All ETDs from UAB

Advisory Committee Chair

Shahid Mukhtar

Advisory Committee Members

Jeffrey Morris

Stephen Watts

Document Type

Thesis

Date of Award

2016

Degree Name by School

Master of Science (MS) College of Arts and Sciences

Abstract

The Yeast-2-Hybrid (Y2H) assay is a heterologous system that is expanding network biology knowledge via in vivo investigations of binary protein-protein interactions. Traditionally, the Y2H protocol entails the mating or co-transformation of yeast in solid agar media followed by visual analysis for diploid selection. Having played a key role in identifying protein-protein interactions for nearly three decades, the Y2H system includes the interaction between two proteins of interest which result in a reconstituted transcription factor allowing a reporter gene to be transcribed. In this thesis, two primary objectives were pursued to expand the knowledge of protein-protein interactions. The primary objective entailed the development of a novel, Y2H protocol veering from traditional methods of mating on agar media to a liquid-assay based method to yield quantitative results in a more cost-effective, time-saving approach. From retesting previously confirmed interacting pairs, to testing media concentration variables, an extensive experimental focus has been devoted to this new protocol which has the potential to enhance numerous aspects of biological research in the future. The protocol set forth here, enables the quantification of protein-protein interaction strength in a 96-well plate format, and spells out a method for enhanced reliability and understanding of interaction types and strengths through the use of quantitative data simultaneously validated for synchrony with testing in solid agar. As a result of many trials conducted in liquid assay in both a Infinite 200 PRO-multimode Tecan spectrophotometer and BioTek Synergy H1 Microplate reader, data collection of Y2H is transformed. Multiple data sets support the hypothesis that liquid assay optical density and possibly growth curve values can help quantify interaction strength from identifying threshold optical density values, delineate strong, weak, and autoactivators, and shed light into the growth patterns of two interactors. The applications of this method are exceptionally broad reaching into fields such as drug discovery, clinical trials for human disease including cancer and neurodegenerative disease, and even into synthetic biology applications and cellular engineering. As the study of protein-protein interactions is incredibly far-reaching, this Y2H application could impact countless realms of science.

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