MICROFLUIDIC APPROACHES FOR QUANTITATIVE ANALYSIS AND SCREENING OF SYNTHETICALLY ENGINEERED MICROBES

Abstract

Department of Biomedical EngineeringRecently, our understanding of complex genomes, proteomes, bio-molecules, and even many metabolic pathways has been developed significantly as growing a fundamental knowledge of the biochemistry of life. These newly revealed findings have incredibly influenced in recent bio-technologies, such as synthetic biology having massive potential to solve the missing connected dots. To have better understanding of microbe, microfluidic approaches were innovatively introduced to the field with the potential to revolutionize high-throughput biological assays. In this study, suggested approach to address limitations of conventional microbiology is microfluidics integrated with synthetic biology. At first, the microbial biosensors will be introduced into microfluidic ratchet platform for a quantitative analysis of microbial bio-signal. The microfluidic device using microfabricated arrowhead-shaped ratchet structures has an intrinsic function that concentrates motile microbes in a microchamber array. Additionally, the ratchet structure provides the concentrated microbes to grow better in a continuous-feed mode. A continuous exposure of detection analytes leads the amplification of fluorescence signal from microbes in a microchamber. Therefore, it was noted that the substantial amplification of bio-signal was achieved from the microfluidic device and measured signals were analyzed in quantitative manner. As a second practical application of the microfluidic approach, for high-throughput screening (HTS) application, a fluid array will be developed by using immiscible character between water and oil for microbial incubation, analysis, selective extraction, recovery process and the demonstration of practical applications. From the characterization of the fluid array platform, HTS will be demonstrated based on two different categories: reporter-gene basis and growth complementation basis. The fluid array device showed not only demonstrations of high-throughput screening, but also advanced screening applications were also demonstrated with higher mutant library screening with 106 and C2C communication screening system. Outstanding mutants were sophisticatedly screened among 106 of a mutant library based on the hybrid type screening method. Also, the proposed C2C screening approach has enabled high-throughput compartmentalization and resulted in 10 possible mutants showing higher extracellular biomolecule secretion performance. The proposed microfluidic approaches can be practically useful combinations showing many advantages: 1) economical and reduced time requirement for real application without complex instruments, 2) facile potentials to enable a multiplex quantitative analysis in a high-throughput manner, and 3) selective, direct and convenient measurement without pre- or post-treatment of sample solutions in near future the entire processes could be fully automated. In this dissertation, different type of microfluidic devices was developed for various collaborative purposes for the bottleneck of conventional microbiology. Therefore, the microfluidic devices have knocked a new door for high-throughput screening application for synthetically engineered microorganisms and quantitative approaches for microbial biosensors. Thus, the research contributions in this doctoral dissertation are the microfluidic approaches to popularize and overcome conventional constraints from biological experimental tools by integration of total analysis system for synthetically engineered microbe cases.ope