Graduation Year

2022

Date of Submission

4-2022

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Biochemistry

Reader 1

Aaron Leconte

Reader 2

Anna Wenzel

Abstract

Much of the current research being conducted in the field of bioluminescence has been directed towards furthering our understanding and development of bioluminescent probes. Firefly Luciferase (FLuc) is one such probe that has been subject to extensive research and advancement in an effort to increase visualization of cell growth and movement in vivo. Recent protein engineering of mutant luciferases with altered selectivity for modified substrates have led to the creation of orthogonal pairs. Such mutant luciferases have minimal cross reactions, only generating light when interacting with their respective substrates. The orthogonal pair derived from FLuc, named Pecan-TS and Cashew-TS, has emerged as the best in vivo multicomponent imaging tools we have to date, but the mutations made for increasing selectivity have resulted in significant decreases in brightness relative to the WT. Building off of past research by Fujii et al. and Noda et al. on the triple mutant LGR (I423L, D436G, L530R), in this project, the LGR mutation is added to Pecan-TS and Cashew-TS in an effort to address the lowered brightness and to further understand the effects of the mutation. The resulting enzymes created not only had increased brightness and luminesce, but increased substrate selectivity as well.

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This thesis is restricted to the Claremont Colleges current faculty, students, and staff.

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