Mahurin Honors College Capstone Experience/Thesis Projects

Department

Chemistry

Document Type

Thesis

Abstract

Transcription activator-like effector (TALE) proteins are important for DNA binding. They bind to specific nucleotide sequences by the use of two residues in each repeat allowing them to target specific DNA sequences. Their modular structure makes TALEs advantageous over other DNA binding proteins such as zinc finger proteins. Zinc finger proteins (ZFP) use a finger like projection to bind 3-4 subsequence base pairs while TALE proteins use two amino acid residues in each repeat to bind one nucleotide. ZFP can use SEER-Lac system for colorimetric detection, while TALE proteins can use fluorescence labeling with Alexa for detection of binding to DNA. The TALE proteins simplistic design makes them easily manipulated and advantageous for genome engineering. They recognize DNA through a tandem array and an RVD (repeat variable diresidue) loop on the 12th and 13th residue in the amino acid chain. Upon binding to DNA, TALEs use fluorescence labeling to emit a signal that can be detected over unbound DNA. This method of targeting DNA is being used for the detection of pathogens which can lead to rapid point-of-care (POC) diagnostic devices. In the research presented, TALE protein purification process has been optimized through techniques that manipulate the cell culture to yield high concentrations of the protein. The methods use a two-column purification technique with His tag and a maltose binding protein linked to the protein. The assay for target DNA detection has been optimized through binding the proteins to the gel surface with interactions strong enough to resist washing steps. The assay used the Alexa bound fluorescence molecules to prove protein bound to target DNA complexes with fluorescence spectroscopy.

Advisor(s) or Committee Chair

Dr. Moon-Soo Kim, Dr. Sigrid Jacobshagen, Dr. Chris Keller

Disciplines

Biochemistry | Chemistry

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