Team:PennState/Project

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  <img src="https://static.igem.org/mediawiki/2008/d/df/Penn_state_igem_logo.JPG" alt="Penn State" />
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     <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/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/Parts">Parts</a> </td>
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    <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>
     <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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  <h4>Diauxie Elimination</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>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/intro">Introduction</a></dd>
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  <dt>Smart Fold</dt>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/TheSystem">The System</a></dd>
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   <dd><a href="smartfold/overview">Overview</a></dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/Strategies">Strategies</a></dd>
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   <dd><a href="smartfold/parts" title="Parts submitted to the registry for this project">Parts</a></dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/progress">Progress</a></dd>
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   <dd><a href="smartfold/references">References</a></dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/conclusions">Conclusions</a></dd>
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  <dt>Nuclear Fusion</dt>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/parts" title="Parts submitted to the registry for diauxie">Parts</a></dd>
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   <dd><a href="fusion/overview">Overview</a></dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/diauxie/references">References</a></dd>
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   <dd><a href="fusion/parts" title="Parts submitted to the registry for this project">Parts</a></dd>
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  <h4>Diauxie Elimination</h4>
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  <h4><acronym title="Nuclear Hormone Receptor">NHR Biosensors</acronym><br/></h4>
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   <dd><a href="diauxie/intro">Introduction</a></dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/NHR/introduction">NHR Introduction</a></dd>
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   <dd><a href="diauxie/overview">Overview</a></dd>
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   <dd><a href="diauxie/parts" title="Parts submitted to the registry for this project">Parts</a></dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/smartfold/overview">Phthalate Biosensor</a></dd>
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  <dd><a href="diauxie/references">References</dd>
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   <dd><a href="https://2008.igem.org/Team:PennState/fusion/overview">BPA Biosensor</a></dd>
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<h4>Project Abstracts</h4>
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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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    <h4>Diauxie Elimination by Xylose Inducible Promoters</h4>
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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 phthalate compounds utilizing the Peroxisome Proliferator Activated Receptor (PPAR) and detecting Bisphenol A (BPA) by the Estrogen Receptor (ER). Recent studies show phthalates cause negative health effects such as damage to the liver and kidneys and birth defects in rodents. Phthalates are introduced into our environment by their use as plastisizers for materials ranging from polyvinyl chloride to nail polish to small toys.  BPA is also found in plastics but instead it is used for 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>
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       <p class="start"><img src="https://static.igem.org/mediawiki/2008/c/c3/Diauxie_curves.JPG" alt="[Graph]" style="float:left; margin:5px;width: 120px;"/>Microorganisms typically preferentially utilize glucose over other sugar carbon sources such as xylose. This is largely regulated through control of gene expression based on the response of regulatory elements to sugars available to the cell. In <em>E. coli</em>, the xylose metabolism operon is controlled by both the xylose-inducible XylR activator protein and the cAMP receptor protein (CRP). In this project we attempt to eliminate glucose control over xylose-inducible gene expression in <em>E. coli</em> by altering the natural transcriptional control region of the xylose operon. Designs constructed and tested include scrambling the CRP binding site, increasing the strength of the xyl promoter, and overexpressing XylR. Xylose-inducible gene expression that functions independently of glucose regulation provides a useful approach to improving microbial utilization of biomass feedstocks containing mixtures of glucose and xylose.</p>
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      <p> We are using two of the natural human nuclear hormone receptor proteins that recognize a 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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    <h4>Hormone Prescreening with <em>E. coli</em></h4>
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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 phthalate compounds using the Peroxisome Proliferator Activated Receptor (PPAR) and detecting Bisphenol A (BPA) by the Estrogen Receptor (ER). The idea is explored in-depth in our <a href="https://2008.igem.org/Team:PennState/hbintro" title="">NHR Biosensors Introduction</a></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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           <td style="padding-top:30px; padding-right:30px" valign="top" width="45%"><h4>"Smart Fold" Pthalate Biosensor</h4>
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             <img src="http://upload.wikimedia.org/wikipedia/en/8/87/PPARg.png" alt="[img]" style="float:left; margin:5px;width: 30%"/>
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             <img src="http://upload.wikimedia.org/wikipedia/en/8/87/PPARg.png" alt="[img]" style="float:left; margin:5px;width: 30%; border: solid 1px #ccc;"/>
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             <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) via the DBD and activates transcription.  Most often a coactivator complex is required for transcriptional activation which involves proteins SRC-1, CBP and others.  </p>
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             <p style="text-indent: 0">This <em>Phthalate Biosensor</em> project uses altered growth conditions so that the entire <acronym title="Human Peroxisome Proliferator Activated Receptor subtype Alpha">hPPARα</acronym> protein is successfully expressed in <em>E. coli</em> and used to transcriptionally report for the presence of phthalates in liquid solution. Rather than changing the receptor and possibly losing its usefulness, we are chemically optimizing the cell environment. Check out our Overview to find out how.
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<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 that 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 need tight requlation 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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           <td style="padding-top:30px; padding-right:30px" valign="top" width="45%"><h4>"Nuclear Fusion" BPA Biosensor</h4>
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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: 150px; border: solid 1px #ccc;"/>
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             <p style="text-indent: 0">The <em>Nuclear Fusion</em> project involves a plasmid construct very generously donated to our iGEM team from David W. Wood, Department of Chemical Engineering at Princeton University.  Research in their lab has constructed a biosensor containing just the ligand binding domain (LBD) of the estrogen receptor (ER). 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 for BPA which has a very different conformation than the natural agonist of the ER system (estradiol).  This difference causes BPA to bind weakly but still disturbs normal ER function.</p><p>We intend 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.</p>
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             <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 of Chemical Engineering at Princeton University.  Research in their lab has constructed a biosensor containing just the ligand binding domain (LBD) of the estrogen receptor (ER). The ER is very similar to the PPAR and other hormone receptors.  Previous attempts at isolating the LBD failed due to the specific folding pattern of this region required to maintain similar binding characteristics to natural ER.  The folding pattern was kept by inserting the LBD 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>
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<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 for BPA which has a very different conformation than the natural agonist of the ER system.  This difference causes BPA to bind weakly but still disturbs normal ER function. One idea is to replace the ER LBD from Wood’s biosensor with the estrogen-related receptor (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, preferentially <em>e. coli</em> 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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Latest revision as of 01:32, 30 October 2008

Home The Team The Project Parts Notebook

Diauxie Elimination

Introduction
The System
Strategies
Progress
Conclusions
Parts
References

NHR Biosensors

NHR Introduction
Phthalate Biosensor
BPA Biosensor

Diauxie Elimination by Xylose Inducible Promoters

[Graph]Microorganisms typically preferentially utilize glucose over other sugar carbon sources such as xylose. This is largely regulated through control of gene expression based on the response of regulatory elements to sugars available to the cell. In E. coli, the xylose metabolism operon is controlled by both the xylose-inducible XylR activator protein and the cAMP receptor protein (CRP). In this project we attempt to eliminate glucose control over xylose-inducible gene expression in E. coli by altering the natural transcriptional control region of the xylose operon. Designs constructed and tested include scrambling the CRP binding site, increasing the strength of the xyl promoter, and overexpressing XylR. Xylose-inducible gene expression that functions independently of glucose regulation provides a useful approach to improving microbial utilization of biomass feedstocks containing mixtures of glucose and xylose.

Hormone Prescreening with E. coli

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 phthalate compounds using the Peroxisome Proliferator Activated Receptor (PPAR) and detecting Bisphenol A (BPA) by the Estrogen Receptor (ER). The idea is explored in-depth in our NHR Biosensors Introduction

"Smart Fold" Pthalate Biosensor

[img]

This Phthalate Biosensor project uses altered growth conditions so that the entire hPPARα protein is successfully expressed in E. coli and used to transcriptionally report for the presence of phthalates in liquid solution. Rather than changing the receptor and possibly losing its usefulness, we are chemically optimizing the cell environment. Check out our Overview to find out how.

"Nuclear Fusion" BPA Biosensor

[img]

The Nuclear Fusion project involves a plasmid construct very generously donated to our iGEM team from David W. Wood, Department of Chemical Engineering at Princeton University. Research in their lab has constructed a biosensor containing just the ligand binding domain (LBD) of the estrogen receptor (ER). 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 Smart Fold Reporter project. The sensitivity will be focused for BPA which has a very different conformation than the natural agonist of the ER system (estradiol). This difference causes BPA to bind weakly but still disturbs normal ER function.

We intend 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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