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University of California, San Diego UNIVERSITY OF CALIFORNIA, SAN DIEGO Application of Microfluidic Device for the Study of Bacteria A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Physics by Chih-Yu Yang Committee in charge: Professor Alex Groisman, Chair Professor Lin Chao Professor Terence Hwa Professor Gürol Süel Professor Massimo Vergassola 2018 Copyright Chih-Yu Yang, 2018 All rights reserved. The Dissertation of Chih-Yu Yang is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2018 iii TABLE OF CONTENTS Signature Page ............................................................................................................ iii Table of Contents ........................................................................................................ iv List of Figures ............................................................................................................. vi List of Tables .............................................................................................................. xii Acknowledgements ..................................................................................................... ix Vita .............................................................................................................................. xi Abstract of the Dissertation ........................................................................................ xii Chapter 1 Introduction ....................................................................................................... 1 Chapter 2 The Aggregation Temperature of E.coli Depends on the thermal gradients ..... 6 2.1 Background ........................................................................................................ 6 2.2 Materials and Methods .................................................................................... 15 2.2.1 Bacterial Culture ................................................................................... 16 2.2.2 Microfluidic Setup ................................................................................ 16 2.2.3 Temperature Control and Video Microscopy Setup ............................. 19 2.3 Results.............................................................................................................. 23 2.4 Discussion ........................................................................................................ 28 2.5 Supplemental Materials ................................................................................... 33 2.5.1 Carbon source in motility medium ....................................................... 33 2.5.2 Revision of device ................................................................................ 34 2.5.3 Concentration gradient caused by temperature gradient ...................... 34 2.5.4 Characterization of flow and cell distribution test ................................ 36 2.5.5 Supplementary Figures ......................................................................... 39 2.5.6 Supplementary Tables .......................................................................... 44 Acknowledgements ................................................................................................... 45 Chapter 3 The Effect of Flow and Peristaltic Mixing on Bacterial Growth in a Gut-like Channel ............................................................................................................................ 46 3.1 Background ...................................................................................................... 47 3.2 Results.............................................................................................................. 51 iv 3.2.1 Model and Analysis .............................................................................. 51 3.2.2 Experimental Setup ............................................................................... 54 3.2.3 Spatiotemporal Density Profiles in the Mini-gut .................................. 57 3.2.4 Effect of Spatial Coupling on Cross-feeding ........................................ 60 3.4 Discussion ........................................................................................................ 63 3.5 Supporting Information ................................................................................... 65 3.6 Supplementary Figures .................................................................................... 84 3.7 Supplementary Tables ..................................................................................... 96 Acknowledgments ..................................................................................................... 98 Chapter 4 Conclusion and Future Works ......................................................................... 99 Appendix A Porous Device Made of Polyethylene Glycol to Study Environmental Stress Response of Bacteria ....................................................................................................... 101 A.1 Background.................................................................................................... 101 A.2 Materials and Methods .................................................................................. 102 A.2.1 Multi-layer Device ............................................................................. 102 A.2.2 Experimental Setup ............................................................................ 103 A.2.3 Microscope and Imaging .................................................................... 104 A.3 Results and Future Work ............................................................................... 104 A.4 Acknowledgement ......................................................................................... 107 Bibliography ................................................................................................................... 108 v LIST OF FIGURES Figure 2.1: (a) Microfluidic device with magnified h-filter. (b) Micrograph of cell experiment near the entrance of gradient channel. (c) Micrograph of cell experiment at downstream ............................................................................. 15 Figure 2.2: Fluorescence measurement of HPTS/Tris ..................................................... 23 Figure 2.3: In shallow gradient (8 oC/mm), E.coli showed thermophilic behavior in 36-40 oC but cryophilic in 39-43 oC ......................................................................... 25 Figure 2.4: (a) E.coli distribution of relative frequency of occurrence with temperature in four temperature gradients. (b) A fitted linear relation between peak temperatures and temperature gradients was found within these four temperature gradients ..................................................................................... 28 Figure 2.S1: (a) Upstream of h-filter, with buffer flowing from left side and immotile cells flowing from right side. (b) Downstream of h-filter. Immotile cells all flowed toward outlet. (c) Upstream of h-filter, with buffer flowing from left side and motile cells flowing from right side. (d) Downstream of h-filter .... 39 Figure 2.S2: Cell distributions were measured at different position along the test channel, which represents different time intervals for them staying in temperature gradient. .................................................................................. 40 Figure 2.S3: (a)(b) Cell distribution versus position and temperature between temperature shifts in 40oC temperature range. (c)(d) Cell distribution versus position and temperature between temperature shifts in 50oC temperature range ............................................................................................................... 41 Figure 2.S4: Temperature gradient was shifted by 10oC ................................................. 42 Figure 2.S5: (a) Temperature profile under different time interval. (b) Molecular diffusion profile under different time interval ............................................... 42 Figure 2.S6: Distribution of beads across temperature gradient (34oC/mm), versus position between 50µm and 450µm ........................................................... 43 Figure 2.S7: Cell distribution versus temperature with 10 μM serine added ........... 43 Figure 3.1: Washout by flow and possible counteracting factors .................................... 49 Figure 3.2: Model predictions for bacterial densities when varying degree of mixing and flow ................................................................................................................ 54 Figure 3.3: Qualitative description of the behaviors of the reaction-diffusion model ..... 57 vi Figure 3.4: Effect of mixing and flow on bacterial growth. ............................................ 60 Figure 3.5: Two strain cross-feeding ecology ................................................................. 63 Figure 3.S1: Hydrodynamic simulations of bacteria growth in a channel with longitudinal flow and recirculation ................................................................ 84 Figure 3.S2: Phase diagrams in the v - D coordinates showing the dependence of washout conditions on the growth rate and channel length ........................... 85 Figure 3.S3: Convergence to steady state. Dynamics of reaching steady states ............. 86 Figure 3.S4: Mini-gut device. (A) Schematic of the mini-gut device
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