Graduation Year

2025

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Reader 1

Aaron M. Leconte

Reader 2

Pete Chandrangsu

Terms of Use & License Information

Terms of Use for work posted in Scholarship@Claremont.

Abstract

Firefly luciferase (FLuc) is a bioluminescent enzyme widely used in biological imaging to track molecular events in real time. While isolated processes can be observed with a single luciferase-luciferin pair, sensitive multicomponent imaging requires enzymes with enhanced brightness and selective substrate recognition. Previous work has identified a number of orthogonal luciferase-luciferin pairs; however, most luciferases emit significantly less light than with the native substrate, limiting their use in imaging. Our high-throughput screening approach increases the likelihood of identifying bright orthogonal pairs by enabling the rapid evaluation of large enzyme libraries for both brightness and selectivity. Using this method, we identified FLuc mutants exhibiting improvement in these two properties with D-Luciferin and 4’-Br-Luciferin as substrates. Several mutations conferring enhancement in both brightness and selectivity for a particular substrate were identified, including F250W, L253W, A477D, A477M, and A482P for D-Luc and I120M, I237S, K372L, H431F, and V519F for 4’-Br-Luc. To complement this approach, we conducted preliminary characterization of FLuc homologs to evaluate their natural brightness and substrate preferences. This analysis revealed natural variation in selectivity that may be leveraged alongside engineered mutants to expand the available luciferases for multicomponent imaging. Collectively, these findings inform avenues for further engineering and optimization of luciferases by granting insight into how specific mutations influence substrate preferences and luminescent output.

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