Team:Hewitt-Trussville

From 2012hs.igem.org

Team

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Project

The United States Environmental Protection Agency and Alabama Department of Environmental Management (ADEM) has declared numerous companies to be in violation of runoff waste laws. Alabama Rivers are affected by high levels of phosphate, which influence the diverse ecosystems inhabited on the rivers, which include 118 species of snails in the Cahaba. There are four that are endangered or threatened. They include the Cylindrical Lioplax, Flat Pebble Snail, Rock Snail, and the Round Rock Snail. (Nijhuis, 2011) There are water testing kits readily available to sample any water system for phosphate levels, though these kits use chemicals. Companies such as Hach and LaMotte provide chemical phosphate testing kits that also require outside components that could potentially be harmful to those handling them. A biological mechanism to test phosphate levels does not exist. We propose to construct an engineered plasmid to use the Pho5 pathway in Saccharomyces cerevisiae as the promoter and it will be attached to other biological parts including: an inverter, red fluorescent protein (RFP), and plasmid backbone. Therefore, it holds minimal to no risk for anyone who works with the new testing method. “Yeast can be grown rapidly on simple media and to high cell density, and its genetics are more advanced than any other eukaryote, so that it can be manipulated almost as readily as E.coli” (Romanos 423). Production of the new plasmid will also be cost effective due to the simple lab procedures needed to quantify the amount. DNA parts were retrieved from partsregistry.org. This website is home to biological parts created from previous science fairs and available for any new research. Just as the biological function of the Pho5 pathway secretes different levels of phosphate into the cell based on environmental levels, the RFP works within that pathway to visually show the different levels of phosphate by glowing red. Throughout the production of the Pho5 Plasmid lab procedures including: Biobrick assembly, Gibson assembly, Polymerase Chain Reaction, electrophoresis, and transformation were used.

Notebook

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Results/Conclusions

We propose to begin the construction phase in March and the testing phase by April. Extensive research has gone into the construction process of this plasmid that utilizes the Pho5 pathway in S. cerevisiae. Construction of the plasmid will begin when the Pho5 DNA sequence and the other parts arrive. Complications concerning the availability of the parts have slowed progress down. To test the completed plasmid it will be immersed in a phosphate solution so that the RFP will be seen at its strongest. The plasmid will then be tested in water samples taken from the Cahaba River, located just behind our school. Water testing guidelines from ADEM will be followed to ensure the most accurate results. Through the course of this year’s research we have found that there are unlimited possibilities within the abilities of DNA. E. coli was thought to be the easiest source for testing for phosphate due to the necessity of inorganic phosphate in its system, but our research has proven differently.

Safety

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Attributions

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Human Practices

What impact does/will your project have on the public?

Fun!

What was your favorite team snack?? Have a picture of your team mascot?

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