Browsing by Author "Bennett, Matthew R"

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    A Synthetic Intercellular Toggle Switch and its Implications on Pattern Formation
    (2021-12-03) de Freitas Magalhaes, Barbara; Bennett, Matthew R
    A genetic toggle switch is a circuit that possesses two stable mutually exclusive states of gene expression caused by cross-repression. It is widely found in natural processes, such as in development, given that cross-repression is important for refining pattern boundaries. Synthetically, many circuits that are applied to biotechnology are also based on toggle switches, such as many biosensors. However, single-cell synthetic genetic toggles are limited by individual noise fluctuations, and functions that do not cause metabolic load to the cell. Here, we propose the creation of an intercellular toggle switch, which is a synthetic toggle with quorum sensing (QS). QS is the mechanism through which bacteria communicate with each other, by producing signaling molecules that can affect gene expression in a density-dependent way. To overcome the main limitations of single-cell toggles, we aim to synchronize a population response, and enable circuit multicellularity through cell communication. Synthetic multicellular genetic circuits can perform complex functions that single cells cannot, and have the ability to better recreate naturally-occurring processes. In the first part, we describe the construction and characterization of a few QS toggle versions. We find that the dynamics of these toggle switches depend on their regulatory topologies, and their gene expression strength and leakiness. In the second part, we explore the aspects of one QS toggle version in a biofilm. We find that the QS toggle can form self-organized patterns when grown in a colony, by spatially segregating cells from different states. Comparatively, a non-QS (NQS) toggle colony does not show spatial-dependent pattern formation until three-dimensional aspects are investigated. NQS toggle shows a vertical segregation of states within colonies. It indicates that the addition of QS causes a directional shift in colony pattern behavior, and makes it more complex and dependent on growth, as shown with the mathematical model. These findings highlight the importance of spatial aspects to a synthetic circuit behavior. They also shed light into natural pattern formation mechanisms, and contribute to the development of synthetic self-organized multicellular systems in bacteria.
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    Design Principles of Cellular Differentiation Regulatory Networks
    (2015-12-01) Narula, Jatin; Igoshin, Oleg A; Tabor, Jeffrey J; Bennett, Matthew R
    To understand cellular differentiation programs is to understand the often large and complex gene regulatory networks (GRNs) that control and orchestrate these programs. The work presented here aims to exploit the wealth of newly available experimental information and methods to identify design principles that relate how GRN structures relate to the functional requirements in three model differentiation/stress-response programs: embryonic hematopoiesis, sporulation and σB general stress response. First we used a statistical thermodynamic approach to characterize the biophysical mechanisms of combinatorial regulation by distant enhancers in eukaryotes and demonstrate how the GRN controlling embryonic hematopoiesis acts as an irreversible bistable switch with low-pass noise filtering properties. We further used our model of the hematopoiesis network to reconcile discrepant experimental observations about the regulator Runx1 and explained how it limits HSC emergence in vitro. In the second project we investigated the Bacillus subtilis sporulation network and showed how a cascade of feed-forward loops downstream of the master regulatory Spo0A~P control cell-fate during starvation. We also identified a rate-responsive network module in the Spo0A regulon to explain why accelerated accumulation of Spo0A~P leads to a dramatic reduction in sporulation efficiency. Further we found that the arrangement of two sporulation network genes on opposite ends of the chromosome ties Spo0A~P activation to the DNA replication status. We were also able to show that the slowdown of cell growth is the primary starvation signal that determines sporulation cell-fates by controlling Spo0A~P activation. For the third project we built a detailed model of the σB network in Bacillus subtilis to mechanistically explain the experimentally observed pulsatile response of this network under stress. We further showed that the same network architecture that enables this pulsatile response insulates the σB network from the effects of competition for cellular resources like RNA polymerase. The design principles identified in the studies of these networks are related to their topological structure and function rather than the specific genes and proteins that comprise them. As a result, we expect them to be widely applicable to and help in the study of a diverse array of other differentiation GRNs.
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    Dynamic Studies of Synthetic Transcriptional Networks
    (2017-12-01) Hirning, Andrew John; Bennett, Matthew R
    Within synthetic biology, orthogonal genetic circuits are created using a small collection of transcription factors. Although the absolute number of transcription factors is small, the number of mutants explored for each transcription factor is greater by orders of magnitude. However, nearly all synthetic networks created utilize the wild-type transcription factor. The large number of published mutants for each transcription factor represent an equal number of variant networks with possibly varied behavior. Study of these networks can increase knowledge about how different aspects of protein structure/function impact the function of genetic networks. For this work, we chose two sets of mutants/variants for the transcriptional repressor LacI - a set of point mutants with varying operator affinity and a library of chimeric repressors that respond to unique inducers. We developed novel microfluidic devices for studying dynamic gene expression in single cells. First, we discuss attempts to alter the function of a two-gene oscillator by mutating LacI to modulate the operator affinity of the protein. We selected mutants with varying affinities for lacO1 and tested the mutant oscillators. We hypothesized the oscillatory period would increase as the affinity of LacI for O1 decreased. Instead, we observed the oscillatory period responded in a non-monotonic fashion to decreasing affinity. This behavior can be explained by a second operator site (lacOsym) in the synthetic promoter used in the oscillator. Our selected mutants do not vary in affinity in the same way for Osym as O1. Second, we discuss efforts to measure temporal responses of genetic logic gates. Utilizing chimeric repressors that bind LacI-responsive promoters, we generated three genetic logic gates (AND, IMPLY, and NOT). To study these gates, we created a bespoke microfluidic device with two independent, time-varying inducer inputs. Each gate was driven with a square wave of inducer across a period range of 20-240 minutes. We find that “ligand” gates (AND, IMPLY) constructed with chimeric repressors give reliable responses over a wide range of driving frequencies, whereas an inverted, “transcriptional” gate (NOT) responds over a narrower range, probably due to excess protein production coupled with degradation steps required for state transition in “transcriptional” gates.
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    Engineering multi-input gene regulation for applications in Synthetic Biology
    (2015-04-17) Shis, David Liu; Bennett, Matthew R; Shamoo, Yousif; Silberg, Jonathan J; Tabor, Jeffrey J
    Synthetic biology offers insight into molecular biology through the design and implementation of synthetic gene networks. One challenge in this effort is implementing transcriptional logic gates that enable synthetic gene networks to make decisions based on multiple inputs. However, the ability to implement transcriptional logic gates is inhibited by a lack of parts available to build them. In this work, we explore strategies for facilitating multi-input gene regulation in prokaryotes. That is, we develop methods for making the expression of a reporter gene dependent on two or more inputs in Escherichia coli. We first demonstrate how fragmentation of T7 RNA Polymerase (T7 RNAP) creates a multi-fragment transcription complex that facilitates AND transcriptional logic. We find split T7 RNAP to be functional in vivo and that both fragments of the split protein must be present for transcription from the T7 Promoter, PT7, to occur. We also find that the specificity of the split protein can be modified to create split protein mutants with orthogonal specificity. In addition to split T7 RNAP, we test the AND transcriptional logic made possible by co-expressing multiple chimeric LacI/GalR transcriptional repressors. We find that each chimeric repressor regulates the operator site of its DNA binding domain (DBD) according to the ligand sensed by its ligand binding domain(LBD). By co-expressing multiple chimeric repressors, we find each repressor independently regulates its DBD's operator. As a result, the number of inputs at a promoter relates directly to the number of species of chimeric repressors with the same DBD. Further, by modifying the DBD we find that we can create chimeras with orthogonal specificities that facilitate an orthogonal open reading frame. We find expression of our chimeric repressors en mass facilitates regulation such as a four-input transcriptional AND gate or two orthogonal transcriptional AND gates. Split T7 RNAP and the coexpression of chimeric LacI/GalR repressors both demonstrate strategies for multi-input gene regulation in prokaryotes. This work also suggest strategies for the engineering of additional components for use in synthetic gene networks.
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    Spatiotemporal Dynamics of Synthetic Microbial Consortia
    (2019-12-06) Alnahhas, Razan Nasser; Bennett, Matthew R
    Synthetic biology traditionally entails the engineering of microbial organisms for a variety of applications such as producing desired products or studying components of gene circuits in nature. For example, synthetic gene oscillators provide information on the function of circadian clocks, and genetic toggle switches are involved in cellular memory networks. As synthetic biology projects become more complex, switching to multiple strains working together becomes advantageous. Multiple engineered strains working together is referred to as synthetic microbial consortia. The first advantage of synthetic microbial consortia is the lessening of the metabolic load on each strain by dividing up tasks. Secondly, splitting up processes makes optimizing one step within each strain a more straightforward task than optimizing several reactions within one strain. Engineered microbial consortia also allows synthetic biologists to study the dynamics of different intercellular and inter-strain interactions. In the first part of this project we establish how a microfluidic environment can affect stability of strains in a consortium. Fluctuations in strain ratios affect the productivity of the engineered circuit, and loss of a strain will break the circuit completely. We determined the ideal environmental factors to ensure for a stable population over time. We also measured signaling distances across communities to determine how close cells need to be in order to communicate or work together. Next, we engineered a consortium with a novel phenotype: gene expression patterns that depend on the ratio of strains within the consortium. This was done by expanding the traditional co-repressive toggle gene circuit to two strains. In our system, there are two engineered strains that each repress the expression of synthetic genes in the opposite strain. This results in a majority wins pattern of gene expression. In nature, bacteria can adjust gene expression based on the overall population size, but our engineered multicellular gene circuit adjusts gene expression based on the ratio of strains within the population. Overall in this work, we have determined ideal environments for synthetic microbial consortia and engineered a consortium with a novel phenotype.
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    Understanding dynamic activities of the yeast galactose utilization network under environmental changes
    (2016-01-27) Nguyen, Truong Huu; Bennett, Matthew R; Beckingham, Kathleen M; Olson, John S; Segatori, Laura; Tao, Yizhi Jane
    Cellular adaptability to environmental changes depends on the collective actions of genes, mRNA, proteins and ligands, all of which are components of a ''genetic network''. To understand the dynamics of a gene network in response to temporally and spatially environmental changes, we focus on the galactose utilization network in the yeast Saccharomyces cerevisiae. This network allows yeast cells to metabolize galactose in the absence of glucose and is tightly repressed when glucose is available in the environment. The main question is how the Gal network is activated when glucose is depleted since both sugars cannot be metabolized simultaneously. Using a microfluidic device, we supplied yeast cells with both glucose and galactose before linearly depleting glucose at different rates. We tracked the onset and accumulation of a yellow fluorescent reporter-tagged Gal1p, the first enzyme of the Gal network. Our data shows that the glucose-depletion rate plays an important role in the activation of the Gal network. The onset of the network's activation depends on the time it takes to pass a specific threshold of the glucose concentration. On the other hand, the full induction of the Gal network, represented by the Gal1-accumulation time, is strongly influenced by the depletion rates. In particular, the mean of the Gal1-accumulation time increases significantly when glucose is depleted instantaneously. Furthermore, the variability of the Gal1-accumulation time also increases in short depletion rates and achieves a minimum at intermediate depletion rates. Using a mathematical simulation, we demonstrate that the increase in the accumulation time is due to the loss of energy when glucose is instantaneously depleted. This loss of energy also correlates with the length of diauxie, a period of catabolic transition from glucose to a secondary carbon source. Thus, changes in the glucose-depletion rate not only affect the dynamics of the Gal network's activation, but can also affect the phenotypic outcomes of the single cells within the population. Our results contribute to growing sets of evidence that a gene network can exhibit complex, dynamic behaviors under environmental changes to shape the fitness and survival of individual and collective members of a microbial population.
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    Understanding the Role of Protein Degradation in Synthetic Gene Circuits
    (2018-04-20) O'Brien, Erin O; Bennett, Matthew R
    Synthetic gene circuits are built with mathematical predictions and are further characterized experimentally. Most synthetic circuits utilize degradation tags to normalize and speed up the rate of degradation of circuit components. Despite their widespread use, the effect of degradation tags on circuit dynamics has not been well studied. This work aims to characterize the degradation rate of the ssrA degradation tag variants on a single substrate level and determine their role in overall network dynamics within a synthetic gene circuit at the single cell resolution. mall differences in the protein degradation rates indicating that the parameter space for the degradation tags is critical for achieving desired circuit dynamics. Mathematically and experimentally, this work demonstrates varying the rate of degradation can ultimately dictate the output oscillations for the circuit dynamics. The ultimate goal of this work is to create a better understanding of the role degradation plays in synthetic gene circuit dynamics.