Team:PennState

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Revision as of 16:47, 25 October 2008

Hormone Biosensors

Introduction
Smart Fold: Overview; Parts; References
Nuclear Fusion: Overview; Parts; References

Diauxie Elimination

PENN STATE iGEM 2008

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 Escherichia Coli. We are also finishing up one of last year's projects which is aimed at creating a more efficient bioproduction process by altering how E. Coli selects between the utilization of 5 and 6 carbon sugars. Please explore our website to find out more about us and our projects!

If there are any questions or comments about the information on this site please contact us at gjt5001@psu.edu.

Hormone Prescreening 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 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.

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 E. Coli. 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 E. Coli.

Smart Fold Reporter

This Smart Fold Reporter 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. 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

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.

Diauxie Elimination: Two spoons full of sugar.

[img] 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.

Quick Links

Table of our Contributions to the Registry

Interactive E. coLisa Schematic

Pennsylvania State University

Penn State Institutes of Energy and the Environment

Center for Molecular Toxicology and Carcinogenesis

Drew Endy On Synthetic Biology

Sponsors for our team! Thanks so much!

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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>
        <hr />
-       <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>
+       <p><img src="picture here" alt="[img]" style="float:left; margin:5px;"/>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>
     </td>
    </tr>