Enzyme Inhibition in Microfluidics for Re-engineering Bacterial Synthesis Pathways

Title of Thesis: ENZYME INHIBITION IN MICROFLUIDICS FOR RE-ENGINEERING BACTERIAL SYNTHESIS PATHWAYS Dean Larios Berlin, Masters of Science, 2009 Directed By: Professor Gary W. Rubloff Department of Materials Science and Engineering and the Institute for Systems Research Enzyme-functionalized biological microfluidic (EF-BioMEMS) systems are an emerging class of lab-on-chip devices that manipulate enzymatic pathways by localizing reaction sites in a microfluidic network. An EF-BioMEM system was fabricated to demonstrate biochemical enzyme inhibition. Further, design optimizations to the EFBioMEM system have been proposed which improve system sensitivity and performance. The pfs enzyme is part of the quorum-sensing pathway that ultimately produces the bacterial signaling molecule AI-2. An EF-BioMEM system was fabricated to investigate the pfs conversion activity in the presence of a transition state analogue inhibitor. A reduction in enzyme conversion was measured in microfluidics for increasing inhibitor concentration that was comparable to the response expected on a larger scale. This EF-BioMEMS testbed is capable of investigating other compounds that inhibit quorum sensing. Design improvements were demonstrates that improve overall system responsiveness by minimizing unintended reactions from non-specifically bound enzyme. EF-BioMEMS signal-to-background performance increased from 0.72 to 2.43. ENZYME INHIBITION IN MICROFLUIDICS FOR RE-ENGINEERING BACTERIAL SYNTHESIS PATHWAYS