Team:IvyTech SouthBend IN

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Team:IvyTech SouthBend IN


"BETA-DATA QUANTATATA"


A device for the rapid detection of human waste contamination of drinking and recreational waters

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We are Team Ivy Tech Community College of Indiana-South Bend!

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What is your project about?

The American Public Health Assay (APHA) for human waste contaminated water is hampered by a dependency on culturing coliform bacteria with a turn-around time exceeding one day. An assay for coliphage detection, as surrogate for coliforms, has been developed which employs the release of the host indicator cell β-galactosidase enzyme (β-gal) activity by lytic coliphage and the conversion of a colorimetric substrate. While significantly shortening the APHA time to 3 hrs, this assay requires laboratory equipment to separate phage-induced released β-gal enzymes from intact cells before addition of the substrate. To optimize this assay we have taken advantage of the α-complementation feature of β-gal. We are engineering cell lines containing either the gene fragments of LacZα or LacZΩ of β-gal on separate plasmids. We predict upon lysis of a mix population of cells by coliphage in the presence of substrate, the gene products will complement in trans reconstituting the β-gal activity thus eliminating a separation step in the assay. In addition we are testing the introduction of a cassette of coliphage cell lysis genes on both plasmids under the control of a coliphage promoter to accelerate the enzyme fragment release thus further reducing test time. Our goal is to design a fast-acting hand-held coliphage biosensor device that can be used by someone without any special technical expertise. We anticipate our device being useful in rapidly reporting the contamination of recreational waters in the U.S. or assuring potable water in the water sources in the developing World.

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How does your project work?

The human pathogens in contaminated water are too varied to reasonably test for their presence. The presence of the normal human enteric bacteria E.coli has been used as a marker for contamination but their are concerns that it might not persist in the environment as long as the human pathogenic virus will. Another dependable marker of water contaminated by human waste is the presence of bacteriophage of the human intestines or so-called "coliphage." These have been found to be more stable under various environmental conditions. Our biosensor is designed to report the lysis of target cells from coliphage by the release of one of two fragments of the enzyme beta-galactosidase (Omega fragment). Upon release, the beta-galactosidase omega fragment joins beta-galactosidase alpha fragments that are suspended by the added contaminated water. After the two fragments join, beta-galactosidase enzyme activity is formed and an enzyme substrate present is converted to its colorimetric form. The amount of color development will be quantified by comparison with a parallel test with known amount of coliphage present.

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Who will your project help?

Our proposed device could not only be used to safeguard lakes, rivers, streams, swimming areas, beaches and public access sites, but potentially municipalities or anywhere clean uncontaminated water is preferred. The rapid detection and the potential low cost of the device would have a positive impact on developing countries as well as developed nations where drinking water safety is a concern.
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Why did you choose this project?

The thought of creating something from the ground up as well as applying education to overcome obstacles that occur in everyday life has been a tremendous motivator. Being able to use science and engineering to make the lives of others safer is our main goal and why we chose this project.
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