To me, the idea of doing something that has never been done before has always fascinated me. With this research opportunity, I had the chance to do just that: develop a novel fluorescent tool to measure a type of ubiquitination, an essential cellular process in which chains of the protein ubiquitin are attached to substrates, signaling them for various fates based on the type of ubiquitin linkage involved. K63 ubiquitination specifically plays a role in the regulation of a cell’s response to oxidative stress. Bimolecular fluorescence complementation (BiFC) can quantitatively measure fluorescence as a proxy for protein-protein interactions, including ubiquitination. In BiFC, two proteins expected to interact with each other are each linked to one half of a fluorophore. When the proteins bind each other, they bring the two fluorophore halves together, allowing it to fold into its functional fluorescent form. Fluorescence can be measured and correlated to protein-protein binding activity. Thus, this research sought to generate a high-throughput tool to measure K63 ubiquitination in live cells and in real time using a modified version of BiFC known as ubiquitin-induced fluorescence complementation (UiFC). In UiFC, the protein-binding domains have a high affinity for K63 polyubiquitin. We expected to see a high correlation between fluorescent activity and K63 ubiquitination. Two UiFC constructs were generated via molecular cloning: the UIM (ubiquitin-interacting motif), linked to the N-terminus of the fluorophore mCherry, and the Npl4 zinc finger (NZF) domain, linked to the C-terminus of mCherry. Polymerase Chain Reaction (PCR) amplified four half-constructs, which were assembled into two separate samples of plasmid backbone via Gibson assembly. These recombinant plasmids were transformed into aliquots of E. coli and extracted after cloning. Confirmation digests and sequencing confirmed successful cloning. Following in vitro specificity testing, human cells can be used to explore the role of ubiquitination in distinct signaling pathways. Through this process, I learned many valuable skills, from the steps of molecular cloning, to how to communicate my science to the public. I learned how to function independently in the lab and follow written instructions from my mentor. It was great training for my upcoming job as a research technician, where I will undoubtedly find myself pulling on many of the tips and practices I learned during my time at Duke. Moreover, the experience itself has helped define me as a scientist. I have, through experience, found what I like most about working in a lab setting.