Team:PennState/Project

[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

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

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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      <h4>Diauxie Elimination by Xylose Inducible Promoters</h4>
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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: 230px;"/>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>
+       <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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