Honors Theses
Date of Award
2005
Document Type
Undergraduate Thesis
Department
Chemistry and Biochemistry
First Advisor
Michael Mossing
Relational Format
Dissertation/Thesis
Abstract
The field of systems biology seeks to engineer systems in order to explore specific dynamic properties as they act in vivo. Cellular activities have been proven to be regulated by a range of DNA binding proteins that bind to specific sequences that activate or repress transcription. Certain phage and bacterial repressor proteins were among the first of these DNA binding proteins to be characterized structurally and functionally. The demand for study of key steps in regulation of protein expression, folding, and repression has created multiple studies into systems of varying complexity. The data in this report explores the lambda system, and more specifically the kinetics of the repressor protein, Cro. The network of regulatory proteins and DNA sites that control a simple developmental switch in bacteriophage lambda have been extensively studied and modeled mathematically.Previous work in our laboratory has shown folding of Cro is very slow in vitro, and this phenomenon has not been included in models to date. The goal is to measure the time of Cro folding and repression in vivo. In order to examine the kinetic properties of the Cro protein, a reporter plasmid containing the gene for the green fluorescent protein is fused to a promoter that is repressible by both Cro and lacR. Several strains of bacteria have been engineered containing lacR, the reporter plasmid, and optionally variants of Cro. Initially GFP and Cro are expressed at a low level. After addition of IPTG, a compound that inactivates lacR, GFP accumulates and the fluorescence of the cell increases. In strains that contain the Cro gene, GFP accumulation is halted after enough active Cro has accumulated to repress transcription of GFP. Several Cro variants have been investigated. Initial observations of fluorescence spectra and induction kinetics of living bacterial cultures are reported.
Recommended Citation
LeBlanc, Zachary Louis, "Engineering Genetic Networks to Measure Real Time Repression by λ Cro in vivo" (2005). Honors Theses. 2232.
https://egrove.olemiss.edu/hon_thesis/2232
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