Team:PennState

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    <td align="center" ><a class="mainLinks" href="https://2008.igem.org/Team:PennState" >Home</a> </td>
    <td align="center" ><a class="mainLinks" href="https://2008.igem.org/Team:PennState/Team" >The Team</a> </td>
    <td align="center" ><a class="mainLinks" href="https://2008.igem.org/Team:PennState/Project" >The Project</a> </td>
    <td align="center" ><a class="mainLinks" href="https://2008.igem.org/Team:PennState/Parts">Parts</a> </td>
    <td align="center" ><a class="mainLinks" href="https://2008.igem.org/Team:PennState/Modeling" >Modeling</a> </td>
    <td align="center" ><a class="mainLinks" href="https://2008.igem.org/Team:PennState/Notebook" >Notebook</a> </td>
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 <h4 style="margin-top: 0;">Hormone Biosensors</h4>
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  <dd><a href="hbintro" title="Intro to Endocrine Disruption, pseudoestrogens, pthalates, nuclear hormone receptors, and our goals">Introduction</a></dd>
  <dt>Smart Fold</dt>
  <dd><a href="smartfold/overview">Overview</a></dd>
  <dd><a href="smartfold/parts" title="Parts submitted to the registry for this project">Parts</a></dd>
  <dd><a href="smartfold/references">References</a></dd>
  <dt>Nuclear Fusion</dt>
  <dd><a href="fusion/overview">Overview</a></dd>
  <dd><a href="fusion/parts" title="Parts submitted to the registry for this project">Parts</a></dd>
  <dd><a href="fusion/references">References</a></dd>
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 <h4>Diauxie Elimination</h4>
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  <dd><a href="diauxie/intro">Introduction</a></dd>
  <dd><a href="diauxie/overview">Overview</a></dd>
  <dd><a href="diauxie/parts" title="Parts submitted to the registry for this project">Parts</a></dd>
  <dd><a href="diauxie/references">References</dd>
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<td valign="top" id="pagecontent"><span style="font-size: 16pt">PENN STATE iGEM 2008</span>
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 <p>Welcome to the Penn State iGEM 2008 team’s website.  We are currently working hard at a few different projects for this year's competition.  In early May we began brainstorming and came up with a couple of ideas to create biosensors that use human nuclear hormone receptors to recognize potentially harmful ligands. These receptor systems occur naturally in the human body, but our goal is to retain and utilize their functions in <em>Escherichia Coli</em>.  We are also finishing up one of last year's projects which is aimed at creating a more efficient bioproduction process by altering how <em>E. Coli</em> selects between the utilization of 5 and 6 carbon sugars.  Please explore our website to find out more about us and our projects!</p>
 <p> If there are any questions or comments about the information on this site please contact us at
 <a href="mailto:gjt5001@psu.edu" title="email us">gjt5001@psu.edu</a>. </p>
    
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        <tr><td style="padding-top:30px; padding-right:30px" valign="top" width="90%"><span style="font-size:14pt">Hormone Prescreening <em>E. coli</em></span>
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      <p>Two of our projects aim to construct biosensors which will ultimately serve as a water prescreening tool.  The focus of these biosensors will be to detect phthalates utilization the Peroxisome Proliferator Activated Receptor (PPAR) and detecting Bisphenol A (BPA) by the Estrogen Receptor (ER).  Recently phthalates have been shown to cause negative health effects such as damage to the liver and kidneys and cause birth defects in rodent studies.  Phthalates are introduced into our environment by their use as plastisizers in materials ranging from polyvinyl chloride to nail polish to small toys.  BPA is also found in plastics but instead is used in the synthesis of hard plastics.  Once BPA enters the human body it is confused for estrogen and parallels the effects of estrogen after attaching to the ligand binding region of the ER.  Analytical detection methods for water contamination are compound specific and very costly.  Having a simple and cheap tool to screen for phthalates or BPA as a general class of compounds would eliminate the cost and time involved in detection.</p>
      <p> We are using two of the natural human nuclear hormone receptor proteins that recognizea large class of ligands, and attempting to express them heterologously in <em>E. Coli</em>.  The complexity of this mammalian protein makes it difficult to express it in a prokaryote.  We have two different strategies to express and use these receptors to detect compounds in <em>E. Coli</em>.  </p>

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          <td style="padding-top:30px; padding-right:30px" valign="top" width="45%"><span style="font-size: 14pt">Smart Fold Reporter</span>
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            <img src="http://upload.wikimedia.org/wikipedia/en/8/87/PPARg.png" alt="[img]" style="float:left; margin:5px;"/>
            <p> The human PPAR has three different types α, β, and γ but only two show any affect by phthalates.  We are using the alpha form which is expressed in the liver, kidney, heart, muscle, adipose tissue, and others.  There are different regions associated with nuclear hormone receptors: N-terminal, DNA binding domain (DBD), Hinge, Ligand binding domain (LBD), and C-terminal.  The LBD is the region that attracts and holds the ligand of interest.  After ligand binding the receptor usually will form a dimer, in our case PPAR will combine with Retinoid X Receptor (RXR) to form a heterodimer.  The RXR protein functions much like the PPAR but in this case it does not need to attach a ligand before dimerization.  The heterodimer will bind to Peroxisome Proliferator Response Element (PPRE) and activates transcription.  Most often a coactivator complex is required for transcriptional activation which involves proteins SRC-1 and CBP and others.  </p>


<p>This <em>Smart Fold Reporter</em> project uses altered growth conditions so that the entire PPAR protein is successfully expressed and used to transcriptionally report for the presence of phthalates.  Expressing the entire PPAR in E. Coli has proven difficult which could be caused by toxicicity to the cells from the DBD.  To overcome this problem we are going to treat the E. Coli with Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) which is an uncoupler of oxidative phosphorylation.  This strategy would correlate to the heat shock proteins involved with synthesis in the human body.  The cells the have the PPAR plasmid will be grown on plates containing Timentin which prevents growth of bacteria without plasmid.  The expression of the PPAR and RXR also needs to have tight control so the arabinose operon will be used.  A green fluorescent protein will be placed after the PPRE to signal transcription after heterodimer binding.  
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          <td style="padding-top:30px; padding-right:30px" valign="top" width="45%"><span style="font-size:18px">Nuclear Fusion</span>
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<img src="https://static.igem.org/mediawiki/2008/d/d9/PSU2008iGEM_BPAimage.png" alt="[img]" style="float:left; margin:5px;width="950px""/>
            <p> The <em>Nuclear Fusion</em> project currently has two similar directions that it may turn but both involve a plasmid construct very generously donated to our iGEM team from David W. Wood, Department r of Chemical Engineering at Princeton University.  Research in their lab has constructed a biosensor containing just the ligand binding domain of the estrogen receptor (ER).  The ER is very similar to the PPAR and other hormone receptors.  Previous attempts at isolating the ligand binding domain (LBD) failed due to the specific folding pattern of this region, therefore similar binding characteristics to natural ER.  This was done by inserting the ligand binding region into a minimal splicing intein domain.  This construct was also made more soluble by addition of a maltose-binding tag.  The ER was fused with a thymidylate synthase enzyme (TS) that remains deactivated until homodimerization of the ER after binding ligand.  The cells are grown on thymine-free plates allowing for recognition of strength and function of ER ligands.</p>

<p>Our plan for this project is to work on the sensitivity of the biosensor in hopes of using this for water prescreens, similar to the <em>Smart Fold Reporter</em> project.  The sensitivity will be focused on BPA which has a very different conformation that the natural agonist of the ER system.  This difference causes the BPA to bind weakly but still causes a disturbance in normal ER function.  One idea is to replace the ER LBD from Wood’s biosensor with the estrogen-receptor related (ERR) LBD.  The ERR is similar to ER and binds many of the same ligands and has a tendency to bind to the estrogen response element (ERE) in the human body.  The one benefit of ERR for our project is that it binds BPA very strongly.  Another direction that this project could take would be to analyze the LBD of ER and perform directed evolution to increase BPA sensitivity.  During directed evolution, certain regions of the ER LBD would be targeted for random mutagenesis providing a library of mutants in the trillions.  The mutant library would be induced with BPA and the best growing colony would be selected, tested, and mutated for further sensitivity.  
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    <td colspan="2" style="padding-top:30px; padding-right:30px" valign="top" width="45%"><span style="font-size: 14pt">Diauxie Elimination: <em>Two</em> spoons full of sugar.</span>
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      <p><img src="picture here" alt="[img]" style="float:left; margin:5px;"/>Cellulosic biomass is an abundant and inexpensive energy source, coming from plant waste: ideal for Ethanol production through fermentation. However, biomass contains glucose and xylose sugars in relatively equal ratios, while <em>e. coli</em> preferentially metabolizes glucose before any other sugar. In this project we attempt to eliminate this phenomenon, called <em>diauxie</em>, and get our cells to utilize both sugars at the same time. Solving this problem will lead to more efficent use of cellulosic biomass including moving towards the future of bioproduction: continous processes.</p>
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   <td style="padding-top:30px; padding-right:30px" valign="top" width="45%"><span style="font-size: 14pt">Quick Links</span>
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            <p><a href="http://parts.mit.edu/igem07/index.php/PennState/final_result#contributions" title="Our Contribution To the Registry">Table of our Contributions to the Registry</a></p>
            <p><a href="http://parts.mit.edu/igem07/images/7/73/IgemDesign.swf" title="Interactive Schematic" target="_blank">Interactive <em>E. co</em>Lisa Schematic</a></p>
            <p> <a href="http://www.psu.edu" title="Pennsylvania State University" target="_blank">Pennsylvania State University</a> </p>
            <p> <a href="http://www.environment.psu.edu/" title="Penn State Institutes of Energy and the Environment" target="_blank">Penn State Institutes of Energy and the Environment</a> </p>
    <p> <a href="http://www.cmtc.psu.edu/" title="Center for Molecular Toxicology and Carcinogenesis" target="_blank">Center for Molecular Toxicology and Carcinogenesis</a> </p>
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          <td style="padding-top:30px; padding-right:30px" valign="top" width="45%"><span style="font-size: 14pt">Drew Endy On Synthetic Biology</span>
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<h4 style="font-size: 14pt;">Sponsors for our team! Thanks so much!</h4>
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<img src="http://ntp.neuroscience.wisc.edu/gwis/gifs/Invitrogen.jpg" alt="invitrogen" style="float:left; clear: left; margin-right: 4%; margin-top: 40px; width: 48%;"/>
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