Poster Title

Pathogen-Specific DNA Detection Using Engineered DNA-Binding Proteins

Grade Level at Time of Presentation

Senior

Institution

Western Kentucky University

KY House District #

20

KY Senate District #

32

Department

Dept. of Chemistry

Abstract

DNA-binding proteins perform some of the most important functions of all biomolecular systems. They are frequent throughout nature, playing roles in cell apoptosis, protein folding, and DNA recognition. Consequently, engineered sequence-specific DNA-binding proteins are one of the most versatile biotechnologies currently being researched. There is high demand in global healthcare for an innovation that can sensitively and selectively detect pathogenic DNA. Our research on DNA-detection via engineered DNA-binding proteins aims to deliver such a technology for use in sensitive medical diagnostic devices as well as for food and water safety assays in parts of the world with limited resources. We previously employed colorimetric Sequence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-Lac) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. The engineered ZFPs were immobilized in the acrylamide gel surface, which allows for protein arrays. The fluorescence detection system was used to improve the sensitivity of our system.

In addition to developing biotechnology with other parts of the world in mind, we are expanding our worldview by collaborating research with a university in Seoul, Korea. During our recent summer in Seoul, we gained valuable experience with the laboratory practices as well as the culture of our fellow Korean lab mates. This opportunity to join hands with fellow researchers has been incredible, and we look forward to progressing global health solutions via our international collaboration provided by the NSF-IRES program.

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Pathogen-Specific DNA Detection Using Engineered DNA-Binding Proteins

DNA-binding proteins perform some of the most important functions of all biomolecular systems. They are frequent throughout nature, playing roles in cell apoptosis, protein folding, and DNA recognition. Consequently, engineered sequence-specific DNA-binding proteins are one of the most versatile biotechnologies currently being researched. There is high demand in global healthcare for an innovation that can sensitively and selectively detect pathogenic DNA. Our research on DNA-detection via engineered DNA-binding proteins aims to deliver such a technology for use in sensitive medical diagnostic devices as well as for food and water safety assays in parts of the world with limited resources. We previously employed colorimetric Sequence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-Lac) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. The engineered ZFPs were immobilized in the acrylamide gel surface, which allows for protein arrays. The fluorescence detection system was used to improve the sensitivity of our system.

In addition to developing biotechnology with other parts of the world in mind, we are expanding our worldview by collaborating research with a university in Seoul, Korea. During our recent summer in Seoul, we gained valuable experience with the laboratory practices as well as the culture of our fellow Korean lab mates. This opportunity to join hands with fellow researchers has been incredible, and we look forward to progressing global health solutions via our international collaboration provided by the NSF-IRES program.