Capillary electrophoresis laser-induced fluorescence (CE-LIF) is widely used to detect both the presence and concentration of fluorescently labeled biomolecules. In CE-LIF, a plug of sample fluid is electrophoretically driven down a microchannel using a high voltage applied between the opposite ends of the microchannel. Molecules of different sizes and charge states travel at different velocities down the channel. Laser light with a wavelength in the excitation band of the fluorophores is focused near the end of the channel. As each species of molecule passes through the laser spot, the fluorophores emit a fluorescence signal which is measured with an optical detector. Commercial CE-LIF systems are available as a complete, expensive package. Custom CE-LIF systems are a collection of commercially available components that meet the specific needs of the end user. Using the custom system in Dr. Woolley's lab as the standard, we hypothesized that 3D printed parts in conjunction with low-cost components could be used to significantly reduce costs and simplify the system, which in turn would make such systems more widely available with a lower barrier to entry. Testing this hypothesis began with five semesters of small teams of senior undergraduate students trying to design and assemble a low-cost CE-LIF system as part of their mandatory one-semester senior project. I was one of the seniors who worked on the system. Although none of the senior project teams were successful, a partially functioning system was ultimately produced. I reference this system as the starting point system throughout this thesis, which is focused on identifying and solving the system's obstacles in order to reach a working state. I re-designed and re-built each sub-system of the starting point system as needed if within the available budget to create a system that was functional. Budgetary constraints were included in evaluating potential improvements. The end goal was to compare the improved system's performance with that of an expensive conventional system (hereinafter referred to as the standard system) available in Dr. Adam Woolley's laboratory on the Brigham Young University campus. The ultimate conclusion of my masters' thesis work is that a low-cost CE-LIF system based on 3D printed and low-cost components results in a system that does not offer repeatable performance. In the course of my work, many lessons were learned as to what would reduce overall system costs while maintaining a user-friendly experience. My analysis is given on a subsystem basis to explain what limited the ability of the system to run consistently or what caused it to fail altogether. Details and methodology of my contributions including circuits designed, code written, components used, and 3D models printed in order to test the hypothesis are documented. Attribution of the work prior to mine is laid out when each subsystem is broken down in detail for the failure modes that prevented consistent operation. Future work is suggested to correct the problems encountered and provide a path forward to implement a next-generation system that can be achieved at a lower cost compared to a conventional system, and yet which does not suffer from the performance problems associated with the version explored in this thesis in which maximum cost reduction was aggressively pursued.

Single molecule studies of enzymes have revealed that nominally identical individual enzyme molecules are functionally heterogeneous. Different individual molecules exhibit different catalytic rates under identical conditions, and individual enzyme molecules show fluctuating rates over broad timescales. The structural basis and the biological sources for such heterogeneity remains poorly understood. Herein, studies are presented of the β-galactosidase from Escherichia coli, using capillary electrophoresis with laser-induced fluorescence (CE-LIF), to investigate the sources of catalytic heterogeneity at the single molecule level. Limited proteolysis as a possible source for single molecule heterogeneity, and for the changes in activity of a population of individual molecules over time, was investigated by inducing enzyme expression in two E.coli strains in the presence of a broad spectrum of protease inhibitors. The effect of protease inhibitors was found to be limited. β-Galactosidase was expressed from a lacZ linear template from two different E. coli strains using an in vitro protein expression system to determine if in vitro synthesized enzyme was identical to its in vivo counterpart. In vitro synthesized enzyme was found to be less active than in vivo sources. The differences were attributed to deficient N-terminal methionine removal and the higher rates of translation error associated with in vitro protein synthesis. Single molecule separations revealed that individual molecules of β-galactosidase were electrophoretically distinct, and that the electrophoretic heterogeneity was independent of source of enzyme, method of measurement, or of capillary coating. Electrophoretic modeling indicated that slight variation of hydrodynamic radius is the most likely source of electrophoretic mobility heterogeneity. The extent of single molecule catalytic variation was reduced in a mutant with a hyperaccurate translation phenotype implying that translation error is a source of the heterogeneity. Streptomycin-induced translation error reduced average activity, but did not lead to an increase in catalytic heterogeneity. No relationship between translation error and electrophoretic heterogeneity was observed. A novel CE-LIF assay was developed for the continuous monitoring of the catalytic activity and electrophoretic mobility of individual β-galactosidase molecules. Thermally-induced catalytic fluctuations were observed suggesting that individual enzyme molecules were capable of conformational fluctuations that supported different catalytic rates.

Single molecule studies of enzymes have revealed that nominally identical individual enzyme molecules are functionally heterogeneous. Different individual molecules exhibit different catalytic rates under identical conditions, and individual enzyme molecules show fluctuating rates over broad timescales. The structural basis and the biological sources for such heterogeneity remains poorly understood. Herein, studies are presented of the β-galactosidase from Escherichia coli, using capillary electrophoresis with laser-induced fluorescence (CE-LIF), to investigate the sources of catalytic heterogeneity at the single molecule level. Limited proteolysis as a possible source for single molecule heterogeneity, and for the changes in activity of a population of individual molecules over time, was investigated by inducing enzyme expression in two E.coli strains in the presence of a broad spectrum of protease inhibitors. The effect of protease inhibitors was found to be limited. β-Galactosidase was expressed from a lacZ linear template from two different E. coli strains using an in vitro protein expression system to determine if in vitro synthesized enzyme was identical to its in vivo counterpart. In vitro synthesized enzyme was found to be less active than in vivo sources. The differences were attributed to deficient N-terminal methionine removal and the higher rates of translation error associated with in vitro protein synthesis. Single molecule separations revealed that individual molecules of β-galactosidase were electrophoretically distinct, and that the electrophoretic heterogeneity was independent of source of enzyme, method of measurement, or of capillary coating. Electrophoretic modeling indicated that slight variation of hydrodynamic radius is the most likely source of electrophoretic mobility heterogeneity. The extent of single molecule catalytic variation was reduced in a mutant with a hyperaccurate translation phenotype implying that translation error is a source of the heterogeneity. Streptomycin-induced translation error reduced average activity, but did not lead to an increase in catalytic heterogeneity. No relationship between translation error and electrophoretic heterogeneity was observed. A novel CE-LIF assay was developed for the continuous monitoring of the catalytic activity and electrophoretic mobility of individual β-galactosidase molecules. Thermally-induced catalytic fluctuations were observed suggesting that individual enzyme molecules were capable of conformational fluctuations that supported different catalytic rates.

Capillary electrophoresis laser-induced fluorescence (CE-LIF) is widely used to detect both the presence and concentration of fluorescently labeled biomolecules. In CE-LIF, a plug of sample fluid is electrophoretically driven down a microchannel using a high voltage applied between the opposite ends of the microchannel. Molecules of different sizes and charge states travel at different velocities down the channel. Laser light with a wavelength in the excitation band of the fluorophores is focused near the end of the channel. As each species of molecule passes through the laser spot, the fluorophores emit a fluorescence signal which is measured with an optical detector. Commercial CE-LIF systems are available as a complete, expensive package. Custom CE-LIF systems are a collection of commercially available components that meet the specific needs of the end user. Using the custom system in Dr. Woolley's lab as the standard, we hypothesized that 3D printed parts in conjunction with low-cost components could be used to significantly reduce costs and simplify the system, which in turn would make such systems more widely available with a lower barrier to entry. Testing this hypothesis began with five semesters of small teams of senior undergraduate students trying to design and assemble a low-cost CE-LIF system as part of their mandatory one-semester senior project. I was one of the seniors who worked on the system. Although none of the senior project teams were successful, a partially functioning system was ultimately produced. I reference this system as the starting point system throughout this thesis, which is focused on identifying and solving the system's obstacles in order to reach a working state. I re-designed and re-built each sub-system of the starting point system as needed if within the available budget to create a system that was functional. Budgetary constraints were included in evaluating potential improvements. The end goal was to compare the improved system's performance with that of an expensive conventional system (hereinafter referred to as the standard system) available in Dr. Adam Woolley's laboratory on the Brigham Young University campus. The ultimate conclusion of my masters' thesis work is that a low-cost CE-LIF system based on 3D printed and low-cost components results in a system that does not offer repeatable performance. In the course of my work, many lessons were learned as to what would reduce overall system costs while maintaining a user-friendly experience. My analysis is given on a subsystem basis to explain what limited the ability of the system to run consistently or what caused it to fail altogether. Details and methodology of my contributions including circuits designed, code written, components used, and 3D models printed in order to test the hypothesis are documented. Attribution of the work prior to mine is laid out when each subsystem is broken down in detail for the failure modes that prevented consistent operation. Future work is suggested to correct the problems encountered and provide a path forward to implement a next-generation system that can be achieved at a lower cost compared to a conventional system, and yet which does not suffer from the performance problems associated with the version explored in this thesis in which maximum cost reduction was aggressively pursued.