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<table width="1059" border="0" align="center" cellpadding="0" cellspacing="0" bordercolor="#03438A">
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<p align="center" class="STYLE6">PROJECT 1 </p>
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     <td height="54" colspan="2" valign="bottom" nowrap="nowrap" bgcolor="#03438A" id="logo"><img src="Logonew1.jpg" width="340" height="116" /><a href="javascript:;" class="STYLE1"> <span class="STYLE2">aaa</span>Home <span class="STYLE2">aa</span></a></td>
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    <td height="15"><p class="STYLE5">Synthetic  Symbiosis Ecosystem </p>    </td>
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     <td height="6" bgcolor="#00CCFF"><span class="STYLE9">Design</span></td>
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    <td height="7"><span class="STYLE7">The list of the ideas generated from the  brainstorm<br />
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The paper-making E coli<br />
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A time relay device (E coli baby sitter) <br />
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An E coli Pipeline <br />
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Torch rely <br />
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Tumor-killer <br />
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E coli neural pathway<br />
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…….</span></td>
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     <td><p class="STYLE4">The coexistence of organisms has been in  the focus of research interests for a while, revealing the interactions between  species not only contributes to the deeper understanding of natural systems but  also provides perspectives for more efficient coculture of organisms that  function in pharmaceutical engineering and soil remediation.</p>
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    <td><p class="STYLE4">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.</p>
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      <p class="STYLE4">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>
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      <p class="STYLE4">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>
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    <td><span class="STYLE4">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. </span></td>
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      <p>&nbsp;</p>
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    <p>&nbsp;</p>      <p>&nbsp;</p></td>
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<p align="center"><span class="STYLE6">PROJECT 2 </span></p>
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    <td width="500" height="17" bgcolor="#33FFFF"><p><span class="STYLE3">Design</span></p>
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        <td class="subHeader" colspan="3"><div align="center" class="STYLE16" style="margin-bottom: 0">A Synthetic Plasmid Self-Assembly system</div>          </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"><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="508" bgcolor="#03438A"><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="512" bgcolor="#03438A"><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="500" height="100" /></a></td>
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            <td bgcolor="#03438A"><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"><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"><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="STYLE12"><strong>Integration of bacteriophage lambda</strong></span></td>
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            <td height="54" colspan="2" bgcolor="#03438A"><p class="STYLE10">              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>. <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="STYLE10">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="STYLE10">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="STYLE10">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="509" 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 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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          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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          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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        <td height="46" 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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        <td width="7" rowspan="4">&nbsp;</td>
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        <td width="468" height="103" bgcolor="#03438A" class="sidebarHeader"><p class="STYLE15"><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.
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        <td valign="top" bgcolor="#03438A" class="bodyText"><p class="STYLE1">Our design</p></td>
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        <td valign="top" bgcolor="#03438A" class="bodyText"><p><span 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</span>.</p></td>
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        <td valign="top" bgcolor="#03438A" class="bodyText"><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 width="348"><img src="https://static.igem.org/mediawiki/2008/d/d1/122111.jpg" width="348" height="200" /></td>
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        <td width="351"><img src="https://static.igem.org/mediawiki/2008/6/6b/222222.jpg" width="351" height="200" /></td>
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        <td colspan="4"><img src="https://static.igem.org/mediawiki/2008/6/63/Wo_cai.jpg" width="1029" height="982" /></td>
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        <td height="59" colspan="3" bgcolor="#03438A" class="subHeader"><p align="center" class="STYLE3" style="margin-bottom: 0">The synthetic convertible  ecosystem</p>
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        <td height="23" colspan="3" bgcolor="#03438A" class="subHeader STYLE5"><p class="STYLE4"><span class="STYLE12">Background</span><br />
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          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>
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            <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>
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          <p class="STYLE4">We aim to build an ecosystem, the  relationship within which could be regulated by culture conditions. </p></td>
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        <td height="30" colspan="3" bgcolor="#03438A" class="subHeader"><span class="STYLE7">The Tools</span></td>
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              <td width="284"><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>
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              <td width="261"><p class="STYLE11"><span class="STYLE4"><strong>Quoru
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                m  sensing</strong>-----Th
 +
               
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                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>
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              <td width="336"><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>
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              <td height="196"><img src="https://static.igem.org/mediawiki/2008/2/2d/Qwe.jpg" width="318" height="195" /></td>
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              <td><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>
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              <td><img src="https://static.igem.org/mediawiki/2008/0/0f/Prison.jpg" width="339" height="194" /></td>
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        <td width="1" rowspan="2" bgcolor="#03438A">&nbsp;</td>
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        <td width="488" height="41" bgcolor="#03438A" class="sidebarHeader STYLE5 STYLE4 STYLE12">Our Design </td>
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        <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>
 +
            <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>
 +
          <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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    <p><span class="STYLE9" 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 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>
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Revision as of 13:51, 26 October 2008

Entertainment - Product Page

    
 
A Synthetic Plasmid Self-Assembly system
Background

Site-specific recombination
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.

Inverted repeats
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:

Direct repeats
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:

The net result is that the segment of DNA between the two recombinogenic sites has inverted with respect to the rest of the DNA molecule.
In other words, recombination at inverted repeats causes an inversion

The net result is that the segment of DNA between the two recombinogenic sites has been deleted from the rest of the DNA molecule and appears as a circular molecule.
In other words, recombination at direct repeats causes a deletion.

Note 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.
Integration of bacteriophage lambda

In order for the lambda prophage to exist in a host E. coli cell, it must integrate into the host chromosome which it does by means of a site-specific recombination reaction.

The E. coli chromosome contains one attachment site which is designated attB. 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 BOB'.

The bacteriophage recombination site - attP - contains the identical central 15 bp region as attB. The overall structure can be represented as POP'.

Integration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is Integration Host Factor. Both of these proteins bind to sites on the P and P' arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned.

The result of recombination is that the integrated prophage is flanked by two attachment sites but now they are slightly different: attL has the structure BOP' and attR has the structure POB'.

Cre-Lox recombination is a special type of site-specific recombination, which is often applied as a gene knockout tool.
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 DNA ligase. 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.

Lox P site
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.
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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 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.

How do they work?
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 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 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.

Where have we been?

The synthetic convertible ecosystem

Background
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.

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.

We aim to build an ecosystem, the relationship within which could be regulated by culture conditions.

The Tools

Toggle switch-----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.

Quoru m sensing-----Th 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.

Prisoners’ Dilemma----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.

  Our Design

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 chloramphenicol as the selective forces.

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.

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.

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 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.

  
       

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