Team:University of Alberta/Plastic Project:The University of Alberta

From 2008.igem.org

Latest revision as of 05:32, 30 October 2008

[http://2008.igem.org/Team:The_University_of_Alberta ]

[http://2008.igem.org/Team:The_University_of_Alberta/Team ]

[http://2008.igem.org/Team:The_University_of_Alberta/Project ]

[http://2008.igem.org/Team:The_University_of_Alberta/Parts ]

[http://2008.igem.org/Team:The_University_of_Alberta/Notebook ]

"How do I love thee, plastic? Let me count the ways. I wake up and glance at my plastic digital cable box to check the time. I go to the bathroom to use my plastic toothbrush, shaking a bit of my “nontoxic” tooth powder from a plastic bottle. I fill the plastic container of my Waterpik with mouthwash from another plastic bottle. I step into the shower—my lacy white curtain is protected by a plastic liner, and my chlorine-free shower water comes to me through a plastic-encased filter.Ah, but in the kitchen I am a bit freer of you, plastic. When I learned that my plastic bowls, dishes, and containers could leach harmful chemicals—especially the ones with that sneaky, practically invisible little recycling triangle embossed with the number 7—I bought Pyrex. My soft-boiled eggs are served up in a Pyrex glass dish. A moment of rebellion against thee, plastic! But the microwave in which I heat water for tea is made of plastic as well as metal. And the refrigerator shelf on which I store my eggs is plastic. The coaster on my desk, on which I place my steaming, oh-so-healthy green tea, is plastic. The 22-inch liquid-crystal computer monitor that seems to be the fulcrum of my entire existence is made of plastic. My keyboard, my mouse, my computer speakers, the CD cases for my music collection, my polycarbonate reading glasses, the remote control for my stereo, my telephone—all plastic. The sun shines through my window onto a riot of green plants in … plastic pots. (I could switch to ceramic, but it’s so heavy and hard to heft when I want to water them.) And I’ve been up for only an hour."

[http://discovermagazine.com/2008/may/18-think-you-can-live-without-plastic/(by Jill Neimark in Discovery Magazine 04.18.2008] 
 See also the article that inspired our project at [http://discovermagazine.com/2008/may/18-the-dirty-truth-about-plastic | Link]

The Project

Estrogen Receptor Mechanism

The aim of our project is to create a biosensor that will detect BPA and other estrogenic compounds in the environment. BPA is a toxin that has been shown to leech from certain types of plastic. Studies have shown this chemical to have detrimental effects in animal studies and is very likely to be harmful to humans as well. Having a biosensor that can detect soil and water contaminated with BPA would be a useful tool indeed.

Our project relies on the mechanism by which BPA affects cells. BPA is an estrogenic compound; i.e. it has the ability to mimic estrogen. When taken into the cell, it can bind to the cytoplasmic estrogen receptors, and activate genes related to estrogen, tricking cells into thinking they are in an environment containing estrogen when they are not.

Our biosensor will take advantage of the estrogen receptor (see diagram to the right)

Schematic of our Project

We aim to move the human estrogen receptor (hER) into E. coli, which will activate a reporter system in the presence of BPA. When BPA is present in the environment, it diffuses through the cell membrane and binds to free cytoplasmic estrogen receptor (1). This causes the estrogen receptors to dimerize (2). Dimerization of the estrogen receptors allows for the receptors to bind to the estrogen responsive element (ERE); this is a small sequence of DNA that the receptors can specifically bind once they are dimerized (3). The ERE is placed immediately upstream of the promotor for a repressor. The binding of the receptor should then block the binding of RNA Polymerase and prevent the transcription of the repressor. Under normal circumstances, the repressor would itself prevent the transcription of a reporter gene (Purple Russian/Blue Ox). Thus, when BPA is in the environment, the repressor is repressed and the reporter gene is actively transcribed, giving us a quick visual way to determine the presence of BPA (4).

Next, we plan on introducing BPA degrading genes from Sphingomonas bisphenolicum to aid in the remediation of BPA contamination. Ultimately, we aim to put this biosensor/degradation system into plants - more on this later.

Estrogen Receptor Toxicity Check

For our project, the estrogen receptor (ERα) was ordered in two separate parts - ERα-1 and ERα-2. ERα-1 consists of the ligand binding domain and ERα-2 consists of the DNA binding domain. The reason why we ordered ERα in two separate parts is because we were not sure if the parts were toxic to the bacterial cells. To test the toxicity of ERα-1 and ERα-2, we ligated the parts to the inducible promoter I0500. Then, using a photo spectrometer, we were able to measure the optical density of the bacterial culture to produce the growth curves.

Estrogen Receptor Toxicity Graph

Note: I0500a is the control.

Status

At this point our operon is nearly complete. All bricks downstream of the estrogen receptor have been assembled, with only the estrogen receptor to be his-tagged and given a promotor before we add the rest onto the end. We have acquired BPA for testing.

Journal Articles of Interest

Synthetic Receptors

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers