Towards Robust, Point-of-Care Biosensing in Cell-Free Systems
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Piorino Macruz De Oliveira, Fernanda
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Abstract
The work described in this thesis enables the development of more robust cell-free biosensors for point-of-care diagnostics by tackling three issues: (1) the limited repertoire of analytes that can be detected in cell-free systems, (2) the resource competition-driven crosstalk between genetic cassettes, and (3) the poorly characterized impact of the degree of cell lysis on cell-free expression. Bacterial cell-free synthetic biology, which harnesses bacterial cell extracts for protein expression, can be an impactful technology in a variety of fields, including biomanufacturing and healthcare. Notably, cell-free biosensors are powerful detection platforms, providing a tool for low-cost, point-of-care diagnostics that can be particularly valuable in resource-limited settings. In this thesis, I describe the development of a cell-free vitamin C (ascorbate) biosensor that harnesses the native ascorbate utilization machinery of Escherichia coli bacteria, including the cross-membrane phosphotransferase system. This biosensor can detect concentrations of ascorbate into the physiologically relevant range in human plasma with color outputs that can potentially be interpreted with the naked eye, obviating the need for additional equipment. Since this sensor requires the membrane-associated proteins of the phosphotransferase system, it also provides a proof of concept for cell-free biosensing via membrane proteins, highlighting the versatility of cell-free systems. I also provide an in-depth investigation of two factors—plasmid crosstalk and the extent of cell lysis—that affect the robustness and reliability of cell-free technologies but are still poorly characterized. I demonstrate that crosstalk generated by resource competition between genetic cassettes in crude extracts has a substantial impact on protein levels and on the performance of cell-free platforms; this crosstalk can be leveraged as a functional feature to improve this performance. In addition, I show that the extent of cell lysis—as measured by the sonication energy input—has unexpected effects on cell-free protein synthesis, and that these effects depend on plasmid dosage, the promoter driving transcription, and backbone components of the expression vector.
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2023-04-25
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Dissertation