Characterizing biological systems: quantitative methods for synthetic genetic circuits in plants and intracellular mechanics

2018 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

Loading as a design parameter for genetic circuits

A significant problem when building complex biomolecular circuits is due to context-dependence: the dynamics of a system are altered upon changes to its context, potentially degrading the system's performance. Here, we study retroactivity, a specific type of context-dependence, by analyzing the effects of loads on a transcription factor applied by the transcription factor's target sites. In particular, we study this loading effect on the model of an activator-repressor oscillator, a widely studied motif in synthetic and systems biology. Our analysis indicates that strong activation and weak repression are key for a stable limit cycle. Repression can be effectively weakened by adding load to the repressor, while activation can be effectively weakened by adding load to the activator. Therefore, loading the repressor can be employed as a design parameter to establish a stable limit cycle. In contrast, loading the activator is deleterious to the clock

How retroactivity impacts the robustness of genetic networks

This paper studies how retroactivity impacts the robustness of gene transcription networks against parameter perturbations. By employing the linearization technique and the real stability radius, we provide comparisons of the robustness between gene transcription networks with retroactivity and ones without retroactivity. Both numerical and analytical results show that retroactivity tends to decrease such robustness. This finding in turn implies that modular genetic networks tend to be more robust against parameter perturbations.National Science Foundation (U.S.) (NSF-CCF-I058127

Nutrition, Hormones, Transcriptional Regulatory Networks and Division of Labor in Honey Bee Colonies

Phenotypic plasticity – one genotype producing alternative phenotypes – is increasingly understood to be an important force in phenotypic evolution, but its mechanistic basis remains poorly understood. This thesis describes research into the molecular mechanisms underlying age-related behavioral and physiological plasticity in worker honey bees. Many animals are able to alter their behavior and physiology in response to changes in the environment. At times, these changes in behavior and physiology are stable for long periods, a phenomenon known as phenotypic plasticity [1]. For instance, short periods of food deprivation stimulate feeding and the mobilization of stored nutrients to meet an individual’s immediate energetic needs. But prolonged food deprivation can also lead to much longer-term effects, causing individuals to enter extended periods of inactivity, alter their reproductive strategy, or lose their position in a dominance hierarchy. In humans, chronic food deprivation early in life may lead to a propensity toward obesity and diabetes in later life (for an expanded and fully-referenced discussion of nutritionally-mediated phenotypic plasticity see Chapter 4). The mechanisms that enable and constrain plasticity in behavior and physiology are not well understood, but it is clear that they often involve coordinated and long-lasting changes in gene expression, brain circuitry, brain chemistry, and endocrine signaling [2]. My doctoral research has focused on understanding the molecular basis for nutritionally- and hormonally-mediated plasticity in the behavior and physiology of worker honey bees. Honey bees are social insects, living together in colonies containing tens of thousands of individuals [3]. Colony life is organized by a complex and sophisticated division of labor. Each colony contains a single queen, who is specialized for reproduction and spends most of her time laying eggs. Males, called drones, are relatively rare, and their sole role is to mate. The vast majority of the individuals in the hive are sterile worker bees that are responsible for all of the other tasks performed by the colony. The tasks performed by worker bees are further divided up among individuals via a process of behavioral maturation that is the focus of this thesis. For the first 2-3 weeks of adult life, worker bees specialize on broodcare (“nursing”). They then switch for a few days to any of a number of more specialized tasks such as building honeycomb cells, storing food in honeycomb cells, or guarding the hive entrance against intruders. Finally, for the remaining 1-2 weeks of their life, worker bees forage outside the hive for nectar and pollen, the colony’s sole sources of food. The work presented in this thesis builds on previous findings demonstrating links between worker honey bee division of labor and nutrition (reviewed in Chapter 4). Behavioral maturation in worker bees is coupled to changes in nutritional physiology, including a dramatic and stable loss of abdominal lipid that occurs prior to the onset of foraging. Moreover, previous studies had demonstrated that nutritional status can have causal influences on the timing of behavioral maturation and manipulations of a few feeding- or nutritionally-related genes accelerates or delays the age at onset of foraging. In the work described here, I first test the hypothesis that worker bee behavioral maturation, a highly derived trait, is regulated, in part, by conserved nutritionally-related hormones (Chapter 1). I demonstrate that genes related to insulin signaling are differentially expressed in the brains and fat bodies of nurses and foragers. Furthermore, I show that manipulation of the insulin-related TOR pathway influences the age at which bees initiate foraging. These results suggest that the evolution of honey bee social behavior involved new roles for ancient nutritionally-related pathways. However, my subsequent work shows that not all nutritionally-related pathways have been coopted in the same way. I describe a more complex, and less resolved, relationship between behavioral state, nutrition and brain gene expression for a second nutritionally-related hormone, Neuropeptide Y (Chapter 2). Next, using transcriptomic experiments, I demonstrate that maturation, as well as age-related stable lipid loss, involve massive changes in gene expression in the fat bodies (Chapter 3). I show that these changes in gene expression involve age-related changes in the responsiveness of hormonally and metabolically related pathways to nutrition, and roles for two evolutionarily novel, non-dietary factors: the storage protein vitellogenin and Queen Mandibular Pheromone, each of which influenced many maturationally-related genes in the fat bodies. These results also suggest the involvement in the responses to all these factors of a single nutritionally-related hormone, juvenile hormone (JH), which had previously been shown to pace behavioral maturation. In Chapter 4, I review my findings from chapters 1-2 of this thesis, and previous studies, and propose a molecular systems biology approach to understanding division of labor. Specifically, I propose that phenotypic plasticity in worker honey bees involves nutritionally- and hormonally-driven changes in transcriptional regulatory networks in the fat bodies (as well in the brain), and I suggest methodologies for their elucidation. Finally, in Chapter 5, I utilize the molecular systems biology approach outlined in Chapter 4 to show that a transcriptional regulatory network in the fat bodies underlies division of labor. I show that a juvenile hormone-related transcription factor, Ultraspiracle (USP), influences the age at onset of foraging. I then use a combination of chromatin immunoprecipitation—genomic tiling microarrays, RNAi and deep mRNA sequencing to develop a model of the USP transcriptional regulatory network in fat cells. My results suggest that JH and USP function together to induce and maintain alternative states of a transcriptional regulatory network. These alternative states may well underlie the two basic phases of worker bee life, the in-hive and foraging phases. Together, the studies presented in this thesis provide insights into the relationship between nutrition, hormones, transcriptional regulation, and phenotypic plasticity. References 1. West-Eberhard, MJ. Developmental Plasticity and Evolution. 2003. Oxford University Press, New York, NY. 794 pp. 2. Robinson, GE, Fernald, RD, Clayton, DF. Genes and social behavior. Science. 2008 Nov 7; 322(5903):896-900. doi:10.1126/science.1159277 3. Winston, ML. The Biology of the Honey Bee. 1987. Harvard University Press, Cambridge, MA. 294 pp

Principles for the design of multicellular engineered living systems

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell–cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the “black box” of living cells

Exploring the intrinsic behaviour of multisite phosphorylation systems as part of signalling pathways

Multisite phosphorylation is a basic way of chemically encoding substrate function and a recurring feature of cell signalling pathways. A number of studies have explored information processing characteristics of multisite phosphorylation, through studies of the intrinsic kinetics. Many of these studies focus on the module in isolation. In this paper, we build a bridge to connect the behaviour of multisite modification in isolation to that as part of pathways. We study the effect of activation of the enzymes (which are basic ways in which the module may be regulated), as well the effects of the modified substrates being involved in further modifications or exiting reaction compartments. We find that these effects can induce multiple kinds of transitions, including to behaviour not seen intrinsically in the multisite modification module. We then build on these insights to investigate how these multisite modification systems can be tuned by enzyme activation to realize a range of information processing outcomes for the design of synthetic phosphorylation circuits. Connecting the complexity of multisite modification kinetics, with the pathways in which they are embedded, serves as a basis for teasing out many aspects of their interaction, providing insights of relevance in systems biology, synthetic biology/chemistry and chemical information processing

Strategies for designing, testing and demonstrating safety : what synthetic biology can learn from retrospective cases

Thesis (S.M. in Technology and Policy)--Massachusetts Institute of Technology, Engineering Systems Division, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 99-101).Synthetic biology is an emerging technology field within the realm of genetic engineering, differing from traditional genetic engineering in that it focuses on the modularization of genetic parts and the creation of de novo organisms. Significant concerns over safety have been expressed. This research explores traditional engineering and biotechnology practices for overarching principles of design, testing and demonstration that address safety concerns. The information is used to assess the current state of design, testing and demonstration in current synthetic biology projects addressing safety. Component and system design literature provide an engineering backbone of safety systems however, biological attributes such as mutation, growth, and multiplication create safety gaps, where biological engineering practices are needed. These principles are organized into categories of design and testing, and testing and demonstration to gain greater insight on where gaps in the literature might lie. Retrospective cases of traditional engineering and current cases of biotechnologies provide external validation and further illustrate which practices address which design, testing and demonstration needs. While most of the traditional engineering cases addressed safety through design and testing, when they were faced with questions of safety, they presented specific efforts to gain public confidence. The pro-biotics case was different in that the safety concerns came from the scientific community since history is being used as the convincing demonstration of safety. The three synthetic biology research projects cross the divide between traditional engineering and biotechnologies, but theses efforts are firmly in the area of design and testing. These efforts begin to show the tradeoff between implementing safety and faster technical results. Strategies for further research are explored.by Neelima Yeddanapudi.S.M.in Technology and Polic