Table Of ContentSingle-Particle Tracking of Proteins in Living Bacteria:
From Single Cells to a Mixed Community
by
Chanrith Siv
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Biophysics)
in the University of Michigan
2017
Doctoral Committee:
Assistant Professor Julie S. Biteen, Chair
Associate Professor Matthew R. Chapman
Professor Victor J. DiRita, Michigan State University
Professor Ari Gafni
Assistant Professor Sarah L. Veatch
Chanrith Siv
To Grandma and my parents, whose sacrifices gave me an opportunity to come to America to
have a chance at the American dream
ii
Acknowledgements
This graduate school journey has been one of the most challenging experiences I have
encountered thus far in my life. Though there were many hurdles to overcome, the rewards at the
end of it all definitely outweighed all of the late nights spent in lab doing experiments. This
academic pursuit was not something I would have ever imagined myself doing growing up, but I
am truly grateful for all of the opportunities that I have received throughout my academic career.
Everything that I have accomplished so far would not have been possible without the support of
some important people in my life. I would like to express my sincerest gratitude towards
everyone who have played a role in shaping my journey.
Finishing this dissertation would not have been possible if it wasn’t for my amazing
advisors, Professors Julie Biteen and Victor DiRita. To Julie, thank you for trusting and
believing in me to take on risky, scary challenges in my projects and in my career endeavors. I
would not have been able to receive the necessary tools to land this dream job if it wasn’t for you
supporting and allowing me to explore different, non-traditional avenues in graduate school. To
Vic, thank you for letting me be a part of your lab and teaching me how to be more confident in
science and in life. Thank you for the pep talks and thoughtful advice you have given me
throughout the years. You two have always been there—even in the midst of your busy
schedules—to reassure me that everything will be all right in the end. I am sorry that I will not be
pursuing an academic career in the future, but I promise to make you both proud in some other
ways. I will not let your many years of training me go to waste.
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Next, I would like to thank my committee members, Professors Sarah Veatch, Ari Gafni,
Kim Seed and Matt Chapman. Thank you for coming to all of the meetings and providing
wonderful feedback. Though you were all hard on me at times, I appreciate the constructive
feedback because it made me a better scientist in the end.
I am also indebted to the lab members who I have worked with during graduate school.
You guys have taught me so much about science and about perseverance. You guys have
provided me with wonderful work environments where I was allowed to be myself. I am
definitely going to miss such a positive work environment. Thank you for all of the fun we have
shared throughout the years; you guys definitely made work a lot easier. I especially want to
thank Yi who have helped me tremendously in the past five years in the lab. To the members of
the Seed and Chapman labs, I want to thank you all for welcoming me and allowing me to do
experiments in your lab space.
I would also like to thank my closest friends. I’m sorry that I am not listing specific
names of people, but I am sure you guys all know who you are. You guys have put up with my
rants about life and have always kept me in check. If it wasn’t for you guys listening and helping
me get through this journey, I probably would’ve had more meltdowns.
Last but not least, I want to thank my family. To Mom and Dad, thank you for your
unconditional love and support. It has been over 9 years since I first left home, but I have never
once felt far away from you guys. To my brother Sotheara, thank you for everything you have
done for me. Though I may be tough on you, just know that it is all coming from a very good
place in my heart. To my older brother Sothearak and his family, thanks for the enjoyable
memories for when I visited home. Though you all may not always understand everything that I
have gone through to get this PhD, you guys were always there for me.
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I am only where I am today because of the many people who have contributed to my success.
Chanrith Siv
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Table of Contents
Dedication………………………………………………………………………………………....ii
Acknowledgements…………………………………………………………………………..…..iii
List of Tables………………………………………………………………………………….....vii
List of Figures…………………………………………………………………………………...viii
List of Appendices………………………………………………………………………………..xi
Abstract…………………………………………………………………………………………..xii
Chapter 1: Introduction……………………………………………………………………………1
Chapter 2: Differences in Labeling, Expression Systems, and Hosts Produce Concealed
Subcellular Phenotypes…………………………………………………………………..36
Chapter 3: Two-Color Super-Resolution Imaging in Live Vibrio Cholerae to Probe TcpP/ToxR
Interactions in the ToxR Regulon…………………………………………………...…...76
Chapter 4: Toward In-vivo Imaging of the Gut Microbiome…………………………………...105
Chapter 5: Final Conclusions and Perspectives………………………………………………...132
Appendix…………………………………………………………………………………...…...148
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List of Tables
Table 1.1 Optical properties and oligomeric states of fluorophores used in this study…………..9
Table 2.1. Strains used in this study…………………………………………………………......42
Table 2.2 Statistics for all strains in different growth conditions………………………………..56
Table 2.3 Statistics for ‘slow’ and ‘fast’ TcpP-PAmCherry for endogenous and ectopic strains in
different growth conditions…………………………………………………………....…56
Table 2.4 Primers used for cloning tcpP-pamcherry………………………………………….....66
Table 2.5 Primers used for qRT-PCR analysis………………………………………………......68
Table 2.6 Raw data for qRT-PCR analysis with CT method……………………………...….68
Table 3.1 Using a 3-term diffusion model to fit to CPD…………………………………….......92
Table 4.1 Classification of resistant starches and food…………………………………………108
Table 4.2 Different methods to grow a B. theta/R. bromii biofilm……………………………..122
Table A.1 List of strains in CS strain box. ……………………………………………………..158
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List of Figures
Figure 1.1 Live Vibrio cholerae imaged using different microscopy techniques…………….......3
Figure 1.2 Model of the virulence cascade in V. cholerae……………………………………….15
Figure 2.1 The ToxR Regulon regulates gene expression of the major V. cholerae virulence
factors CTX and TCP through ToxT…………………………………………………….39
Figure 2.2 In vitro characterization of the O395 V. cholerae strains reveals differences in
transcription and expression levels………………………………………………………43
Figure 2.3 mRNA levels in the fusion strains relative to the wildtype strain……………………45
Figure 2.4 Transcript levels of toxT, tcpP, toxR, and aphB were determined for cultures………47
Figure 2.5 Single-molecule tracking of TcpP-PAmCherry in the endogenous and ectopic V.
cholerae strains reveals differences in dynamics………………………………………...49
Figure 2.6 Immunoblot with antibodies against TcpP and TcpA for the wt, endogenous and
ectopic V. cholerae strains……………………………………………………………….52
Figure 2.7 Diffusion coefficients and population weights of TcpP-PAmcherry as a function of pH
and temperatures…………………………………………………………………………54
Figure 2.8 Characterization of ectopically expressed TcpP-PAmCherry from a second plasmid
induced by IPTG…………………………………………………………………………58
Figure 2.9 Dynamics of plasmid-expressed TcpP-PAmCherry from a second IPTG-induced
plasmid…………………………………………………………………………………...59
viii
Figure 2.10 Fluorescence intensity of ectopically expressed TcpP-PAmCherry in V. cholerae and
in a heterologous host…………………………………………………………………....61
Figure 3.1 Virulence signaling cascade in V. cholerae and fluorescent labeling of TcpP and
ToxR……………………………………………………………………………………..79
Figure 3.2 Endogenous expressions of ToxR and TcpP protein fusions in V. cholerae…………82
Figure 3.3 Immunoblots of ToxR and of TcpP…………………………………………………..84
Figure 3.4 Immunoblot of toxT-regulated toxin coregulated pilus protein TcpA………………..85
Figure 3.5 Cholera toxin ELISA of the V. cholerae strains used in this study with and without
inducers……………………………………………………………………………..……87
Figure 3.6 Coomassie stain of cell lysates grown with and without inducers…………………...88
Figure 3.7 Imaging live V. cholerae cells with high resolution……………………………...…..89
Figure 3.8 Single-molecule protein tracking in live cells……………………………………......91
Figure 3.9 Cumulative probability distributions of ToxR-mCitrine and TcpP-PAmCherry
motions…………………………………………………………………………….…..92
Figure 3.10 Dual band pass filter for laser excitation utilizing a 488 nm and 561 nm lasers…..100
Figure 4.1 Model for starch catabolism by the B. thetaiotaomicron Sus…………………....…107
Figure 4.2 Imaging live anaerobic bacterial cells on a conventional benchtop microscope……111
Figure 4.3 Growth curves from B. theta, R. bromii, and B. theta/R. bromii co-culture………..113
Figure 4.4 Growth of co-cultures on a coverslip……………………………………………….114
Figure 4.5 Contamination of cultures grown in the anaerobic chamber………………………..115
Figure 4.6 Fluorescence detection of BtCreiLOV in E. coli………………………………………..116
Figure 4.7 Fluorescence excitation and emission spectra of purified CreiLOV and VafLOV from
Chlamydomonas reinhardtii and Vaucheria frigida………………………………………..118
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