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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Food Science

Year Degree Awarded

2016

Month Degree Awarded

September

First Advisor

Sam R. Nugen

Second Advisor

Vincent M. Rotello

Subject Categories

Food Microbiology

Abstract

One of the major safety causes in drinking water is from the bacteria contamination, especially in developing countries and resource-limited settings. Although many of these Escherichia coli (E. coli) strains in drinking water are nonpathogenic, they sever as the indicator for bacterial contamination. And, the more widely used method to detect E. coli in drinking water is to determine the activity of β-galactosidase (β-gal), which is released by E. coli. Rapid, sensitive and inexpensive detection of E. coli in drinking water can reduce the risk of food-borne bacteria infection and stop the disease widely spreading.

In this degree research, phage-based colorimetric methods were developed to detect viable Escherichia coli (E. coli) concentration in drinking water. During the phage infection cycle, phages can specifically recognize target E. coli cells and release intracellular β-gal enzyme. Using our proposed novel biosensor strategies, the β-gal enzymatic activity as the indictor for the presence of E. coli in drinking water was measured. Firstly, T7 phage covalently conjugated to magnetic beads were used to capture, separate, and purity E. coli cells from drinking water. The released β-gal was determined using commercial colorimetric substrates. Due to the specific chemical and physical properties of nanomaterials, T7 phage were immobilized on magnetic nanoparticle to improve the capture efficiency of bacteria separation. Next, a novel enzyme-induced metallization multi-colorimetric assay was developed to monitor and measure β-gal activity, which was further employed for high-resolution colorimetric phage-enabled detection of E. coli. In order to improve the limit of detection and decrease the effect of the β-gal vary of in different E. coli strains, engineered phage (T7lacZ) carrying lacZ gene was furthermore built to force overexpression of β-gal in E. coli cells during phage infection. The uses of T7lacZ phage have been demonstrated to become a rapid, sensitive, and reliable colorimetric detection of viable E. coli.

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