Team:PennState

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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) and activates transcription.  Most often a coactivator complex is required for transcriptional activation which involves proteins SRC-1 and CBP and others.  </p>
             <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>

Revision as of 20:22, 27 June 2008

Home The Team The Project Parts Modeling Notebook

Hormone Biosensors

Introduction
Smart Fold
Overview
Parts
References
Nuclear Fusion
Overview
Parts
References

Diauxie Elimination

Introduction
Overview
Parts
References
 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 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.

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 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
[img]

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.

This Smart Fold Reporter 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.

Nuclear Fusion
[img]

Nuclear Fusion is our second approach to constructing a phthalate detection system in E. coli. In this project we use just the ligand binding domain of hPPARα fused to thymidylate synthase (TS). Binding of the phthalate ligand to this chimeric protein activates TS. When this construct is placed in a TS diffident strain, only E. coli in the presence of a hPPARalpha agonist will survive.

Diauxie Elimination: Two spoons full of sugar.

[img]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 e. coli preferentially metabolizes glucose before any other sugar. In this project we attempt to eliminate this phenomenon, called diauxie, 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.
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!

invitrogen Dupont

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