Date of Award
Doctor of Philosophy
Dr. Gary S. Sayler
Dr. Robert Moore, Dr. Jeff Becker, Dr. Patricia Tithof, Dr. Yun You
This study describes the optimization of expression of the bacterial luciferase enzyme in mammalian cells. Previous attempts to express this heterodimeric enzyme complex in mammalian cells have been met with only modest success. In this research effort, several vector formats were evaluated to fully determine the optimal format for the expression of these genes. It was determined that the bacterial luciferase enzyme produced optimal bioluminescence in mammalian cells when the genes were cloned and expressed as a bicistronic transcript fused with an internal ribosomal entry site (IRES). To optimize the enzyme expression further, a novel approach to codon optimize the genes was performed. To accomplish this task, completely synthetic versions of the codon optimized sequences were generated. This codon optimization, led to an increase in bioluminescence levels greater than two orders of magnitude versus the wild type genes. Additionally, the availability of the FMNH2 substrate was evaluated and determined to be a limiting substrate for the reaction. In an attempt to alleviate this limitation, a flavin oxidoreductase gene (frp) from Vibrio harveyi was cloned and expressed along with the codon optimized luxA and luxB genes. Although the expression of this enzyme enhanced the bioluminescence significantly, FMNH2 remains the limiting substrate for optimal bioluminescence. To produce a usable reporter cell line, the reporter must remain stable within the cells for long periods of time. The overall stability of the engineered cells was assessed to determine the persistence of the reporter for long-term monitoring applications. These data revealed that the luciferase genes were stable in HEK293 cells for more than forty passages (five months) in culture in the absence of antibiotic, indicating that these cell lines would be stable enough for relatively long term monitoring projects and applications.
Patterson, Stacey Swiger, "Optimization of Bacterial Luciferase for Expression in Mammalian Cells. " PhD diss., University of Tennessee, 2003.