Publication Date

Fall 2020

Advisor(s) - Committee Chair

Dr. Moon-Soo Kim (Director), Dr. Kevin Williams and Dr. Matthew Nee

Degree Program

Department of Chemistry

Degree Type

Master of Science

Abstract

A faster method of quantitative detection of specific dsDNA of pathogenic bacteria such as the Shiga toxin 2 gene (stx2) present in E. coli O157:H7 is of great importance. There is a need for a simple and facile method for sensitive detection of pathogenic gene which is crucial for the prevention and earlier treatment of any infectious diseases. A Transcriptional Activator-Like Effector (TALE) is a novel class of DNA-binding proteins with the unique modularity, flexibility and easy programmability compared to previously discovered DNA- binding proteins. TALEs can bind to any DNA sequences through its unique variable di- residues (RVDs) present in each repeat in the DNA-binding region of the protein. Graphene oxide (GO) is a 2D-nanosheet formed by the oxidation of graphite. It has many oxygen- containing functional groups such as carboxyl, hydroxyl, carbonyl and epoxide in its structure, allowing for interaction with biomolecules such as DNA and proteins. The GO has gained wide popularity because of its unique properties such as larger surface area, high water dispersibility and excellent biocompatibility. Quantum dots (QDs) are metallic, semi-conducting nanoparticles possessing unique electrical, physical and optical properties.

Our goal was to develop a rapid method to quantitatively detect pathogenic double stranded (ds) DNA in the stx2 gene encoding the Shiga toxin present in E. coli O157:H7. Novel TALEs were designed to detect the stx2 gene and were cloned into the pMAL c2x vector system replacing AvrBs3 TALE protein for bacterial BL-21 E. Coli expression. The TALE proteins were purified by His-tag affinity chromatography using Ni-resin. TALE proteins were then labeled with CdSe/ZnS QDs using EDC/NHS bioconjugation to create a stable peptide bond between the QDs and the proteins. Labeling efficiency was higher, > 90% compared to the previously discovered Zinc Finger Proteins (ZFPs). The GO quenching assay was performed to determine the optimal concentration of GO that quenches QD signals via fluorescent resonance energy transfer (FRET). The optimal sensitivity conditions were determined to be 20 nM of QD-labeled proteins and 3 μg/mL of GO. The GO assay took in less than 30 minutes in a simple laboratory setting. The limit of detection of TALE stx2 236 was determined to be 200 pM which is equivalent to 40 fmol of the target oligonucleotide. In summary, we have demonstrated the novel application of a new class of DNA binding protein TALEs and 2D nanosheet GO as a sensing platform for detecting pathogen-specific dsDNA in a simple assay.

Disciplines

Analytical Chemistry | Biochemistry, Biophysics, and Structural Biology | Chemistry

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