abstract: Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin. I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions. SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug. Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019

The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion-based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, and PelB) that operate via type I and II secretory machinery. GFP translocation was investigated in parallel due to its relative ease of monitoring to gather information about the functionality of signal peptide sequences. The translocation of phasin was investigated because of its physical binding interaction with the PHA granule surface. Genetic fusion of phasin with targeting signal peptides creates a PHA-phasin-signal peptide complex that can then be potentially used for cellular export. An important design aspect of this investigation is that synthetic biological engineering principles and standardized technical formats BBF RFC 10 and BBF RFC 23 were applied for more efficient construction of genetic devices. As an additional part of this study, an 1H NMR-based PHA quantification method was developed to facilitate analysis of intracellular PHAs. Overall, this study demonstrated that the BioBrick model can be used to construct functional devices that promote secretion of cellular compounds. The information gathered from this work can be further optimized and applied to more complex cellular manufacturing systems.

Exploring and understanding how the smallest scale features of a cell affect biochemical reactions has always been a challenge. Nanoscale fabrication advancements have allowed scientists to create small volume reaction containers that resemble the physical scale of cell membranes. Engineers seek to use biological design principles to manipulate information and import new functionality to such synthetic devices, which in turn, play a crucial role in allowing them to explore the effects of physical transport and extreme conditions of temperature and pH on reaction systems. Engineered reaction containers can be physically and chemically defined to control the flux of molecules of different sizes and charge. The design and testing of such a container is described here. It has a volume of 19 pL and has defined slits of 10-200 nm. The device successfully contains DNA and protein molecules and has been used to conduct and analyze enzyme reactions under different substrate concentrations and a continuous cell-free protein synthesis. The effect of DNA concentration and slit size on protein yield is also discussed. Glucose oxidase and horseradish peroxidase were loaded in the small volume container and fed with a solution containing glucose and Amplex Red™ to produce Resorufin. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Enzyme kinetics were characterized and compared with conventional scale results. Continuous cell free protein synthesis in arrays of nanoporous, picoliter volume containers has also been achieved. A multiscale fabrication process allows for the monolithic integration of the containers and an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the nanoporous containers continues beyond 24 hours and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. These picoliter, nanoporous containers provide new ways for quick determination of enzyme kinetics and continuous protein synthesis in microfluidic systems. They can be used in a wide variety of applications such as drug discovery, clinical diagnostics and high-throughput screening.

The growing incidences of coronary artery bypass graft surgeries have triggered a need to engineer a viable small diameter blood vessel substitute. An ideal tissue engineered vascular graft should mimic the microenvironment of a native blood vessel, while providing the adequate compliance post-implantation. Current vascular graft technologies lack the ability to promote vascular ECM deposition, leading to a compliance mismatch and ultimately, graft failure. Hence, in order to engineer suitable vascular grafts, this thesis describes the synthesis and characterization of novel elastin mimetic peptides, EM-19 and EM-23, capable of promoting vascular ECM deposition within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel. By combining the material properties of a synthetic and bio-inspired polymer, a suitable microenvironment for cell growth and ECM deposition can be engineered, leading to improved compliance. As such, characterization of EM-19 and EM-23 was conducted in human vascular smooth muscle cell (SMC) cultures, and the peptides self-assembled with a growing elastic matrix. After grafting the peptides onto the surface of PEG-DA hydrogels, EM-23 increased SMC adhesion by 6000% over PEG-RGDS hydrogels, which have been the gold standard of cell adhesive PEG scaffolds. Moreover, EM-23 grafted surfaces were able to promote elastin deposition that was comparable to tissue cultured polystyrene (TCPS) surface even though TCPS had roughly 4.5 times more SMCs adhered. Once translated to a 3D model, EM-23 also stimulated increased elastin deposition and improved the mechanical strength of the scaffold over time. Moreover, degradation studies suggested that EM-23 may serve as a template that not only promotes ECM deposition, but also allows ECM remodeling over time. The characterization studies in this thesis suggest that this peptide is an extremely promising candidate for improving vascular ECM deposition within a synthetic substrate, and that it may be beneficial to incorporate EM-23 within polymeric scaffolds to engineer compliant vascular grafts.

Two major roadblocks in synthetic biology are the difficulties associated with the physical assembly of gene regulatory networks (GRNs) and the lack of characterized biological parts. In this work we aimed to address both of these issues. We developed a novel method for the assembly of GRNs called Brick- Mason assembly. We have shown that the method can assemble a 6 part network in a single day and provides significant advancements over traditional cloning methods. We used BrickMason to assemble GRNs that would allow us to compare natural yeast mechanisms of repression to the steric hindrance based mechanisms that are commonly used in synthetic GRNs in yeast. Our results show that the two mechansisms of repression are not equivalent. This finding opens possibilities for using a new class of repressor in a synthetic context in yeast.

The field of Synthetic Biology seeks to develop engineering principles for biological systems. Modular biological parts are repurposed and recombined to develop new synthetic biological devices with novel functions. The proper functioning of these devices is dependent on the cellular context provided by the host organism, and the interaction of these devices with host systems. The field of Systems Biology seeks to measure and model the properties of biological phenomena at the network scale. We present the application of systems biology approaches to synthetic biology, with particular emphasis on understanding and remodeling metabolic networks. Chapter 2 demonstrates the use of a Flux Balance Analysis model of the Saccharomyces cerevisiae metabolic network to identify and construct strains of S. cerevisiae that produced increased amounts of formic acid. Chapter 3 describes the development of synthetic metabolic pathways in Escherichia coli for the production of hydrogen, and a directed evolution strategy for hydrogenase enzyme improvement. Chapter 4 introduces the use of metabolomic profiling to investigate the role of circadian regulation in the metabolic network of the photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942. Together, this work demonstrates the utility of network-scale approaches to understanding biological systems, and presents novel strategies for engineering metabolism.

The field of synthetic biology seeks to revolutionize the scope and scale of what is currently feasible by genetic engineering. By focusing on engineering general signal processing platforms using modular genetic parts and devices rather than `one-off' systems, synthetic biologists aim to enable plug-and-play genetic circuits readily adaptable to different contexts. For mammalian systems, the goal of synthetic biology is to create sophisticated research tools and gene therapies. While several isolated parts and devices exist for mammalian systems there are few signal processing platforms available. We addressed this need by creating a transcriptional regulatory framework using programmable zinc finger (ZF) and TALE transcription factors and a conceptual framework for logical T-cell receptor signaling. We first engineered a large set of ZF activator and repressor transcription factors and response promoters. ZFs are scalable elements as they can be engineered to bind to given DNA sequences. We demonstrated that we could ‘tune’ the activity of the ZF transcription factors by fusing them to protein homo-dimerization domains and by modifying their response promoters. We also created OR and NOR logic gates using hybrid promoters and AND and NAND logic gates by reconstituting split zinc finger activators and repressors with split inteins. Next, using a computational algorithm we designed a series of TALE transcriptional activators and repressors predicted to be orthogonal to all 2kb human promoter regions and thus minimally interfere with endogenous gene expression. TALEs can be designed to bind to even longer DNA sequences than ZFs, however off-target binding is predicted to occur. We tested our computationally designed TALEs in human cells demonstrating that they activated their intended target genes, but not their likely endogenous off-target genes, nor synthetic promoters with binding site mismatches. Finally, we created a conceptual framework for logical T-cell-mediated killing of target cells expressing combinations of surface antigens. The systems consist of conventional and novel chimeric antigen receptors (CARs) containing inhibitory or co-stimulatory cytoplasmic signaling domains. In co-incubation assays of engineered T-cells with target cells we demonstrated a functioning OR-Gate system and progress toward development of a functional NOT-Gate system using the CD300a and CD45 inhibitory receptor domains.

Kinetically controlled isothermal growth is fundamental to biological development, but it remains challenging to rationally design molecular systems that self-assemble isothermally into complex geometries via prescribed assembly and disassembly pathways. By exploiting the programmable chemistry of base pairing, sophisticated spatial and temporal control have both been demonstrated in DNA self-assembly, but largely as separate pursuits. This dissertation extends a new approach, called developmental self-assembly, that integrates temporal with spatial control by using a prescriptive molecular program to specify the kinetic pathways by which DNA molecules isothermally self-assemble into well-defined three-dimensional geometries. / Chemistry and Chemical Biology

Synthetic biology aims to engineer biological systems to meet new challenges and teach us more about natural biological systems. These pursuits range from the building of relatively simple transcriptional circuits, to engineering the metabolism of an organism, to reconstructing entire genomes. While we are still emerging from the foundational stages of this new field, we are already using engineered cells to discover underlying biological mechanisms, develop new therapeutics, and produce natural products. In this dissertation, we discuss the application of synthetic biology principles to the development of memory and pulse-detecting genetic circuits. In Chapter 2, we use novel transcriptional positive-feedback based memory devices integrated in human cells to study heterogeneous responses to cellular stresses. We built doxycycline, hypoxia, and DNA damage sensing versions of the device, demonstrating its modularity. In Chapter 3, we discuss further applications of the memory device in the study of long-term responses to hypoxia, gamma radiation, and inflammation. Finally, in Chapter 4 we describe work leading to the future construction of a pulse-detecting genetic circuit integrated in the E. coli genome. The work presented here illustrates the general applicability of synthetic biology in the study of biological phenomena and brings us one step closer to achieving a more exquisite understanding and control of natural systems.

To maintain high average fitness, populations must effect selection against the deleterious mutations that continuously arise de novo. Theoretical models of mutation-selection balance predict that the maximum tolerable mutation rate is much lower for organisms growing in colonies than for those in well-mixed liquid media due to drift imposed by competition for position along the growing colony front. Simplifying assumptions made in these models, including the irreversibility and fixed fitness cost of mutations, do not strictly hold in extant species. To explore the applicability of these models in natural contexts, we have constructed a yeast strain which undergoes recombinase-mediated irreversible gene excision at a single locus with tunable fitness cost, but also possesses the random genomic mutation profile characteristic of yeast. We find that several theoretical predictions hold for our strain, including the dependence of maximum tolerable mutation rate on growth condition and selective coefficient. These results constitute the first direct biological test of mutation-selection balance theory.