ABSTRACT Title of Dissertation: BIOENGINEERED CONDUITS FOR DIRECTING DIGITIZED MOLECULAR-BASED INFORMATION Jessica L. Terrell, Doctor of Philosophy, 2015 Directed By: Professor William E. Bentley Fischell Department of Bioengineering Molecular recognition is a prevalent quality in natural biological environments: molecules- small as well as macro- enable dynamic response by instilling functionality and communicating information about the system. The accession and interpretation of this rich molecular information leads to context about the system. Moreover, molecular complexity, both in terms of chemical structure and diversity, permits information to be represented with high capacity. Thus, an opportunity exists to assign molecules as chemical portrayals of natural, non-natural, and even non-biological data. Further, their associated upstream, downstream, and regulatory pathways could be commandeered for the purpose of data processing and transmission. This thesis emphasizes molecules that serve as units of information, the processing of which elucidates context. The project first strategizes biocompatible assembly processes that integrate biological componentry in organized configurations for molecular transfer (e.g. from a cell to a receptor). Next, we have explored the use of DNA for its potential to store data in richer, digital forms. Binary data is embedded within a gene for storage inside a cell carrier and is selectively conveyed. Successively, a catalytic relay is developed to transduce similar data from sequence- based DNA storage to a delineated chemical cue that programs cellular phenotype. Finally, these cell populations are used as mobile information processing units that independently seek and collectively categorize the information, which is fed back as ‘binned’ output. Every development demonstrates a transduction process of molecular data that involves input acquisition, refinement, and output interpretation. Overall, by equipping biomimetic networks with molecular-driven performance, their interactions serve as conduits of information flow. BIOENGINEERED CONDUITS FOR DIRECTING DIGITIZED MOLECULAR-BASED INFORMATION By Jessica L. Terrell Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2015 Advisory Committee: Professor William E. Bentley, Chair Professor Gregory F. Payne Assistant Professor Christopher Jewell Associate Professor Edward Eisenstein Professor Herman O. Sintim, Dean’s Representative © Copyright by Jessica Terrell 2015 Dedication To my family and loved ones for their unconditional support and to my earliest mentors in scientific research, Mrs. M. Turpyn and Dr. D. Shipp. ii Acknowledgements This work would not have been possible without the instrumental exchange of ideas and recommendations by my colleagues, mentors, and committee members. The people with whom I have interacted closely in pursuit of this research willingly engaged in my work, providing inspiration and enthusiasm. I have benefited from the talented people of the Bentley lab for biomolecular and metabolic engineering and the interdisciplinary Biochip Collaborative, both past and present members. In particular, Dr. Hsuan-Chen Wu, Dr. Chen Yu Tsao, Dr. Varnika Roy, and Dr. Karen Carter were exceptional role models for me; they invested time in my instruction, guidance, and inclusion on their projects and collaborations from the beginning. I am deeply grateful for this support. My advisor, Dr. William Bentley, has provided tremendous mentorship and professional opportunities to improve my abilities as a scientist- especially to strategize projects, to recognize profound impacts, to explore concepts beyond their boundaries, and to establish vision. I have also been grateful for the mentorship of Dr. Gregory Payne, who has provided much guidance in the development of scientific skills, in particular, in experimental organization and scientific communication. This research has allowed me to pursue international collaboration- Dr. Sheref Mansy and his lab group, especially Dr. Roberta Lentini, graciously hosted me as a visiting scientist in Trento, Italy, where I learned new perspectives and techniques toward synthetic biology. Finally, the Fischell Department of Bioengineering at the University of Maryland has provided a rich environment- due to engaged faculty, administration, and students- to learn, grow, and to contribute to this interdisciplinary field. iii Table of Contents Dedication…………………………………………………………………………………….. i Acknowledgements…………………………………………………………………………… iii Table of Contents……………………………………………………………………………… iv List of Figures…………………………………………………………………………………. vii List of Tables………………………………………………………………………………….. viii Chapter 1: Introduction 1.1Motivation……..…………………………………………………………………………….……. 1 1.1.1 Theories of biological interactions for nano-machination and computation………. 1 1.1.2 Integratable componentry for harnessing biological interactions…………….…… 3 1.2Background ...……………………………………………………....……………………………... 6 1.2.1 Biofabrication……………………………..………………………………………………… 6 1.2.2 Physical information storage in biopolymers…….…………….………………………... 8 1.2.3 Molecular Automata for robust information processing in vitro……………….......... 10 1.2.4 Cell populations for categorizing molecular information………………………...……. 11 1.3 Dissertation outline…………………………………………………………………..……... 14 Chapter 2: Functionalization of biofabricated scaffolds for interactive biological networks 2.1 Introduction……………………………………………………………………………….. 16 2.2 Materials and Methods…………………………………………………………………… 20 2.3 Results and Discussion……………………………………………………………………. 24 2.3.1 Adjacent electroaddressability of chitosan and alginate films…………………… 24 2.3.2 Rapid Monitoring of mAb Production from NS0 cells………………………….. 26 2.3.2-1 Production address characterization and scalable assembly…………….. 26 2.3.2-2 Productivity Performance: Antibody production and release from alginate hydrogels………………………………..………………………………... 29 2.3.2-3 In-film bioprocessing utility: Incubation of entrapped NS0 cells and recovery ……….……………………………………………………….. 30 2.3.3 HG3T functionalized chitosan films as receptive surfaces for antibody capture.……….. 31 2.3.3-1 Capture address characterization by fluorescence analysis……………… 31 2.3.3-2 Receptive Performance: Antibody exchange across addresses ..………….. 33 2.3.3-3 Scalable relationship between addresses………………………………… 34 2.3.3-4 In-film bioprocessing utility: Semi-quantification of antibody titer……… 35 2.4 Concluding Remarks……………………………………………………………………... 36 Chapter 3: Dual use of DNA for embedded data storage within a functional genetic framework iv 3.1 Introduction……………………………………………………………………….………. 37 3.2 Materials and Methods………………………………………………………………….. 39 3.3 Results and Discussion……………………………………………………………….….. 44 3.3.1 Mapping of GATC mutagenesis targets on plasmids…………………………...…. 44 3.3.2 Investigation of ∆GATC impact within the origin of replication ………….……. 45 3.3.3 Investigation of antibiotic resistance due to ∆GATC-introduced rare codons within b-lactamase………………………………………………………………………. 48 3.3.4 Efficient assembly of GATC-free segments onto a single plasmid……………… 51 3.3.5 Investigation of gene expression due to ∆GATC-introduced amino acid substitutions in LacI.………….…………………………………………..………….. 52 3.3.6 ∆GATC accumulation in plasmid series toward pGATCfree………………….... 53 3.3.7 Embedding text characters as binary data in egfp gene ..……………………….. 55 3.3.8 Selective accession of embedded data by exploiting pGATCfree architecture….. 58 3.4 Conclusions …………………………………………………………………….………… 62 Chapter 4: Digitally-programmed information relay from DNA to a chemical signal 4.1 Introduction…………………………………………………………………….…………. 63 4.2 Materials and Methods…………………………………………………………………… 67 4.3 Results and Discussion…………………………………………………………………… 70 4.3.1 Dpn enzymes for tracking the state of DNA…………………………………….. 70 4.3.2 Methylation activity of EcoDam…………………………………………………. 72 4.3.3 Characterization and rate kinetics of methylation-coupled AI-2 synthesis……… 75 4.3.4 Bioluminescence as a quantifiable quorum sensing response by Vibrio harveyi to autoinducer-2…….…………………………………………………………………………… 78 4.3.5 GATC-dependency of AI-2 production during methyl-initiated synthesis ……... 79 4.3.6 Assembly of designer GATC-rich DNA blocks as gradated inscriptions………. 81 4.3.7 Inscription of plasmid DNA with GATC-enriched patterns.……………………. 83 4.3.8 Inscribed plasmid DNA for AI-2 delineation with post-read documentation..…. 85 4.3.9 Repetitive extension and pattern creation of GATC-rich templates………….…. 86 4.4 Concluding Remarks…………………………………………………………………….. 89 Chapter 5: Nano-guided cell networks as conveyors of molecular communication 5.1 Introduction…………………………………………………………………….………… 92 5.2 Materials and Methods…………………………………………………………………… 97 5.3 Results……...…………………………………………………………………….……………….. 101 5.3.1 Coexpression of surface-localized SBP and fluorescent marker protein……….. 101 5.3.2 Cell hybridization with magnetic nanoparticles through affinity interaction……. 104 5.3.3 Linking SBP-fluorophore expression to autoinducer-2 recognition…………….. 105 5.3.4 Establishing parallel ranges of cell interrogation for quorum sensing v information………………………………………………………………………. 106 5.3.5 Consensus feedback by combining biotic and abiotic processing……………….. 110 5.3.6 System response patterns delineated through the relationship between parallel populations……………………………………………………………………….. 114 5.4 Discussion…………………………………………………………………….……………