Multicellular circuits control the development of multicellular organisms, through programming processes such as cell proliferation, cell differentiation, cell movement, and cell signaling. A fundamental goal of biology is to understand the design principles of these multicellular circuits, and use these principles to design synthetic multicellular systems for therapeutic purposes. Top-down approaches, for example analyzing embryos bearing genetic mutations, have identified key genes in many multicellular circuits, but are challenging to study these circuits in an isolated context and in a quantitative and systematic manner. An alternative, complementary approach is to engineer or reconstitute multicellular circuits from bottom-up, which allows us to overcome the limitations of top-down approach and gain quantitative insights into multicellular circuit design. In this thesis, we use this bottom-up approach to explore the design principles of two multicellular circuits. In the first project, we took inspiration from two prevalent features from natural multistable circuits, namely competitive protein-protein interactions and positive autoregulation, to design a synthetic multistable circuit architecture called MultiFate. Both in the model and in the experiment, MultiFate circuits generate multiple cellular states, each stable for weeks, allow control over state-switching and state stability, and can be easily expanded to generate more states. In the second project, we use a gradient reconstitution system to systematically analyze a gradient modulation circuit consisting of BMP4 and its modulators, Chordin, Twsg and BMP-1. We found that the circuit can give rise to diverse gradient modulation capabilities. In particular, the full circuit is sufficient for active ligand shuttling and generation of non-monotonic displaced gradient. These multicellular circuits could provide a foundation for engineering synthetic multicellular systems in mammalian cells.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/w0q1-7s17, author = {Ma, Yitong}, title = {Multicellular Synthetic Biology in Mammalian Systems}, school = {California Institute of Technology}, year = {2023}, doi = {10.7907/w0q1-7s17}, url = {https://resolver.caltech.edu/CaltechTHESIS:04132023-015900885}, abstract = {In multicellular organisms, different types of cells use intercellular signals to communicate and regulate population dynamics, and further coordinate complex behaviors. This presents a rarely tapped into potential for mammalian synthetic biology, which was largely restricted to engineering a single cell type in the past to mimic and use similar multicellular designs to achieve more functionalities. However, with current synthetic biology tools and designs, there are several major challenges to achieve a multicellular circuit. Challenges include precise and tunable control over cell type switching, having an orthogonal cell-cell communication signal, and robust control of cell populations.
To address these challenges, this thesis presents a system for tunable regulating of gene expression with DNA methylation, an auxin-based module for mammalian cell-cell communication, and a robust circuit for population control in mammalian cells. I further applied these work to engineering immune cells to show the potential of multicellular circuits in immunotherapies. Together, these works demonstrated the possibility of constructing multicellular circuits in mammalian systems, and that multicellular circuit can further extend the scope of synthetic biology to achieve more complex functions.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/a4m3-m603, author = {Edmonds, KeHuan Kuo}, title = {Imaging Cell Lineage with a Synthetic Digital Recording System}, school = {California Institute of Technology}, year = {2022}, doi = {10.7907/a4m3-m603}, url = {https://resolver.caltech.edu/CaltechTHESIS:11272021-193744399}, abstract = {In multicellular organisms, the lineage history and spatial organization of cells both play pivotal roles in cell fate determination during development, homeostasis, and disease. Investigating lineage relationships alongside cell state and space would provide a fundamental understanding of these biological processes. Current lineage tracking approaches rely on the progressive accumulation of either naturally-occurring somatic mutations or experimentally introduced markers. In most cases, these marks are then read out by sequencing, discarding the spatial information of the cells. To address this vital gap in our toolkit, we developed a new synthetic lineage tracking system that allows us to image single-cell lineage history. This system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), uses serine integrases to stochastically and irreversibly edit a synthetic memory array, generating up to 59,049 different outcomes that can be unambiguously distinguished by fluorescence in situ hybridization (FISH). We evaluated the reconstruction accuracy of our system in mouse embryonic stem (mES) cells and disentangled the relative contribution of lineage and space to cell fate determination in Drosophila brain development, establishing the foundation for an expandable synthetic microscopy-readable system. In this thesis, Chapter 1 introduces the importance of cell lineage and spatial organization to cell fate determination, and includes a brief history of the existing technologies of the lineage tracking field. Chapter 2 describes our characterization and demonstration of the intMEMOIR system. Finally, Chapter 3 discusses design principles for robust, serine-integrase-based recording systems and suggests future directions for intMEMOIR.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/z7dv-m192, author = {Su, Christina Janet}, title = {Principles of Addressing Specificity in Promiscuous Ligand-Receptor Systems}, school = {California Institute of Technology}, year = {2022}, doi = {10.7907/z7dv-m192}, url = {https://resolver.caltech.edu/CaltechTHESIS:06022022-032024376}, abstract = {In multicellular organisms, a relatively small number of highly conserved signaling pathways are used to enable intercellular communication. While the underlying molecular components and interactions are increasingly well understood, a fundamental mystery is how the diverse cell types of the body can be so precisely coordinated by so few pathways. It has long been known that different cell types exhibit varied responses to molecular signals, and it is unclear how this cell type specificity arises. In this work, we take a different perspective on this question and explore how cell type specificity can be generated at the level of intracellular signal. We refer to this ability to selectively activate different cell types as “addressing.” By eliminating the complexity of considering downstream pathway effectors, we are able to more comprehensively understand how cell type specificity can arise in spite of—or because of—promiscuity in ligand-receptor interactions. We focus on the bone morphogenetic protein (BMP) pathway as an ideal example. This pathway is essential in development, is of therapeutic interest in an array of pathologies, and has proven amenable to theoretical and experimental analysis. We first describe a minimal model of the pathway and identify what types of response functions can be achieved. We show that each layer of computation, from the formation of signaling complexes to the activation of downstream second messenger, can provide nontrivial integrations of ligand inputs. We then extend this analysis to systems with multiple cell types that may vary in receptor expression profile. The diverse response functions of this pathway enable systems in which different cell types or sets of cell types may be addressed with high specificity. In particular, the BMP pathway can address multiple cell types with high capacity, flexibility, and robustness. Taken together, these results provide a framework for understanding how molecular promiscuity in signaling pathways can, in fact, enable cellular specificity in pathway responses.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/3vv4-bk06, author = {Klumpe, Heidi Elizabeth}, title = {Context-Dependent, Combinatorial Logic of BMP Signaling}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/3vv4-bk06}, url = {https://resolver.caltech.edu/CaltechTHESIS:05312021-221223611}, abstract = {Evolution generated diverse signaling proteins for the control of multicellular patterns and organ- isms. These include the proteins of the Bone Morphogenetic Protein (BMP) pathway. Nearly a dozen BMPs activate the BMP pathway to promote the formation of tissues as diverse as bone, cartilage, blood vessels, and the kidney, making them attractive therapeutics for regenerating those tissues in adults. During development, the response to a given BMP depends heavily on context, such as which other BMPs are present and which BMP receptors are expressed on the cell being ac- tivated. However, despite knowing that context matters, the overall logic of this context-dependent signal processing, including the roles of specific ligands and receptors in shaping context and how this logic arises from biochemical features of specific pathway components, remains unclear. Inspired by maps of gene epistasis and drug interactions that functionally classify members of complex biological systems, we comprehensively measured responses to all pairs of ten BMP homodimers (BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP10, GDF5, GDF6, and GDF7), combining robotic liquid handling with a high-throughput fluorescent reporter of pathway activa- tion. These data functionally classify ligands into “equivalence groups,” or ligands that combine in the same way with all other ligands across combinations. Surprisingly, the functional groupings do not correlate with similarity of ligand sequence and can change with cell context. Together, the context-dependent equivalence groups summarize the diverse responses to combinations of BMP ligands and their dependence on specific BMP receptors. The experimentally observed pairwise responses are also consistent with a mathematical model where BMP ligands compete for limited BMP receptors with different affinities and then produce outputs with different ligand-specific activ- ities. Ultimately, these results provide a useful reference for explaining the unique effects of BMP combinations in different tissues or time points in development, as well as highlighting counter- intuitive mechanisms for this complex signal processing. Chapter 1 provides an introduction to how and why we study cell-cell signaling. Chapter 2 provides a summary of the determination of equivalence groups, their dependence on receptor context, and fitting the mathematical model of receptor competition. Chapter 3 provides suggestions for future work, including recommendations for improved model fitting as well as crucial extensions to the definitions of BMP “combinations” and “context” to deepen our understanding and control of this critical pathway.}, address = {1200 East California Boulevard, Pasadena, California 91125}, } @phdthesis{10.7907/jdhe-by95, author = {Chong, Lucy Shin}, title = {Engineering and Delivery of Programmable Protein Circuits as Potential Therapeutic Devices}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/jdhe-by95}, url = {https://resolver.caltech.edu/CaltechTHESIS:06072021-211522720}, abstract = {Cell-specific targeting of therapeutics is a fundamental challenge in biomedicine. The use of engineered proteins that interact with one another as designed, synthetic circuits represents a promising solution to this challenge. These circuits can be constructed to directly sense endogenous cell signals, act on these signals to classify cellular state, and produce a specific response such as conditional triggering of cell death or targeted expression of a reporter. Synthetic protein circuits can also be delivered in mRNA vectors transiently to avoid permanent gene modification.
We recently showed viral proteases can be engineered to regulate one another in a composable manner, permitting the construction of diverse protein-level circuits (Circuits of Hacked Orthogonal Modular Proteases). CHOMP could perform a wide range of computations including Boolean logic, analogue signal processing, and dynamic signal processing. Using this system we were also able to directly sense key cellular pathways and conditionally respond to trigger apoptosis in cancer-like cells. Further expansion of synthetic protein circuits to include nonlinear signal processing enables new system-level behaviors.
Protein-based circuits are compatible with innovative delivery methods including mRNA encapsulated in lipid-nanoparticle formulations and engineered viruses. As a proof of principle, we were able to develop a controllable, transient RNA-virus delivery system that allowed for targeted delivery to defined cell populations. This paradigm requires control over multiple aspects of the viral delivery system, including (1) production and release of viral particles, (2) target cell entry based on cell-surface proteins, (3) replication within the cell depending on intracellular proteins, and (4) drug-dependent elimination of the virus. Here, we integrate each of these distinct levels of control can into a single system based on the well-characterized negative stranded RNA virus. This RNA-virus platform will enable synthetic protein circuit delivery.
Combining viral engineering and protein circuit construction, the work described here suggests a roadmap towards “smarter” circuit-based therapies that can integrate multiple cues to maximize therapeutic specificity and establishes a role for post-translational circuits as future therapeutic devices.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/CT0B-Q853, author = {Park, Jin}, title = {Circuits of Dynamically Interacting Sigma Factors in Single Cells}, school = {California Institute of Technology}, year = {2018}, doi = {10.7907/CT0B-Q853}, url = {https://resolver.caltech.edu/CaltechTHESIS:12312017-234413682}, abstract = {How do cells integrate multiple, dynamic genetic circuits? I study this question in the context of the alternative sigma factors of B. subtilis.
The first project proposes a novel mode of gene regulation called timesharing. The key idea is that a limited resource is shared dynamically in time. Here we show that the alternative sigma factors of B. subtilis use dynamic sharing to share a limited supply of core RNA Polymerase (RNAP). We show that 5 alternative sigma factors activate in pulses, and that these pulses operate in a competitive regime. Interestingly, we found that pairwise correlations between these sigma factors contained a mixture of positive and negative correlations, whereas one may naively expect all correlations to be negative. We show with a mathematical model that competitive pulsing can lead to non-intuitive sets of mixed correlations.
The second project take a closer, quantitative look at sigma factor competition. Although competition between the housekeeping sigma and a single alternative sigma has been well studied, competition between alternative sigmas themselves has been relatively unexplored. To address this issue, we systematically investigated the pairwise competitive relationships between 7 alternative sigma factors in B. subtilis. The main experimental tool was a 7x7 ‘deletion’ matrix of strains, where every matrix strain was deleted for one sigma, and reported on another sigma via a fluorescent reporter. The deletion matrix revealed that competition is highly asymmetric. Deletion of any given sigma factor increased σW activity, but did not affect other sigma factors. These results are recreated by a minimal mathematical model of sigma factor competition, where importantly σW is relatively high in abundance but weak in affinity for core RNAP. We used the model to predict how overexpressing sigma factors affect each other, and these predictions were matched by experiments.
The third project reports a novel activator for alternative sigma factors. Alternative sigmas factors are activated by many forms of stress, such as nutrient limitation, temperature shifts, and molecular stresses like antibiotics. Here we show that surprisingly, cell lysis causes adjacent cells to specifically activate σX. This cell lysis-σX response is a general phenomenon, as it is observed under multiple experimental conditions. We show this relationship between cell death and σX is causal, since harvested cell extract activates σX. Finally, we hypothesize that cell death and σX play an important role in biofilm wrinkle formation.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/Z98050TB, author = {Nandagopal, Nagarajan}, title = {New Capabilities of the Notch Signaling Pathway}, school = {California Institute of Technology}, year = {2018}, doi = {10.7907/Z98050TB}, url = {https://resolver.caltech.edu/CaltechTHESIS:12282017-140713708}, abstract = {Animal cells use a conserved repertoire of signaling pathways to exchange information during and after development. The constituent molecules of these pathways and their individual interactions are now well-characterized. However, it is becoming clear that pathways often possess unexpected signal-processing capabilities, which are typically collective, systems-level, features. Recent work shows that these capabilities are best investigated using quantitative, single-cell, dynamic analyses of pathway behavior. Here, we used this approach to study Notch signaling pathway, which is widely utilized for juxtacrine signaling during the development and maintenance of most tissues. Our work reveals two new capabilities of this pathway. First, the receptor Notch1 is capable of discriminating between two similar ligands, Dll1 and Dll4, and can use this ability to enact ligand-specific developmental programs. To enable this, the pathway encodes ligand identity in the dynamics of Notch1 signaling, and later decodes it for controlling gene expression. We show that dynamic encoding by Dll1 and Dll4 results from different requirements for ligand-receptor clustering during activation. Second, the pathway is capable of cell-autonomous signaling (cis-activation). This mode of signaling is general to multiple ligand-receptor combinations, and possesses many attributes of intercellular signaling. We show that cis-activation occurs in natural stem-cell contexts, where it could be important for self-renewal and prevents premature differentiation. These new capabilities of this central signaling pathway have implications for understanding the role of Notch in development and homeostasis, diagnosing and treating its misregulation in disease, and controlling it for tissue engineering and regeneration.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/Z9ZC80VX, author = {Yong, John}, title = {Dynamics and Heterogeneity of Gene Expression and Epigenetic Regulation at the Single-Cell Level}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z9ZC80VX}, url = {https://resolver.caltech.edu/CaltechTHESIS:04292015-153123844}, abstract = {The ability of cells to establish and remember their gene expression states is a cornerstone of multicellular life. This thesis explores how gene expression states are regulated dynamically, and how these regulations differ in individual cells even under the same conditions. These properties underlie cellular state decisions and often determine the balance between different cell types in a multicellular system, but are typically inaccessible to conventional techniques that rely on static snapshots and population averaging. We address these issues in two separate contexts, one natural and one synthetic, using time-lapse imaging and other single-cell techniques.
In the first context, we use embryonic stem cells (ES), which were shown to exist in a mixed population of at least two cellular states with distinct differentiation propensities, as a model to study natural dynamics of cellular states. These cells display rare, stochastic, and spontaneous transitions between the two states, as well as more frequent fluctuations in gene expression levels within each state. Our system enables us to further investigate how these dynamics are modulated under a cell signaling environment that enhances pluripotency, and the role DNA methylation plays in maintaining these states.
In the second context, we investigate how chromatin regulators (CRs), part of a complex system that enables cells to modulate gene expression and epigenetic memory, operate dynamically in individual cells. We build a synthetic platform to measure the isolated effect of recruitment and de-recruitment of four individual CRs. In contrast to conventional transcription factor control, all CRs tested regulate gene expression in all-or-none events, controlling the probability of stochastic transitions between fully active and silent states rather than the strength of gene expression. The qualitative and quantitative responses of a cell population are determined by the set of event rates associated with each CR, as well as the duration of CR recruitment. These results provide a framework for understanding and engineering chromatin-based cellular states and their dynamics.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/Z9ST7MS1, author = {Singer, Zakary Sean}, title = {Metastability and Dynamics of Stem Cells: From Direct Observations to Inference at the Single Cell Level}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z9ST7MS1}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262015-152206802}, abstract = {Organismal development, homeostasis, and pathology are rooted in inherently probabilistic events. From gene expression to cellular differentiation, rates and likelihoods shape the form and function of biology. Processes ranging from growth to cancer homeostasis to reprogramming of stem cells all require transitions between distinct phenotypic states, and these occur at defined rates. Therefore, measuring the fidelity and dynamics with which such transitions occur is central to understanding natural biological phenomena and is critical for therapeutic interventions.
While these processes may produce robust population-level behaviors, decisions are made by individual cells. In certain circumstances, these minuscule computing units effectively roll dice to determine their fate. And while the ‘omics’ era has provided vast amounts of data on what these populations are doing en masse, the behaviors of the underlying units of these processes get washed out in averages.
Therefore, in order to understand the behavior of a sample of cells, it is critical to reveal how its underlying components, or mixture of cells in distinct states, each contribute to the overall phenotype. As such, we must first define what states exist in the population, determine what controls the stability of these states, and measure in high dimensionality the dynamics with which these cells transition between states.
To address a specific example of this general problem, we investigate the heterogeneity and dynamics of mouse embryonic stem cells (mESCs). While a number of reports have identified particular genes in ES cells that switch between ‘high’ and ‘low’ metastable expression states in culture, it remains unclear how levels of many of these regulators combine to form states in transcriptional space. Using a method called single molecule mRNA fluorescent in situ hybridization (smFISH), we quantitatively measure and fit distributions of core pluripotency regulators in single cells, identifying a wide range of variabilities between genes, but each explained by a simple model of bursty transcription. From this data, we also observed that strongly bimodal genes appear to be co-expressed, effectively limiting the occupancy of transcriptional space to two primary states across genes studied here. However, these states also appear punctuated by the conditional expression of the most highly variable genes, potentially defining smaller substates of pluripotency.
Having defined the transcriptional states, we next asked what might control their stability or persistence. Surprisingly, we found that DNA methylation, a mark normally associated with irreversible developmental progression, was itself differentially regulated between these two primary states. Furthermore, both acute or chronic inhibition of DNA methyltransferase activity led to reduced heterogeneity among the population, suggesting that metastability can be modulated by this strong epigenetic mark.
Finally, because understanding the dynamics of state transitions is fundamental to a variety of biological problems, we sought to develop a high-throughput method for the identification of cellular trajectories without the need for cell-line engineering. We achieved this by combining cell-lineage information gathered from time-lapse microscopy with endpoint smFISH for measurements of final expression states. Applying a simple mathematical framework to these lineage-tree associated expression states enables the inference of dynamic transitions. We apply our novel approach in order to infer temporal sequences of events, quantitative switching rates, and network topology among a set of ESC states.
Taken together, we identify distinct expression states in ES cells, gain fundamental insight into how a strong epigenetic modifier enforces the stability of these states, and develop and apply a new method for the identification of cellular trajectories using scalable in situ readouts of cellular state.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/MM53-XC23, author = {Tan, Frederick Eng How}, title = {Brfl Post-Transcriptionally Regulates Pluripotency and Differentiation Responses Downstream of Erk MAP Kinase}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/MM53-XC23}, url = {https://resolver.caltech.edu/CaltechTHESIS:05292014-234047875}, abstract = {FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/RGPT-RS80, author = {Lakhanpal, Amit}, title = {Experimental and Theoretical Studies of Notch Signaling-Mediated Spatial Pattern}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/RGPT-RS80}, url = {https://resolver.caltech.edu/CaltechTHESIS:10312013-102814054}, abstract = {Notch signaling acts in many diverse developmental spatial patterning processes. To better understand why this particular pathway is employed where it is and how downstream feedbacks interact with the signaling system to drive patterning, we have pursued three aims: (i) to quantitatively measure the Notch system’s signal input/output (I/O) relationship in cell culture, (ii) to use the quantitative I/O relationship to computationally predict patterning outcomes of downstream feedbacks, and (iii) to reconstitute a Notch-mediated lateral induction feedback (in which Notch signaling upregulates the expression of Delta) in cell culture. The quantitative Notch I/O relationship revealed that in addition to the trans-activation between Notch and Delta on neighboring cells there is also a strong, mutual cis-inactivation between Notch and Delta on the same cell. This feature tends to amplify small differences between cells. Incorporating our improved understanding of the signaling system into simulations of different types of downstream feedbacks and boundary conditions lent us several insights into their function. The Notch system converts a shallow gradient of Delta expression into a sharp band of Notch signaling without any sort of feedback at all, in a system motivated by the Drosophila wing vein. It also improves the robustness of lateral inhibition patterning, where signal downregulates ligand expression, by removing the requirement for explicit cooperativity in the feedback and permitting an exceptionally simple mechanism for the pattern. When coupled to a downstream lateral induction feedback, the Notch system supports the propagation of a signaling front across a tissue to convert a large area from one state to another with only a local source of initial stimulation. It is also capable of converting a slowly-varying gradient in parameters into a sharp delineation between high- and low-ligand populations of cells, a pattern reminiscent of smooth muscle specification around artery walls. Finally, by implementing a version of the lateral induction feedback architecture modified with the addition of an autoregulatory positive feedback loop, we were able to generate cells that produce enough cis ligand when stimulated by trans ligand to themselves transmit signal to neighboring cells, which is the hallmark of lateral induction.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/83PA-9833, author = {LeBon, Lauren E.}, title = {The Logic of Receptor-Ligand Interactions in the Notch Signaling Pathway}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/83PA-9833}, url = {https://resolver.caltech.edu/CaltechTHESIS:05212014-094412950}, abstract = {The Notch signaling pathway enables neighboring cells to coordinate developmental fates in diverse processes such as angiogenesis, neuronal differentiation, and immune system development. Although key components and interactions in the Notch pathway are known, it remains unclear how they work together to determine a cell’s signaling state, defined as its quantitative ability to send and receive signals using particular Notch receptors and ligands. Recent work suggests that several aspects of the system can lead to complex signaling behaviors: First, receptors and ligands interact in two distinct ways, inhibiting each other in the same cell (in cis) while productively interacting between cells (in trans) to signal. The ability of a cell to send or receive signals depends strongly on both types of interactions. Second, mammals have multiple types of receptors and ligands, which interact with different strengths, and are frequently co-expressed in natural systems. Third, the three mammalian Fringe proteins can modify receptor-ligand interaction strengths in distinct and ligand-specific ways. Consequently, cells can exhibit non-intuitive signaling states even with relatively few components.
In order to understand what signaling states occur in natural processes, and what types of signaling behaviors they enable, this thesis puts forward a quantitative and predictive model of how the Notch signaling state is determined by the expression levels of receptors, ligands, and Fringe proteins. To specify the parameters of the model, we constructed a set of cell lines that allow control of ligand and Fringe expression level, and readout of the resulting Notch activity. We subjected these cell lines to an assay to quantitatively assess the levels of Notch ligands and receptors on the surface of individual cells. We further analyzed the dependence of these interactions on the level and type of Fringe expression. We developed a mathematical modeling framework that uses these data to predict the signaling states of individual cells from component expression levels. These methods allow us to reconstitute and analyze a diverse set of Notch signaling configurations from the bottom up, and provide a comprehensive view of the signaling repertoire of this major signaling pathway.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/93MF-Q808, author = {Young, Jonathan Wan}, title = {Architecture, Dynamics, and Function of the General Stress Response System in B. subtilis}, school = {California Institute of Technology}, year = {2012}, doi = {10.7907/93MF-Q808}, url = {https://resolver.caltech.edu/CaltechTHESIS:05292012-120719001}, abstract = {Cells exhibit diverse and dynamic responses to stress. However, in many cases it remains unclear what the dynamics are, how they are generated, and why they are beneficial to the cell or organism. To investigate these issues we studied the General Stress Response in B. subtilis, a critical, conserved stress signaling pathway, mediated by the alternative sigma factor, σB. First, we find that σB activates with stochastic, frequency modulated pulses in response to energy stress. We explore the mechanism behind this striking response and find that a small, compact circuit facilitates this behavior. Second, we find that σB activates with a single-homogenous pulse of activity exposed to environmental stress, in contrast to energy stress dynamics. We also find that activation is rate-responsive, and show how this property may separate broad and specific regulatory modes. Lastly, we present some preliminary work toward a synthetic sigma factor activation circuit. Combined, these results present a comprehensive study of σB activation and generate a platform by which other dynamic stress response systems can be understood.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/35A7-K421, author = {Levine, Joseph H.}, title = {Genetic Regulatory Circuit Dynamics: Analysis and Synthesis}, school = {California Institute of Technology}, year = {2012}, doi = {10.7907/35A7-K421}, url = {https://resolver.caltech.edu/CaltechTHESIS:06052012-121432503}, abstract = {How can cells shape and utilize dynamic gene regulation to enable complex cellular behaviors? I study this question in natural and synthetic contexts.
The first project studies how a natural genetic network can imbue cells with a sense of ‘time’. It has long been known that environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable ‘polyphasic’ positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a timer that operates over time scales much longer than a cell cycle.
The second project proposes a method to rapidly generate and test complex genetic network dynamics in living cells. Existing microorganisms have evolved genetic circuitry to meet diverse challenges and maximize their survival and fitness. These challenges arise from external environmental pressures, or internal evolved constraints. Furthermore, these challenges may be either static or dynamic in nature. While existing circuits have likely evolved to be ‘good enough’ to respond to historical challenges, it remains unclear if they can be improved upon, and whether they respond well to novel situations. Synthetic biology seeks to engineer organisms with complex novel phenotypes, both to harness these novel organisms for a function and to understand their underlying biology. Dynamic gene expression strategies may be necessary to successfully generate these phenotypes. Unfortunately, generating novel dynamic gene expression patterns with conventional genetic engineering remains a challenge. Here I propose and describe progress towards a computerized feedback control setup to enable the programming and rapid testing of dynamic gene regulatory patterns in living cells. Small sets of genes will be regulated optogenetically based on programmed control laws, and past and present cellular state. This setup will enable us to explore the functions and limits of engineered dynamic gene regulation, while hopefully, in the process, providing lessons about the underlying biology.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/NPZD-G382, author = {Sen, Shaunak}, title = {Regulatory Consequences of Bandpass Feedback in a Bacterial Phosphorelay}, school = {California Institute of Technology}, year = {2011}, doi = {10.7907/NPZD-G382}, url = {https://resolver.caltech.edu/CaltechTHESIS:05252011-222115269}, abstract = {Under conditions of nutrient limitation, Bacillus subtilis cells terminally differentiate into a dormant spore state. Progression to sporulation is controlled by a genetic circuit structured as a phosphorelay embedded in multiple transcriptional feedback loops, and which is used to activate the master regulator Spo0A by phosphorylation. These transcriptional regulatory interactions are ‘bandpass’-like, in the sense that activation occurs within a limited band of Spo0A~P concentrations, and have recently been shown to pulse in a cell-cycle-dependent fashion. Additionally, the core phosphorelay is an architectural variant of the canonical two-component signaling system, which allows signal integration from a larger number of inputs, including two types of phosphatases that act on different protein components. However, the impact of these pulsed bandpass interactions on the circuit dynamics preceding sporulation and the utility of two types of phosphatases remains unclear. In order to address these questions, we measured key features of the bandpass interactions at the single-cell level and analyzed them in the context of a simple mathematical model. The model predicted the emergence of a delayed phase shift between the pulsing activity of the different sporulation genes, as well as the existence of a stable state, with elevated Spo0A activity but no sporulation, embedded within the dynamical structure of the system. To test the model, we used time-lapse fluorescence microscopy to measure dynamics of single cells initiating sporulation. We observed the delayed phase shift emerging during the progression to sporulation, while a re-engineering of the sporulation circuit revealed behavior resembling the predicted additional state. The core phosphorelay model also showed a post-translational bandpass response, and we find that the two types of phosphatases can independently tune the two bandpass thresholds. These results show that periodically-driven bandpass feedback loops can give rise to complex dynamics in the progression towards sporulation, and that similar inputs can tune different response features.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/MHSR-9875, author = {Dalal, Chiraj Kiran}, title = {Causes and Consequences of Gene Expression Noise}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/MHSR-9875}, url = {https://resolver.caltech.edu/CaltechTHESIS:03312010-150904525}, abstract = {Genetically identical cells harvested in the same environment exhibit heterogeneity in gene expression. This phenomenon, termed gene expression noise, has been measured in several model organisms under various conditions. However, we still do not have a clear understanding of (1) the factors responsible for generating gene expression noise, or (2) the potential consequences noise can have on cellular processes. In an attempt to investigate these issues, we have determined the effects of 1) directional selection, 2) promoter mutation and 3) fluctuations in transcription factor localization on gene expression noise. First, we have used analytic and computational modeling of the effects of directional selection on gene expression noise to discover that, assuming expression can be described by up to two independent parameters, μ, mean, and σ, noise, strong directional selection yields an increase in noise. Next, we generated mutant promoter libraries and measured gene expression to determine the effects of cis-regulatory mutations on gene expression noise. Here we found that the expression noise can indeed be modulated by mutation independent of mean expression levels, lending credence to the previously mentioned analytical result. Based on this result, that mutations can harness noise, we wanted to determine whether the binding and unbinding of transcription factors to promoter regions also contributed to gene expression noise. To do so, we analyzed the localization dynamics of a transcription factor Crz1. We determined that Crz1 translocates to the nucleus in coherent bursts of localization in response to calcium. The frequency, but not the duration, of these bursts increases with the concentration of extracellular calcium. This frequency modulation propagates downstream of Crz1, enabling proportional regulation of target genes. Intrigued by this result, we characterized different types of localization dynamics used by the yeast proteome. We have found several classes of localization behavior, including proteins that burst on several timescales, exhibit static heterogeneity, and show amplitude modulation. Strikingly, several of these dynamic localization systems must co-exist in the same cell under the same conditions. Amongst the proteins that burst on a fast timescale like Crz1, Msn2 and Mig1 are transcription factors that both burst when deprived of glucose. Furthermore, both regulate a common set of target genes. Interestingly, when imaged together, the proteins exhibit correlations on two timescales, a positive correlation that typically lasts for an hour and an anti-correlation that lasts a few minutes. We are continuing to investigate the potential regulatory impact of these correlations by measuring the expression of their combinatorial target genes.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, } @phdthesis{10.7907/KRW6-DH88, author = {Cox, Robert Sidney, III}, title = {Transcriptional Regulation and Combinatorial Genetic Logic in Synthetic Bacterial Circuits}, school = {California Institute of Technology}, year = {2008}, doi = {10.7907/KRW6-DH88}, url = {https://resolver.caltech.edu/CaltechETD:etd-03042008-130011}, abstract = {We engineered several synthetic regulatory circuits to study transcriptional regulation in bacteria. We developed a new technique for DNA construction, built and characterized in vivo a library of genetic logic gates, examined the role of genetic noise transcriptional regulation using a fluorescent multi-reporter system, and characterized a synthetic circuit for the control of population density.
We used synthetic duplex DNA fragments and very short cohesive overhangs to direct ordered assemblies of diverse combinatorial libraries. Multiple DNA fragments were simultaneously ligated in a single step to produce random concatemers, without the need for amplification or product purification. We characterized the assembly process to identify optimal cohesive overhangs. We showed that the method was 97% efficient for assembling 100 base-pair concatemers from three duplex fragments.
We constructed a library of 10,000 prokaryotic promoters, containing over 1,000 unique 100 base-pair sequences. These promoters responded to up to three inputs, and contained diverse architectural arrangements of regulatory sequences. We characterized the logical input functions of 288 promoters in Escherichia coli, and analyzed the relationship between promoter function and architecture. We defined promoter function in terms of regulatory range, logic type, and input symmetry; and identified general rules for combinatorial programming of gene expression.
We built a synthetic three-color fluorescent reporter framework. This construct was non-toxic and extensible for synthetic and systems biology applications. Three spectrally distinct and genetically isolated reporter proteins allowed independent monitoring of genetic signals at the single-cell level. We showed that the simultaneous measurement of multiple genes can exploit genetic noise to sensitively detect transcriptional co-regulation.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Elowitz, Michael B.}, }