Mahurin Honors College Capstone Experience/Thesis Projects
Department
Chemistry
Additional Departmental Affiliation
Biology
Document Type
Thesis
Abstract
Antibiotic resistance is a rapidly spreading global threat to human health. Previously in our lab, we developed a simple method for facile detection of antibiotic resistance genes (ARGs) in bacteria. This DNA detection system takes advantage of the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeat and CRISPRAssociated Protein). We designed CRISPR in complex with nuclease deactivated Cas9 (dCas9) on graphene oxide (GO) sheets conjugated with Cas9 antibodies. Single guide RNA (sgRNA) was customized to program dCas9 for target DNA binding. sgRNA was covalently labeled with fluorescein to provide fluorescence signal. Graphene oxide (GO) is utilized for its quenching properties in this fluorescence resonance energy transfer (FRET) based DNA detection system and is based on the fluorescence quenching mechanism of antibody-GO (Ab-GO) in the absence and presence of target DNA. Cas9 antibodies (Abs) are first chemically conjugated to the GO surface, and conjugation was confirmed by atomic force microscopy (AFM). Previously, we validated the approach by performing a sensitivity assay to detect dsDNA within the tetracycline (tetM) resistance gene. This study is an extension of our prior work, which aimed to improve dCas9 protein purification and perform a quenching efficiency assay to confirm the optimal Ab-GO concentration as 2 μg/mL.
Advisor(s) or Committee Chair
Moon-Soo Kim, Ph.D.
Disciplines
Biochemistry | Biotechnology | Molecular Biology
Recommended Citation
Cecil, Wendy, "Development of a DNA Biosensor Using CRISPR/dCas9 on a Graphene Oxide Surface for Detecting Antibiotic Resistance Genes" (2021). Mahurin Honors College Capstone Experience/Thesis Projects. Paper 948.
https://digitalcommons.wku.edu/stu_hon_theses/948