Date of Defense

4-20-2012

Date of Graduation

4-28-2012

Department

Biological Sciences

First Advisor

John R. Geiser

Second Advisor

Vanessa Revindran

Third Advisor

Rachel C. Chimner, Biological Sciences

Keywords

alamarBlue, liquid assay

Abstract

Yersinia outer proteins (yops) are effector proteins that have been known to cause cytolysis to host cells. Using a yeast genetic model, Yop genes from Yersinia enterocolitica can be inserted and expressed in Saccharomyces cerevisiae. With the utilization of alamarBlue® indicator, we hope to create a multi-functional liquid assay with the intent of further analysis and quantitative investigation on the effects of galactose induction of the effector protein genes. AlamarBlue® is a REDOX colorimetric/flourometric indicator specialized in detecting metabolic activity. As Saccharomyces cerevisiae metabolized, the indicator is added to monitor cell viability. In developing an effective assay, we determine that to properly prep the samples, sample cells must be washed and reintroduced in fresh media before proceeding with addition of sugars. Calculation of reduction percentages of alamarBlue® is utilized to analyze metabolic activity to identify viability of cells due to sugar induction. Galactose induction of several effector proteins displayed inhibition of growth consistent with YopT. Through an additional supplement of glucose, we tested for the suppression of inhibition of growth due to YopT expression. Results identified that the assay is successful in determining the threshold of glucose concentration falling between 0.05% and 0.10% in order to counteract the effects of YopT.

Access Setting

Honors Thesis-Open Access

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