Additional Departmental Affiliation
Our objective was to develop a faster method of quantitively detecting double stranded (ds)DNA of pathogenic bacteria such as the Shiga Toxin 2 gene present in E. coli O157. Transcription Activator-Like Effectors (TALEs) are a new class of DNA-binding proteins which selectively bind to dsDNA with the 12th and 13th amino acids of each repeat, called repeat variable diresidues (RVDs). Novel TALE proteins were designed to target the stx2 gene and were cloned into existing AvrBs3 TALE protein in the pMAL c2x vector system for bacterial BL-21 E. Coli expression. The protein’s DNA-binding region was then subcloned pEAQ vectors for expression in N. Benthamiana expression systems. Protein expression was comparable between systems, as plant expression had a higher yield at the cost of additional time and resources. After expression, TALE proteins were purified using affinity chromatography and characterized. TALE proteins were then labeled with CdSe/ZnS Quantum Dots (QDs) using EDC and NHS labeling method to create a stable peptide bond between the QD and protein. Labeling efficiency was very high ~90% with relatively low initial protein concentration. Labeled proteins were then used in Graphene oxide (GO) quenching and sensitivity arrays to observe if GO could quench QD signal and if target DNA could restore QD signal. In both experiments, QD were excited with 300 nm light and 515 nm endpoint fluorescence data was collected. Optimal sensitivity array conditions were determined to be 10 nM protein and 2 μg/ml GO, and DNA sensitivity arrays could be completed in under 30 minutes. It is important to further investigate the binding capabilities of the TALE proteins and the sensitivity of this system in the presence on nontarget DNA and complex biosamples.
Advisor(s) or Committee Chair
Dr. Moon-Soo Kim, Dr. Claire Rinehart, Dr. Chris Keller
Biochemistry | Biotechnology | Molecular Biology
Jackson, Colleen, "Investigation of Transient Expression System in Nicotiana bethamiana to Produce Novel TALEs and the Development of (ds)DNA Detection with Quantum Dot-Labeled Proteins in Graphene Oxide Quenching Arrays" (2019). Mahurin Honors College Capstone Experience/Thesis Projects. Paper 833.