Engineering Bacterial Populations Via Dna Messaging
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ENGINEERING BACTERIAL POPULATIONS VIA DNA MESSAGING ADISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOENGINEERING AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Monica Elise Ortiz June 2013 © 2013 by Monica Elise Ortiz. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/ht577gs4955 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Andrew Endy, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Daniel Fisher I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Hans Riedel-Kruse Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii iv Abstract Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. We observe cell-cell communication throughout every scale in na- ture, from simple single-celled bacteria to complex multicellular mammals. Engineers have begun to repurpose elements of natural communication systems to coordinate their own population-level behaviors, including oscillations and programmed pat- tern formation. Existing engineered systems, however, rely on small, system-specific biomolecules to send messages among cells. However, such molecules are capable of sending only a single message, typically “regulate transcription.” Thus, the informa- tion transmission capacity of such biological communication systems is fundamentally limited. Through this thesis, I demonstrated the decoupling of messages from a common communication channel via the autonomous transmission of numerous arbitrary ge- netic messages. To do so, I engineered a cell-cell communication platform using bacte- riophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. Further, I increased the range of engineered DNA messaging across semisolid media by coupling message transmis- sion or receipt to active cellular chemotaxis. Through this coupling, I demonstrated that our system is adaptable to di↵erent contexts by creating simple patterns. Fi- nally, using recombinase-mediated logic gates developed within the Endy laboratory, Idemonstratedtheabilitytoprogrambacteriabytransmittinglogicgatestosur- rounding cells. Overall, this work significantly enhances the suite of cell-cell communication tools available to engineers. I have shown that a variety of DNA messages can be trans- mitted among cells and have moved the field of synthetic biology closer to designing synthetic ecologies with more complex communication schemes and varied behaviors. v Acknowledgements This dissertation would not have been possible without the love and support of my family. To my parents, Brenda and Edgar Ortiz, for instilling in me a confidence and drive without which I would not be where I am today. To Nicole, my sister and first roommate, for being a force of nature and an inspiration. To my grandparents and extended family for always making me feel special, even when I was actually a brat. To Stephen, my partner and best friend, for his support over the better part of the last decade. To Olive and Sprout for their unconditional love and incessant purring. Iwouldnextliketothankmyadvisor,DrewEndy,foracceptingmeintohislab when I had no academic home, and for fostering an environment where good work is valued above all. Drew embodies the “dream it and achieve it” mentality better than anyone I know, and I am a better engineer for joining Drew’s group. I am grateful to Daniel Fisher and Ingmar Riedel-Kruse for serving on my disser- tation reading committee. I thank Daniel Fisher, who has provided hours of advice and wonderful conversation on a myriad of topics. I will particularly miss Daniel’s insight and booming voice. I thank Ingmar Riedel-Kruse, who was unselfish with his time and provided a number of interesting research avenues. I also thank Jan Skotheim, chair of my dissertation defense committee, and Christina Smolke, also a member of my dissertation defense committee, for their time and counsel. I thank past and present members of the Endy Lab—Sara Aguiton, Jerome Bon- net, Paul Jaschke, Xiaofan Jin, Linda Kahl, Lance Martin, Megan Palmer, Je↵Quinn, C´esar Rodriguez, Pablo Schyfter, Fran¸cois St.-Pierre, Pakpoom (Ton) Subsoontorn, Brent Townshend, Peter Yin—and Smolke Lab for not only providing guidance and discussion to help push forward this work, but also for preserving my sanity. The “En- dolke Lab” has been a wonderful place to work. I am sad to lose these co-workers, but glad to have gained many wonderful friends. Teaching has been an integral part of my graduate education. For this, I thank my past mentors—Jennifer Cochran, Christina Smolke, and Daniel Fisher. I also thank my fellow TAs—Aakash Basu, Ryan Bloom, and Kathy Wei. Finally, I thank Norbert Pelc, Isis Trenchard, Jennifer Lahti, and Hedi Razavi, all of whom I had the pleasure of working with as a TA coordinator. vi As a member of the fifth incoming Ph.D. class to Stanford Bioengineering, I have been fortunate to witness the transformation of an initially small department into a powerhouse of high-impact research and home to many talented students. I thank the faculty for the education I was provided and for their continuous support. I thank Olgalydia Urbano-Winegar profusely for being supportive, for always having her door open, and for tolerating my perpetually late forms. I also thank my tal- ented Bioengineering classmates, especially Amy Lam, Jayodita Sanghvi, and Tony Schindler. Finally, I felt fortunate to have a supportive group of friends spread around the country who were always just a phone call away. Thanks to Disha Shah, Julie Young, Elissa and Ben Cosgrove, Emily Schwartz, David Ritchie, D. Paul Golden, Kevin Lar- son, Amanda Cannata, Andrew Chow, Arup Chakrabarti, Michael Dini, and Gian Merlino for providing a constant stream of encouragement and entertaining voice- mails. vii Contents Abstract v Acknowledgements vi 1 Introduction 1 1.1 Anintroductiontocell-cellcommunication . 1 1.2 A Shannon framework . 4 1.3 Anoverviewofthisdissertation . 13 1.4 Collaborations . 15 1.4.1 Chapter 2 . 15 1.4.2 Chapter 3 . 15 1.4.3 Chapter 4 . 15 2 Achieving decoupled and scalable cell-cell communication via M13 16 2.1 Introduction . 16 2.2 Materials and Methods . 18 2.2.1 Strains and media . 18 2.2.2 Messaging phagemid and plasmid construction . 19 2.2.3 Preparation of sender and receiver cells . 22 2.2.4 Liquid-based experiments . 23 2.3 Results . 27 2.3.1 Requirements of M13-based message transmission . 27 2.3.2 Multiple messages can be sent through a single channel . 32 2.4 Discussion . 35 viii 3 Expanding phage-based communication to 2-D space 37 3.1 Introduction . 37 3.2 Materials and methods . 40 3.2.1 Di↵usion experiments . 40 3.2.2 Motility experiments . 41 3.3 Results . 45 3.3.1 Information channel capacities for AHL- and M13-based com- municationsystems. 45 3.3.2 Di↵usion analysis for AHL- and bacteriophage M13-based cell- cell communication systems . 49 3.3.3 E↵ective range of a DNA message transmission coupled to bac- terial chemotaxis . 51 3.4 Discussion . 57 4 Bacterial programming by transmission of DNA logic gates 60 4.1 Introduction . 62 4.2 Materials and Methods . 66 4.2.1 Molecular biology . 66 4.2.2 Site specific chromosomal integration . 67 4.2.3 Cell culture and gate operating conditions . 67 4.2.4 Transmission experiments . 68 4.3 Results . 72 4.3.1 Uniform populations can be programmed with di↵erent logic elements . 72 4.3.2 Intermediate states of logic elements can be transmitted . 76 4.3.3 Logic elements can be remotely activated . 76 4.4 Discussion . 78 5 Future Work and Final Remarks 81 5.1 Future work . 81 5.1.1 Improvements to the M13-based platform. 82 5.1.2 Next steps with respect to pattern formation . 85 ix 5.1.3 Firstapplications . 86 5.2 Conclusions . 88 A Vector maps and DNA sequences 90 A.1 VectormapsforChapters2and3 . 90 A.2 VectormapsandsequencesforChapter4. 92 A.2.1 pWSK29mod XOR........................ 92 A.2.2 pWSK29mod AND . 96 A.2.3 Othersequencesandaddedinformation. 99 Bibliography 100 x List of Figures 1.1 Existing cell-cell communication systems are limited by message-channel coupling .................................. 3 1.2 Generic communication systems have five components . 5 2.1 Biological communication systems can be represented formally . 18 2.2 Targeted and autonomous communication of arbitrary DNA messages via a reusable cell-cell communication channel . 28 2.3 Growth and fluorescence measurements of co-cultures under antibiotic selection . 30 2.4 M13-based cell-cell communication does not occur in absence of the F-plasmid.................................. 31 2.5 Additional DNA messages sent via M13-based cell-cell communication. 34 3.1 Channel capacity and transmission distances di↵er between AHL- and M13- based cell-cell communication systems. 48 3.2 Activation of beta-galactosidase activity in receiver cells via message particle di↵usion alone. 52 3.3 Viable M13 particles are produced during active chemotaxis . 53 3.4 Active chemotaxis enables DNA messaging across centimeter lengths 55 4.1 Using transcriptors to implement three-terminal Boolean integrase logic gates .