Graduation Year

2020

Date of Submission

5-2020

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

W.M. Keck Science Department

Reader 1

Aaron Leconte

Reader 2

Erin Jones

Terms of Use & License Information

Terms of Use for work posted in Scholarship@Claremont.

Abstract

Bioluminescence imaging (BLI) harnesses biochemical light emission by living organisms as an imaging tool. Perhaps the most commonly used reaction in BLI is that between firefly luciferase (Fluc) and D-luciferin (D-luc), which steadily emits light at 562 nm. A limitation of the Fluc/D-luc system is its lack of multicomponent imaging capabilities. To address this problem, researchers have developed orthogonal luciferase-luciferin pairs. Each mutant luciferase only reacts with a specific modified luciferin, allowing for various in vivo processes to be tracked at the same time. Prior work identified Fluc 93 and Fluc 51, an orthogonal pair of luciferase mutants specific for D-luc and 4’Br-luc, respectively. However, both of these mutants are far less thermostable than wild-type Fluc. Findings suggest that adding mutations T214A/A215L/I232A/F295L/E345K, also called the Mutant E set, to wild type Fluc can improve its heat tolerance. We hypothesized that adding the Mutant E set to new versions of Fluc 51 and Fluc 93 would generate luciferases that are both substrate specific and thermostabilized. Here, we successfully designed, cloned, and screened new Fluc 51 and Fluc 93 variants with the Mutant E set added. We found that the mutants derived from Fluc 93 lost all activity with the addition of the Mutant E mutations. We have yet to screen the mutants derived from Fluc 51. This work suggests that the incorporation of a large set of mutations may not be the best approach to thermostabilizing Fluc 51 and Fluc 93, and alternative methods are likely necessary.

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