is introduced to the culture, LuxPr or Prhl  will be activated to produce the auto-inducers required by their partners, and  express the anti-biotic resistant genes to ensure each other’s survival. During  the process, PBad/arac and Plac will be repressed because of the repressors  Arac and LacI expressed by LuxPr and PrhI. </p>
+
            <param name="quality" value="high">
-
          <p>However,  upon the introduction of IPTG or arobinose, the repressors will stop  functioning, so that Aiia will be expressed, and therefore AHL and BHL will be  degraded, so that there will be no communications any more, and the  relationship between the two strains will enter the phase of competition. </p></td>
+
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-
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-
    </table>    
+
          </object></td>
-
    <p><span class="STYLE9" style="margin-bottom: 0">This idea was inspired by the theory of  Prisoner’s Dilemma.
+
        </tr>
-
    As in prisoners’ dilemma, the bacteria in  our design are faced with two solutions for coexistence, they could either  choose to cooperate with one another by providing inducers to express their  partners’ antibiotics-resistance genes or they could take a foe strategy in  which no cooperation is needed for both strains’ survival.</span></p></td>
+
        <tr>
-
  <td bgcolor="#03438A">&nbsp;</td>
+
          <td bgcolor="#03438A"><object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=7,0,19,0" width="610" height="515">
 +
            <param name="movie" value="https://static.igem.org/mediawiki/2008/3/32/Ah11l.swf">
 +
            <param name="quality" value="high">
 +
            <embed src="https://static.igem.org/mediawiki/2008/3/32/Ah11l.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="610" height="515"></embed>
 +
          </object></td>
 +
          <td bgcolor="#03438A"><p class="STYLE19"><span class="STYLE12"><strong>Mutualism</strong></span> </p>
 +
            <p class="STYLE19"><br>
 +
              In this state the  two kinds of cells communicate with each other by sensing the signal molecular sent  by the counterpart.<br>
 +
              It seems that  with the help of each other, both of them can live better in the harsh  environment and the fact is the capacity of the LuxPr and PrhI are higher than that of the Plac and Pbad/araC promoters. With higher expression of the ampicillin and chloromycetin resistant protein  both of them can survive in the ultra-high antibiotic concentration. </p></td>
 +
        </tr>
 +
      </table>
 +
      <table width="556" height="433" border="0" align="center">
 +
        <tr>
 +
          <td bgcolor="#03438A"><span class="STYLE11"><span class="STYLE4"><strong>
 +
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 +
              <param name="movie" value="https://static.igem.org/mediawiki/2008/e/eb/222.swf" />
 +
              <param name="quality" value="high" />
 +
              <embed src="https://static.igem.org/mediawiki/2008/e/eb/222.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="577" height="523"></embed>
 +
            </object>
 +
            </strong></span></span></td></tr>
 +
      </table>    
 +
      <p><span class="STYLE25" style="margin-bottom: 0">This idea was inspired by the theory of  Prisoner’s Dilemma.
 +
      As in prisoners’ dilemma, the bacteria in  our design are faced with two solutions for coexistence, they could either  choose to cooperate with each other by providing inducers to express their  partners’ antibiotics-resistance genes or they could take a foe strategy in  which no cooperation is needed for both strains’ survival.</span></p>
 +
    </td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td width="8">&nbsp;</td>
+
     <td colspan="2" bgcolor="#03438A"><table width="973" border="0">
-
    <td width="471">&nbsp;</td>
+
        <tr>
-
    <td width="558">&nbsp;</td>
+
          <td width="477" bgcolor="#03438A"><div align="center"><span class="STYLE17"><a href="https://2008.igem.org/Experiment">Experiment</a></span> </div></td>
-
<td width="7">&nbsp;</td>
+
          <td width="493" height="80" bgcolor="#03438A"><div align="center"><a href="https://2008.igem.org/Model" class="STYLE18">Model</a></div></td>
 +
        </tr>
 +
    </table></td>
   </tr>
   </tr>
</table>
</table>

Latest revision as of 05:11, 30 October 2008

Entertainment - Product Page

PROJECT 1

Foolproof Plasmid Self-Assembly system

Objectives:

Bacterial assembly is aimed to be achieved based on the mechanism of site-specific recombination systems, So that the expensive reagent as well as the laboring tasks could be saved in gene cloning experiments.

Our design

We have innovatively utilized the site-specific systems to build a foolproof bacterial assembly system to future reduce the labor and cost involved in gene cloning experiments. We have designed three standardized vectors which perform as the donors, receptor vector respectively.

Background

Parts contributed to the registry

How do they work?


First, we define the Receptor as the vector that has already existed in the cell (E.coli.), and the Donor as the vector containing the desired gene that we intend to integrate into the Receptor. The gene circuits for these plasmids are illustrated below.

When the Donor vector carrying the gene of interest GENE1 was introduced to the E Coli which contains the Receptor vector, the site-specific recombination will occur between the attB1 site and the attP1 site, so that the two sequences will be integrated into one circular DNA.

The recombinant DNA then could be selected in the liquid culture containing both ampicillin and kanamycin. Then, under inducible conditions, Cre will be expressed and the recombined sequence will be divided into two separate plasmids; one will retain the desired gene 1, while the other will preserve the killer gene ccdB, which is under the control of another inducible promoter. Because the two plasmids have shared origin site, plasmid incompatibility will occur thus the two kinds of plasmids will be separated into different cells.

When induced, the expressed CCDB will lead to the Programed Cell Death (PCD).

In order to realize the linkage of GENE 1 with GENE 2, we will introduce the new plasmid containing the desired GENE2 to the survival cells, in which the plasmids containing GENE 1 will behave as the new Receptor plasmid. Very similarly recombination between the attB2 and attP2 and the cleavage between the two loxp sites will be performed, and plasmids containing the linked GENE1 and GENE2 will be selected when the promoter expresses CCDB is induced.

The reason for us to use two sets of attB/attP specific sites is to avoid the combination within one module.

The whole process

a
a

PROJECT 2

Synthetic convertible ecosystem


There is no mono-culture in nature! And in industry, coculture of species/strains are widely used to either improve productivity or lower the cost. The manufacturing of Vitamin C in China, which has contributed to 60 percent of its world production, could serve as an excellent example to validate the significance of coculture in industry. Thus to understand the interactions between coexistent ecosystems will not only contribute to human’s perception of nature but also to human practices in engineering.

Background

Our Design

The design for the cell 1: The genetic circuit can be divided into three different functional sections.
The first one in the graph is the detecting Section. By using this section you can detect the cell density according to the intensity of the red fluorescence. The detecting section is especially useful when you incubate two different kinds of E.coli in a coculture.

The second one is the Helper section. We call it helper section because the LuxR protein is the prerequisite for the activation of PLux. Here we used a constitutive promoter to regulate the expression of the LuxR protein.

The core section is the convertible switch. Convertible switch is a genetic device that can switch between two convertible states, which, here, represents a different survival strategy for the cells each.

When adding Arabinose/AHL different genes will get expressed behind the two mutually-repressive promoters. That means when added into the culture, AHL will diffuse into the cell bind the LuxR protein and form a complex which can activate the LuxPr promoter and then the genes of rhII capR and araC will express. Then the AraC protein will bind to the PBad/araC promoter and repress the expression of the aiiA and another capR gene. However, you can turn the switch to the other side by adding Arobinose. When adding Arobinose into the culture, the repression functional molecular AraC protein will get released from the PBad/AraC promoter. With the expression of the aiiA gene the signal molecular will get digested and therefore decrease to a proper level which is not high enough to activate the LuxPr promoter.
The most important thing in this section is the capacity of the two different promoters luxPr and PBad/araC are quite different. When the luxRr promoter is activated, its higher capacity will express more chloromycetin resistant protein and another important thing is by sensing the AHL which is sent out by cell 2 it can produce another kind of signal molecular BHL.
Cell 2 is similarly designed as Cell 1

Mutualism and Competition

Competition

In the culture that both ampicillin and chloromycetin are available, it requires the expression of the both resistant genes for both antibiotics for a strain’s survival. Without adding any signal molecular as the initially inducing factor into the culture, the two kinds of E.coli can not communicate with each other so they will keep on the competitive stage. In this stage each kind of cell must survive all by it self in some method as assimilating the nutrition in the culture. As a result the two different kinds of E.coli fight with each other for the space and nutrient ingredient.

By adding some AHL into the culture, the luxPr promoter will get activated by the AHL and LuxR complex. And then the expression product of the rhlI gene, BHL will diffuse into the cell 2 which can sense BHL-RhIR complex by binding to the PrhI promoter and turning on the expression of luxI kanR and lacI genes. The LacI protein will bind to the PBad/araC promoter and therefore stop the digestion of the signal molecular by the expression of the aiiA gene. At the same time the expression of the luxI gene will send out AHL. By using a very similar mechanism the cell 1 can sense the AHL molecular.

Mutualism


In this state the two kinds of cells communicate with each other by sensing the signal molecular sent by the counterpart.
It seems that with the help of each other, both of them can live better in the harsh environment and the fact is the capacity of the LuxPr and PrhI are higher than that of the Plac and Pbad/araC promoters. With higher expression of the ampicillin and chloromycetin resistant protein both of them can survive in the ultra-high antibiotic concentration.

This idea was inspired by the theory of Prisoner’s Dilemma. As in prisoners’ dilemma, the bacteria in our design are faced with two solutions for coexistence, they could either choose to cooperate with each other by providing inducers to express their partners’ antibiotics-resistance genes or they could take a foe strategy in which no cooperation is needed for both strains’ survival.