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Fitzpatrick-Dissertation-2018 Copyright by Mignon Denise Fitzpatrick 2018 The Dissertation Committee for Mignon Denise Fitzpatrick Certifies that this is the approved version of the following dissertation: Investigating Sociomicrobiology by Integrating Micro 3D Printing with Quantitative Analytical Techniques Committee: Jason B. Shear, Supervisor Jennifer S. Brodbelt Sean T. Roberts Eric V. Anslyn David W. Hoffman Investigating Sociomicrobiology by Integrating Micro 3D Printing with Quantitative Analytical Techniques by Mignon Denise Fitzpatrick Dissertation Presented to the Faculty of the Graduate School of the University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December 2018 Dedication I would like to dedicate this dissertation to Ann Crane, who taught me French, patience, perseverance, and is still trying to hammer self-worth into my head. She is an epic human being, and an inspiration. Acknowledgements First, I would like to thank my advisor, Dr. Jason Shear, for allowing me to complete the work necessary to earn this Ph.D. in Chemistry. Secondly, I would like to thank my committee members, Drs. Jenny Brodbelt, Sean Roberts, Eric Anslyn and David Hoffman. Thank you to all the Shear lab members past and present, who have contributed directly to my success in this endeavor. In particular, I’d like to thank Drs. Eric Ritschdorff and Jodi Connell for their guidance, and Allison Myers for all her help and friendship, without which this dissertation would not have been able to be completed. Thank you also to Dr. Janine Elliott and Olja Simoska for their help with Project Beam a Laser through the Wall, and for all of Olja’s help with setting up the new lab optical tables. In addition, I’d like to thank Tim Hooper for his constant support and contribution to keeping our instrumentation running and our sanity intact. Thank you also to Dwight Romanovicz, Julie Hayes, and Anna Webb in the core facility for all their help and patience. I’d also like to thank Steven Moore for helping us keep the lab from falling apart and Cory Konieczny for doing the same, in addition to helping us complete the enormously challenging building move. Thank you to Betsy Hamblen, and my therapist Dr. Bradbury, for their kindness and support. Finally, thank you to all my friends and family, especially my parents who have been here though it all, Michael and Maureen Fitzpatrick. v Investigating Sociomicrobiology by Integrating Micro 3D Printing with Quantitative Analytical Techniques by Mignon Denise Fitzpatrick, Ph.D. The University of Texas at Austin, 2018 SUPERVISOR: Jason B. Shear Antibiotic resistant polymicrobial infections have become a source of great concern in recent years both in clinical settings as well as in basic and medical research. Incidence of resistance and increased virulence, which typically emerge within small, dense cellular ensembles on picoliter scales, is on the rise and scientists are just beginning to understand the complexity of these dangerous bacterial populations. To that end, the research in this dissertation has sought to analyze the complex social interactions of micro 3D (µ3D) printed bacterial colonies with a variety of analytical techniques. Through characterization of the µ3D printed hydrogels themselves, and by pairing this technology with fluorescence and confocal microscopy, electrochemical studies, and mass spectrometry, important insights regarding the sociomicrobiology of these bacterial communities emerge. The Shear lab has previously employed µ3D printing of bacterial aggregates to study microbial populations in environments that reproduce attributes associated with complex spatiotemporal in vivo conditions to a much greater extent than vi traditional culture techniques. Combining this technology with advanced imaging approaches has enabled a detailed investigation into properties of intra- and inter-species cooperation, including factors that influence antibiotic resistance and virulence. The goal of the work presented here is to integrate quantitative and qualitative analytical techniques with µ3D printing technology to enable novel approaches for studying interactions, both within and between small bacterial aggregates in complex microbial environments. This information will be vital in the next steps toward designing better and more efficient strategies for combatting complex pathogenic communities that exist within polymicrobial infection environments. vii Table of Contents List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1: Introduction .......................................................................................................1 Sociomicrobiology ......................................................................................................3 Human Pathogens and Pseudomonas aeruginosa ..........................................5 Unculturable Bacteria and Vibrio fischeri ......................................................6 Multiphoton Excitation and µ3D Printing ..................................................................7 Fluorescence and Confocal Microscopy ...................................................................12 Fluorescence Microscopy .............................................................................13 Laser Scanning Confocal Microscopy ..........................................................15 Electrochemistry of Cells..........................................................................................16 Mass Spectrometry ...................................................................................................18 Ultraviolet Photodissociation (UVPD)-MS ..................................................19 Desorption Electrospray Ionization (DESI)-MS...........................................20 Conclusion ................................................................................................................21 Chapter 2: Micro 3D Printing and Optical Table Setup ...................................................23 Introduction ...............................................................................................................23 Multiphoton Excitation and µ3D Printing ....................................................23 Reagents ....................................................................................................................25 Fabrication Conditions ..............................................................................................26 General Optical Table Setup and Components .........................................................26 Importance of Components to Hydrogel Material Fabrication .................................29 viii Conclusion ................................................................................................................40 Chapter 3: Designing a Platform for Studying Unculturables ..........................................42 Introduction ...............................................................................................................42 Unculturable Bacteria and Vibrio fischeri as a Model Organism .................42 Protein Conjugated Photosensitizers ............................................................43 Experimental Design and Methods ...........................................................................45 Reagents ........................................................................................................46 Bacterial Strains, Plasmids, and Culture Conditions ....................................47 Conjugation Protocol ....................................................................................47 Optimization of Fabrication Conditions .......................................................48 Results and Discussion .............................................................................................51 Successful Culturing of Vibrio fischeri ........................................................51 Conjugation Troubleshooting .......................................................................53 Proposed Future Studies ...........................................................................................58 Conclusion ................................................................................................................59 Chapter 4: Characterizing Materials for Micro 3D Printing .............................................60 Introduction ...............................................................................................................60 Melting Temperature of Hydrogel Precursor Solutions ...............................60 Porosity of Hydrogels ...................................................................................60 Design and Methods .................................................................................................61 Melting Temperature Determinations ...........................................................61 Reagents ...............................................................................................61 Experimental Setup ..............................................................................62 ix Porosity Studies ............................................................................................64 Reagents ...............................................................................................65
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