Date of Award
Doctor of Philosophy
Gary S. Sayler
Alison Buchan, Timothy E. Sparer, Seung J. Baek, John P. Biggerstaff
Despite its extensive use as a prokaryotic bioreporter, only recently has the bacterial bioluminescence (lux) system been adapted to express at a functional level in the mammalian cellular background. While this novel strategy has the potential to contribute to the fields of high throughput screening and non-invasive in vivo imaging due to its autonomous substrate production, it is still constrained because its signal intensity is lower than that of other bioluminescent reporters. This work demonstrates the development of strategies that optimize human cell lux-based bioluminescence to overcome this detriment for advancement towards a fully functional lux reporter system. To enhance lux gene expression, a single vector construct has been developed that improves upon the initial two plasmid expression system. This construct separates the lux genes using viral-derived 2A elements, allowing for simultaneous expression of the six genes from a single promoter in eukaryotic cells. This strategy results in increased bioluminescent output compared to the previous two plasmid system. Additionally, it is demonstrated that gene copy number is the primary limiting factor for bioluminescent output following expression in human cell lines. This limitation has been overcome through the development of a two-step transfection strategy that yields significantly higher transfection efficiency and improves both transgene integration and bioluminescence output. An optimized HEK293 cell line with enhanced bioluminescent production has been constructed using the two-step transfection strategy. The enhanced signal intensity allows bioluminescent detection from a smaller population size in cell culture compared to their un-optimized counterparts, as well as detection using alternative lower cost instruments. A developmental effort towards lux-based biosensing in human cell lines is also reported in this study. It has been shown that bioluminescence emitted constitutively from lux-expressing human cell lines can be utilized for monitoring population dynamics in a non-invasive manner and that, by regulating the expression of the lux genes, the lux system can function as a fully autonomous bioreporter for continuous monitoring of targets of interest.
Xu, Tingting, "Optimization of Bacterial Bioluminescence (lux) Expression and Development of Autonomous lux-Based Reporters in Human Cell Lines. " PhD diss., University of Tennessee, 2012.