Team:LCG-UNAM-Mexico/Project

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     <td colspan="3" rowspan="2"><img src="http://kabah.lcg.unam.mx/~larriola/mm_health_photo.jpg" alt="Header image" width="524" height="143" border="0" /></td>
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     <td height="50" colspan="3" id="logo" valign="bottom" align="center" nowrap="nowrap">LCG-UNAM-Mexico</td>
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     <td height="50" colspan="3" id="logo" valign="bottom" align="center" nowrap="nowrap"><a name="top"></a>LCG-UNAM-Mexico</td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Team" class="navText">About Us</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project" class="navText">Our project</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project" class="navText">Our Project</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Modeling" class="navText">Modeling</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Parts" class="navText">Parts Submitted to the Registry</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Experiments" class="navText">Wet Lab</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Modeling" class="navText">Modeling</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Parts" class="navText">Parts Submitted to the Registry</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook" class="navText">Notebook</a></td>
           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook" class="navText">Notebook</a></td>
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          <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Story" class="navText">Our story</a></td>
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          <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Team" class="navText">About us</a></td>
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      &nbsp;<a href="#description"><img src="https://static.igem.org/mediawiki/2008/d/df/Boton_about1.jpg" width="190" height="31" border="0" /></a> <a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Relevance"><img src="https://static.igem.org/mediawiki/2008/e/e5/Boton_about2.jpg" width="190" height="31" border="0" /></a> <a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project_results"><img src="https://static.igem.org/mediawiki/2008/f/f1/Boton_about3.jpg" width="190" height="31" border="0" /></a><br>
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      <a name="modeling"></a><img src="https://static.igem.org/mediawiki/2008/9/99/Ribbon435773498.gif" alt="ribbon" width="579" height="9" /><br />
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           <td class="pageName"><strong>Our Project</strong></td>
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           <td class="pageName"><div align="center">
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            <p class="calHeader"><span class="style6"><br>
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              &#9834; Singing bacteria! &#9835; </span><span class="pageName"><br>
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              <br>
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              Controlling <em>E. coli</em>'s nickel efflux pump</span> </p>
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           <td class="bodyText"><p align="justify">I bet you're thinking we're insane... so why don't you take a look at out project? Below you can watch a Flash movie summarizing the whole idea: </p>
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<strong>Singing Bacteria!</strong><br><br>
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            <p align="center">
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The overall idea in our project for the 2008 iGEM competition is to make bacteria sing. <strong>And how can we possibly achieve that?</strong><br><br>
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              <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="550" height="400" title="Movie: Controlling E. coli's nickel efflux pump">
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The main idea behind this is to control <i>Escherichia coli</i>’s nickel efflux pump (RcnA). This way we’ll be able to predict the extracellular nickel concentration at any time in function of the controling signal (AHL). Electrodes will be sensing the electrical conductivity in the extracellular medium and sending that information to a computer. The computer will interpret this information and emit a sound depending on the nickel concentration at that time. This way, bacteria are “singing”.<br><br>
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                <param name="movie" value="https://static.igem.org/mediawiki/2008/8/87/Controlling_Ecolis_nickel_efflux_pump.swf">
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The proteins and compounds involved in our model are the following:<br>
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                <param name="quality" value="high">
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<li><b>RcnA.</b> This is <i>E. coli</i>’s nickel efflux pump. We’re using a RcnA-mutant <i>E. coli</i> strain with a plasmid with RcnA under the control of cI* and RcnR (both repressors).<br>
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                <embed src="https://static.igem.org/mediawiki/2008/8/87/Controlling_Ecolis_nickel_efflux_pump.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="550" height="400"></embed>
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<li><b>RcnR.</b> The natural repressor of RcnA. When there’s nickel present, RcnR changes its conformation and stops repressing RcnA. <br>
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<li><b>cI*.</b> The λ phage repressor. The transcription of cI* is under the control of the activator dimer AHL:LuxR. cI is modified with a LVA tail so it is quickly degraded (hence the *).<br>
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            </p>
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<li><b>LuxR.</b> Used here as an activator of cI*. It is under a constitutive promoter (pTetR).<br>
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            <p align="justify" class="style2"><br class="pageName">
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<li><b>AHL.</b> This is the input signal. AHL forms a dimer with LuxR and starts the transcription of cI*. <br>
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              <span class="pageName"><a name="description"></a></span><img src="https://static.igem.org/mediawiki/2008/9/99/Ribbon435773498.gif" alt="ribbon" width="579" height="9" /></p>
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<li><b>AiiA.</b> This protein mediates the degradation of AHL. It is placed under a weak promoter (modified pLacZ).<br><br>
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            <p align="center" class="pageName">Project description</p>
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Here is a simplified diagram of the whole process:
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</p>
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              The aim of this project is to make bacteria sing,  and it will be done by modifying the extracellular medium's resistivity through the modulation of the RcnA (<em>E. coli'</em>s natural efflux pump) which will in turn change the concentration of nickel  outside the cell. By doing this we expect to gain insight into a  fundamental aspect of ecological dynamics which is currently not well  understood. On the other hand, we expect to show that measuring changes  in resistivity is an effective way to determine the activity of the  efflux pump, and that this can become an efficient indicator of real  time transcription for <em>in vivo</em> experiments.   </p>
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<img src="https://static.igem.org/mediawiki/2008/b/b9/Our_Project_01.jpg" width="580">
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            <p align="justify" class="bodyText"> To achieve this, we designed a system where <em>rcnA</em> is under  the regulation of the lambda repressor CI which itself is under the regulation  of <em>Vibrio fisheri</em>’s quorum sensing core components: LuxR and AHL. The former is constitutively produced and the latter is our input signal.  Further regulation of the system is achieved by constitutively  synthesizing AiiA, which degrades AHL, and by RcnR, RcnA's natural repressor which is inactivated in the presence of nickel.  All  the protein components of the system are encoded in <a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Parts">two devices</a>:  the first one contains the efflux pump, and the second one the  regulatory cascade. The reason for this partitioning is that in this  way RcnA can be integrated with any desired regulatory upstream signal.   </p>
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<p align="justify">So, here is the formal definition:<br>
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            <p align="justify" class="bodyText"> Once the system is built and implemented on <em>E.  coli</em>, we will measure the medium's resistivity on a real time basis  through a set of copper electrodes connected to a multimeter. The  monitoring device will be connected to a computer which will filter the noise in the signal and return a sound depending on the resistivity. <a href="http://en.wikiquote.org/wiki/Frankenstein_(1931_film)">They sing! They sing! Now we know what it’s like to be God!</a></p>
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The objective of this project is to modulate the extracellular nickel concentration through the regulation of its efflux pump. We plan to measure the change in the nickel concentration, and this can be achieved by measuring the change in the conductivity of the medium. These data will be interpreted and converted into a sound depending on the concentration read.<br><br>
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            <p align="justify">&nbsp;</p>
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Our system is conformed by two regulation mechanisms. The first of these is the one controlled by us through AHL. AHL enters the cell and forms a dimer with LuxR, which is under a constitutive promoter. This dimer serves as an activator of cI*, which represses RcnA. In this way we can express the concentration (and therefore the activity) of RcnA as a function of AHL. The second of these mechanisms is the natural regulation of the pump (RcnA) in response to the intracellular nickel concentration. When there is no nickel inside the cell, RcnR represses RcnA. However, when nickel enters the cell, it forms a dimer with RcnR and changes its conformation so it no longer represses RcnA. RcnA is then free to start pumping Ni out of the cell. We are keeping this because it is damaging to the bacteria to have the pump always on, and otherwise it would need a constant supply of AHL.<br><br>
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            <p align="justify"><a name="parameters"></a><a href="#top"><img src="https://static.igem.org/mediawiki/2008/c/cd/Boton_back.jpg" alt="Back to top" border="0"></a><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Relevance"><img src="https://static.igem.org/mediawiki/2008/e/e5/Boton_about2.jpg" alt="Parameters&amp;Kinetics" width="190" height="31" border="0"></a><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project_results"><img src="https://static.igem.org/mediawiki/2008/f/f1/Boton_about3.jpg" alt="Simulation &amp; Analysis" width="190" height="31" border="0"></a><br>
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The final result will be a biological system capable of modifying its surrounding medium. In addition, our measurements will allow us to model the dynamic behavior of the pump and the intra and extracellular nickel concentrations.<br>
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          <td class="pageName">Project Details</td>        
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          <td class="subHeader"><li>Part 1</li></td>
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<td class="bodyText"><p align="justify">--- - -  --- -- -- -- -- -- -- --  - ------ -----. --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ---- --- --- -- - ------ -- ---- ---  ---  --- -- -- ----- ---, --- - -  --- -- -- -- -- -- -- --  - ------ -----.
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--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----. </p>
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--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----. </p>
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          <td class="subHeader"><li>Part 3</li></td>  
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--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----. </p>
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Latest revision as of 22:18, 28 October 2008

LCG-UNAM-Mexico:Modeling

Header image
iGEM 2008 TEAM
line decor
  
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ribbon


♪ Singing bacteria! ♫


Controlling E. coli's nickel efflux pump

I bet you're thinking we're insane... so why don't you take a look at out project? Below you can watch a Flash movie summarizing the whole idea:


ribbon

Project description


The aim of this project is to make bacteria sing, and it will be done by modifying the extracellular medium's resistivity through the modulation of the RcnA (E. coli's natural efflux pump) which will in turn change the concentration of nickel outside the cell. By doing this we expect to gain insight into a fundamental aspect of ecological dynamics which is currently not well understood. On the other hand, we expect to show that measuring changes in resistivity is an effective way to determine the activity of the efflux pump, and that this can become an efficient indicator of real time transcription for in vivo experiments.  

To achieve this, we designed a system where rcnA is under the regulation of the lambda repressor CI which itself is under the regulation of Vibrio fisheri’s quorum sensing core components: LuxR and AHL. The former is constitutively produced and the latter is our input signal. Further regulation of the system is achieved by constitutively synthesizing AiiA, which degrades AHL, and by RcnR, RcnA's natural repressor which is inactivated in the presence of nickel.  All the protein components of the system are encoded in two devices: the first one contains the efflux pump, and the second one the regulatory cascade. The reason for this partitioning is that in this way RcnA can be integrated with any desired regulatory upstream signal.  

Once the system is built and implemented on E. coli, we will measure the medium's resistivity on a real time basis through a set of copper electrodes connected to a multimeter. The monitoring device will be connected to a computer which will filter the noise in the signal and return a sound depending on the resistivity. They sing! They sing! Now we know what it’s like to be God!

 

Back to topParameters&KineticsSimulation & Analysis

ribbon