Author Date

2023-08-04

Degree Name

BS

Department

Chemistry and Biochemistry

College

Physical and Mathematical Sciences

Defense Date

2023-08-01

Publication Date

2023-08-04

First Faculty Advisor

Dr. Christopher M. Tracy

First Faculty Reader

Dr. Julianne Grose

Honors Coordinator

Dr. Walter F. Paxton

Keywords

polyubiquitinated condensates, Tnk1, UBA, cellular liquid-liquid phase separation, LLPS

Abstract

Cellular liquid-liquid phase separation (LLPS) is a mechanism by which membraneless organelles such as polyubiquitinated condensates form and function. A non-receptor tyrosine kinase, Tnk1, binds to the polyubiquitin of polyubiquitinated condensates that are labeled for the proteasome for autophagy through its ubiquitin association (UBA) domain. This tyrosine kinase has links to a subset of hematological cancers, so identifying the protein contents within polyubiquitinated condensates is essential for understanding their significance in cancer pathways and to eventually identify potential therapeutic targets. This honors thesis focuses on the cloning of a fusion gene, pcDNA3.1 mNeonGreen–V5–UBA, as well as its deactivated L36D variant, to utilize the interaction between the ubiquitin association (UBA) domain of Tnk1 and polyubiquitin. Methods included cloning, PCR, restriction digest, HiFi DNA assembly, site-directed mutagenesis, blunt-end ligation, E. coli transformations, plasmid extractions, gel electrophoresis, sequence alignment, cell culture, vertical electrophoresis, and western blot. The successful cloning and deactivating site-directed mutagenesis of the L36D variant allow for further investigation, specifically the identification of protein interactors via separation techniques and proteomics.

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