Fabrication, and Characterization of Chemotaxis and Quorum Sensing Al

115 Pages·2014·3.01 MB·English

Development of Bacteria-Based Bio-Hybrid Delivery Systems: Fabrication,
and Characterization of Chemotaxis and Quorum Sensing

Ali Akbar Sahari

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State
University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy
in
Biomedical Engineering

Chair: Bahareh Behkam
Lissett R. Bickford
Rafael V. Davalos
Birgit E. Scharf
Ann M. Stevens

September 2, 2014
Blacksburg, VA

Keywords: bio-hybrid systems, microfluidics, drug delivery, chemotaxis, quorum
sensing
Development of Bacteria-Based Bio-Hybrid Delivery Systems: Fabrication,
and Characterization of Chemotaxis and Quorum Sensing

Ali Akbar Sahari

Abstract
Bio-hybrid approaches have recently provided a possible solution to address the challenge of on-
board actuation, control and communication modules for micro/nanoscale cargo-carrying
vehicles by integrating live prokaryotic or eukaryotic cells with synthetic objects. More
specifically, because micro/nanoparticles are able to transport cargos efficiently and bacteria can
play the role of targeted and selective delivery agents, a hybrid of these two can advance the
current strategies for environmental monitoring, drug delivery and medical imaging. The main
goal of this dissertation was to fabricate, assemble, and characterize different components of a
mobile network of bacteria-based bio-hybrid systems for long-term applications in drug delivery
and biosensing. First, a new library of bacteria-enabled delivery systems was developed by
coupling live engineered bacteria with non-spherical particles and the transport of these bacteria-
based systems was investigated in the absence and presence of chemical cues using microfluidic
platforms. Next, a quorum-sensing (QS) based bacterial cell-cell communication network was
characterized in a high-throughput manner in order to understand the coordinated behavior of the
bacterial species ferrying the cargoes. Lastly, the QS behavior of a chemotactic population of the
bacterial species in response to the endogenously produced signaling molecules was studied. The
work presented in this dissertation lays the foundation for a well-characterized generation of
bacteria-assisted cargo delivery devices with enhanced transport properties and capable of

executing pre-programmed multi-agent coordinated tasks upon their arrival at the target site.
Dedication

I dedicate this dissertation to my parents, Sherafat and Esmaeil. I hope
this achievement has completed the dream that you had for me many
years ago.

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Acknowledgements
I would like to sincerely thank my advisor and mentor, Dr. Bahareh Behkam, for giving me the
opportunity to work in her lab, and the constant and relentless support of my Ph.D. work with her
insightful advice, patience, understanding and enthusiasm. The conversations we have had, both
work-related and unrelated, have been stimulating, challenging and most importantly, enjoyable.
I am also very grateful to my committee members; Dr. Birgit E. Scharf deserves special
recognition, her advice, encouragement, training and relentless support have greatly helped me
during my intellectual journey as a graduate student. Dr. Rafael Davalos, Dr. Ann Stevens and Dr.
Lissett Bickford deserve many thanks for their helpful discussions, support and encouragement
along the way.
On a more personal note, I would like to thank my very special friends, Dr. Omidreza Shoghli, Dr.
Behnam Bahrak, Arash Jahangiri, Sahar Ghanipoor, Yahya Hosseini and Dr. Nima Toosizadeh,
whom have helped me along the way and I consider more than friends. I am grateful for their
friendship and moral support throughout all the years in graduate school and away from home. I
also would like to thank my past and present colleagues at the MicroN BASE laboratory, especially
Aziz Traore, Brian Geuther, Chris Suh, Ahram Kim, Zhou Ye, Devon Headen, and Meghan Canter
for making the day to day life in the lab something to look forward to and for offering support and
intellectual input during the past five years. Special thanks also go to all members of Dr. Biswarup
Mukhopadhyay’s lab, especially Jason Rodriguez, for their help. I am also very grateful to Revathy
Ramachandran from Dr. Ann Stevens’ lab, for her helpful discussions and experimental protocols.
Virginia Tech was a wonderful place for me to complete my graduate studies. I have immensely
profited from this rich and safe environment to conduct my Ph.D. research. I would like to
specifically acknowledge the facilities provided by the Micro and Nanofabrication laboratory at
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Virginia Tech, Virginia Bioinformatics Institute and the funding sources provided by the
Department of Mechanical Engineering and the National Science Foundation, without which, this
research would not have been possible.

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Table of Contents
Abstract………………………………………………………………………………………….ii
Dedication…………………………………………………………………………………….…iii
Acknowledgements……………………………………………………………………………...iv
List of Figures…………………………………………………………………………………...ix
List of Tables………………………………………………………………...…………………xiv
Chapter 1: Introduction
1.1 Motivation and problem statement…………………………………..…..…………...….…....1
1.2 Background………………………………………………………………………….…….......4
1.2.1 Non-spherical polymeric particles for drug delivery applications……………………....4
1.2.2 Bacteria-based drug delivery systems for cancer treatment applications…………….....5
1.2.3 Cell-cell communication through diffusible signals…………………………………….7
1.3 Organization of the dissertation…………………………..……………….......………..…....10
Chapter 2: Effect of body shape on the motile behavior of bacteria-powered swimming
microrobots (BacteriaBots)
Abstract…………………………………………………………………………………………..13
2.1 Introduction……………………………...…………………….……………….……….…....14
2.2 Materials and methods…………………………………...………………………….…….....17
2.2.1 Fabrication of non-spherical particles………………………….………….…...……....17
2.2.2 Preparation of bacteria…………………….…………………………………..…….…19
2.2.3 BacteriaBot assembly………………..…………………..……………...…….………..20
2.2.4 Two-dimensional particle tracking……………………………….………….……….21
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2.2.5 Statistical analysis…………………………………………………....………………..22
2.3 Results and discussion………………………………………………......….………...……..23
2.3.1 Theoretical analysis…………………………………………………..………….…....24
2.3.2 Effect of body shape on directionality……..…………….…………….…….…..…....27
2.3.3 Effect of body shape on the number of attached bacteria and propulsion speed……...29
2.3.4 Effect on number of the attached bacteria on propulsion speed and directionality…...30
2.4 Conclusions………...……………………………………………………………………..….33
Chapter 3: Directed Transport of Bacteria-Based Bio-Hybrid Delivery Vehicles
Abstract…………………………………………………………………………………………..35
3.1 Introduction……..………...…………………………………………………….…………....36
3.2 Materials and methods…………………….……..…………………………………….….....38
3.2.1 BacteriaBot construction………………………………...………………….…….…....38
3.2.2 Microfluidic device fabrication and assembly………………...……………...……..…41
3.2.3 Bacteria/BacteriaBot chemotaxis assay..……………………..……………...….……..40
3.2.4 Data analysis……………………………………..……………………………….……44
3.3 Results and discussion…………………………………......………………………....……...45
3.4 Conclusions………………..…….….……………………………………………………….52
Appendix A………………………………….…………………………………………………..54
Chapter 4: Spatiotemporally High-Throughput Characterization of Bacterial/BacteriaBot
Quorum-Sensing Response………….…………………………………………………………56
Appendix B…………...……………………...…………………………………………………..67
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Chapter 5: Microfluidic-based characterization of quorum-sensing response in a
chemotactic sender-receiver network
Abstract…………………………………………………………………………………………..76
5.1 Introduction………………….………………………………………………….…………....77
5.2 Materials and methods…………………….…..……………………………………….….....77
5.2.1 Microfluidic assay…………………………..………...………………...…….……..…77
5.2.2 Bacteria and plasmids……………….....………..…..…………………….…….……..78
5.2.3 Data analysis…………………………..………………………………………….……79
3.3 Results and Discussion……………………….……….……………………………....……..80
3.4 Conclusions………………..……………………………………………………………...….83
Chapter 6: Conclusions and future directions
6.1 Concluding remarks………………….………………………………………….…………...84
6.2 Future Directions…….……………...………………………………………………….…....87
Bibliography…………………………………………………………………………………….89

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List of Figures
Figure 1.1 Bacteria carrying particles in the form of a bio-hybrid system (i.e. BacteriaBots); (a)
Serratia marcescens, (b) Escherichia coli, and (c) Salmonella Typhimurium carrying micron-sized
polystyrene particles…………...……………………………………………………………7
Figure 1.2 (a) The LuxIR network in Vibrio fischeri. AHL molecules are produced, released and
taken up by the bacterium. (b) Bioluminescence of Vibrio fischeri in the light organs of marine
animal……………………….……………………………………………………………………..9
Figure 2.1 Fabrication of non-spherical polystyrene (PS) particles. (a) Step-by-step fabrication
process for making non-spherical PS particles. Particles embedded in a PVA film are stretched in
one dimension, liquefied in an oil bath and then solidified at room temperature; (b) Scanning
electron microscopy (SEM) representative images of spherical and non-spherical PS particles.
Scale bars are 2 µm…………………………………………………...………………………….19
Figure 2.2 General steps in constructing BacteriaBots. a) A group of microparticles with different
shapes are selected for the body of the BacteriaBots. b) The surfaces of the particles are coated
with Poly- -lysine (PLL). c) Bacteria are attached on the surface of the microparticles via
L
controlled self-assembly………………………………………………...……………………….21
Figure 2.3 BacteriaBots with different body geometries and different numbers of attached bacteria.
Each individual representative image gives a snapshot of a fabricated geometry with one or more
bacteria attached on the surface. All scale bars are 2 µm…………..………………23
Figure 2.4 The geometric factors G for a prolate spheroid with major and minor axes lengths of 2a
and 2b as a function of aspect ratio……………………………………..…………………….26
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Figure 2.5 Experimental results indicating the motile behavior of BacteriaBots with different
geometries. a) The directionality values of non-spherical robots are higher than that of the spherical
ones (*, P<0.0001) and are dependent on the robot body aspect ratio. b) There is no statistically
significant difference between the average speeds the BacteriaBots with different body geometries
(α=0.0001)……………………………………………………………..………28
Figure 2.6 Representative trajectories of BacteriaBots. a) A prolate spheroid BacteriaBot is pushed
by a single bacterium. b) A bullet-shaped BacteriaBot is pushed by a single bacterium. c) In one
control experiment, the bullet-shaped particle does not displace significantly since there is no
bacterium attached on the surface. All scale bars are 2 µm……………………..……………30
Figure 2.7 (a) Speed, and b) directionality of the spherical BacteriaBots as a function of the number
of bacteria adhered on the surface of the microparticle…………………………..……..32
Figure 2.8 (a) Speed, and (b) directionality of the barrel-shaped BacteriaBots as a function of the
number of bacteria adhered on the surface of the microparticle…………………………………32
Figure 2.9 (a) Speed, and (b) directionality of the bullet-shaped BacteriaBots as a function of the
number of bacteria adhered on the surface of the microparticle…………………………..……..32
Figure 2.10 (a) Speed, and (b) directionality of the prolate spheroidal BacteriaBots (A.R. =
1.4±0.2) as a function of the number of bacteria adhered on the surface of the
microparticle………………………………………………………………………………….….33
Figure 2.11 (a) Speed, and (b) directionality of the prolate spheroidal BacteriaBots (A.R .=
2.0±0.1) as a function of the number of bacteria adhered on the surface of the microparticle….33
Figure 3.1 (a) Schematic of a spherical BacteriaBot; (b) Illustration of the BacteriaBot
components: GFP-expressing E. coli bacteria are decorated with a cell membrane specific antibody
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