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Publication Date

2024-5

First Advisor

Nathan D. Derr

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

GFP. RFP, Fluorescence microscopy, genetic recombination, S. cerevisiae, Cre recombinase, fluorescent fusion protein, protein expression

Abstract

Protein dynamics can be observed through the construction of fluorescent fusion proteins and subsequent fluorescence microscopy. Typically, individual proteins are indistinguishable and observations are limited to the entire population of that protein. Recent advancements have been made which can distinguish proteins by inducing a fluorescent color change to already synthesized proteins. These proteins can be differentiated between those produced before and after the color change event. However, induction of this color change must be controlled extracellularly. This thesis explores a system that uses intracellularly controlled, inducible genetic recombination to change the color in a fluorescent fusion protein expressed in S. cerevisiae for fluorescence microscopy. This work aims to construct a system for observing protein partitioning in dividing cells, where reversible recombination is induced via cellular control with every cell cycle and proteins are distinguished between “old” or “new” during development and division. Additionally, this work aims to further characterize a previously constructed system with extracellular galactose-induced irreversible recombination to elucidate the molecular dynamics of recombination. These findings will be used in the cell cycle dependent system to ensure recombination is precisely timed. This system will provide a tool to gain insight into the partitioning of cellular contents during division via fluorescence microscopy, which has broad implications in cellular biology and biochemistry.

Rights

©2024 Abigail Wilcox. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Comments

80 pages: color illustrations, charts. Includes bibliographical references (pages 64-73).

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