35 research outputs found

    Mitigation of ribosome competition through distributed sRNA feedback (extended version)

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    This paper is an extended version of a paper of the same title accepted to Proceedings of the 55th IEEE Conference on Decision and Control (2016).A current challenge in the robust engineering of synthetic gene networks is context dependence, the unintended interactions among genes and host factors. Ribosome competition is a specific form of context dependence, where all genes in the network compete for a limited pool of translational resources available for gene expression. Recently, theoretical and experimental studies have shown that ribosome competition creates a hidden layer of interactions among genes, which largely hinders our ability to predict design outcomes. In this work, we establish a control theoretic framework, where these hidden interactions become disturbance signals. We then propose a distributed feedback mechanism to achieve disturbance decoupling in the network. The feedback loop at each node consists of the protein product transcriptionally activating a small RNA (sRNA), which forms a translationally inactive complex with mRNA rapidly. We illustrate that with this feedback mechanism, protein production at each node is only dependent on its own transcription factor inputs, and almost independent of hidden interactions arising from ribosome competition.AFOSR grant FA9550-12-1-0129 and ONR grant N00014131007

    Robustness of networked systems to unintended interactions with application to engineered genetic circuits

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    A networked dynamical system is composed of subsystems interconnected through prescribed interactions. In many engineering applications, however, one subsystem can also affect others through "unintended" interactions that can significantly hamper the intended network's behavior. Although unintended interactions can be modeled as disturbance inputs to the subsystems, these disturbances depend on the network's states. As a consequence, a disturbance attenuation property of each isolated subsystem is, alone, insufficient to ensure that the network behavior is robust to unintended interactions. In this paper, we provide sufficient conditions on subsystem dynamics and interaction maps, such that the network's behavior is robust to unintended interactions. These conditions require that each subsystem attenuates constant external disturbances, is monotone or "near-monotone", the unintended interaction map is monotone, and the prescribed interaction map does not contain feedback loops. We employ this result to guide the design of resource-limited genetic circuits. More generally, our result provide conditions under which robustness of constituent subsystems is sufficient to guarantee robustness of the network to unintended interactions

    Principles of genetic circuit design

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    Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

    Towards engineering microbial consortia using RNA-based genetic controllers

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    In nature, quorum sensing is a mechanism used by microbes to communicate and coordinate behaviours at the population level. Over the last two decades, synthetic biologists have used the unique property of quorum sensing to sense population density for coordinating cellular behaviours of single and mixed cultures. This gave rise to the development of multicellular biosynthesis systems for metabolic engineering and of spatially distributed systems for synthetic biology. However, robustly controlling the composition of multicellular systems remains a challenge and limits its wide adoption by the metabolic engineering community. Current strategies for controlling synthetic microbial communities vastly rely on engineer- ing metabolic dependencies between microbial species in a process called syntrophy. While syntrophy guarantees the survival of all strains in the coculture, it does not provide a way to control community composition. Existing genetic circuits that can dynamically control community composition often impose too much burden on their hosts for division of labour to be a viable solution to improve yields and titers of valuable metabolic products. Here we investigate the potential of using RNA-based gene circuits to reduce the cost of express- ing heterologous genes for the control of community composition in a two-member E. coli coculture. In this work, we present the development of three genetic modules that rely on RNA species to detect changes in population density and to regulate growth rate when community composition becomes unstable. Together, the modules work in concert to stabilise community composition around a ratio set by the intrinsic properties of the circuit’s genetic components. We identify the key parameters of the circuits that enable tuning of the composition ratio. We characterise the cost of expressing each module of the genetic controller by measuring its impact on the host growth rate and on consumption of free cellular resources. Together these findings highlight the importance of developing host-aware circuits to control community composition so as to enable their wide adoption by metabolic engineers.Open Acces

    Stress & Granules : regulation of Cauliflower mosaic virus disease in Arabidopsis thaliana

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    Viruses exist in intimate relationships with the organisms they are infecting and are just as dependent on compatibility with their host’s cellular components as they are on subverting them. During any virus infection, the cells are flooded with foreign nucleic acids (DNA and/or RNA) and have learned to recognize, disarm, or eliminate them. In turn, viruses have evolved to use the cellular transcription and RNA regulatory machinery for their own benefit. Cytoplasmic RNA granules, namely Processing bodies (PBs) and Stress granules (SGs) are at the forefront of RNA regulation as they contain and store untranslated RNA and are responsive in number, size, and composition to various stresses, including virus infection. For this thesis, we have explored the role of Arabidopsis thaliana RNA granules during infection with the pararetrovirus Cauliflower mosaic virus (CaMV). We show that PB components aid virus accumulation through shielding of the viral RNA from the antiviral RNA silencing machinery (Paper I). In addition, we find that the cytoplasmic viral replication factory contains several RNA granule proteins during infection, including both, PB components and SG components. Opposite to PBs, SGs are likely antiviral and CaMV subverts their biogenesis through its multifunctional protein P6 (Paper II). In an effort to uncover novel disease determinants, we explore the variation of CaMV disease in naturally occurring populations of Arabidopsis thaliana and uncover the importance of the plant hormone abscisic acid and its homeostasis for CaMV infection, as well as a novel CaMV susceptibility factor, the ABA synthesis gene NCED9 (Paper III)

    Toward Multiscale Models of Cyanobacterial Growth

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    Oxygenic photosynthesis dominates global primary productivity ever since its evolution more than three billion years ago. While many aspects of phototrophic growth are well understood, it remains a considerable challenge to elucidate the manifold dependencies and interconnections between the diverse cellular processes that together facilitate the synthesis of new cells. Phototrophic growth involves the coordinated action of several layers of cellular functioning, ranging from the photosynthetic light reactions and the electron transport chain, to carbon-concentrating mechanisms and the assimilation of inorganic carbon. It requires the synthesis of new building blocks by cellular metabolism, protection against excessive light, as well as diurnal regulation by a circadian clock and the orchestration of gene expression and cell division. Computational modeling allows us to quantitatively describe these cellular functions and processes relevant for phototrophic growth. As yet, however, computational models are mostly confined to the inner workings of individual cellular processes, rather than describing the manifold interactions between them in the context of a living cell. Using cyanobacteria as model organisms, this contribution seeks to summarize existing computational models that are relevant to describe phototrophic growth and seeks to outline their interactions and dependencies. Our ultimate aim is to understand cellular functioning and growth as the outcome of a coordinated operation of diverse yet interconnected cellular processes.Peer Reviewe

    Abundance, distribution and functional characterisation of gut-associated Type II toxin-antitoxin systems

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    Prokaryotic toxin-antitoxin (TA) systems (also known as addiction modules), are ubiquitous genetic modules first discovered due to their role in stabilising vertical transmission of plasmids. Generally they are two-gene systems encoding a stable toxin (Tx) and an unstable antitoxin (ATx). Loss of the TA module leads to rapid ATx degradation and depletion, leaving the Tx free to interact with cellular targets and inhibit growth. For plasmid encoded TA systems, this leads to the death of plasmid free daughter cells, ensuring plasmid maintenance in a population, and gives rise to the term "addiction module". More recently, the expansion in microbial genome data has highlighted the prevalence and diversity of TA systems, and demonstrated that they are common features of many bacterial chromosomes. In addition, metagenomic surveys have pointed to the enrichment of some TA families in particular microbial ecosystems; a prime example from surveys of the human gut microbiome and RelBE TA family. Collectively, these observations indicate a wider role for TA modules in bacterial function, with numerous roles for TA systems now hypothesised. These include: i) Stabilisation of TA associated chromosomal DNA during vertical transmission; ii) Formation of "persister" cells resistant to environmental stresses, and; iii) Population level resistance to bacteriophage attack. Additionally, some Tx components have shown activity in eukaryotic cells, raising the potential for a role in prokaryote-eukaryote interaction. Here we undertook a systematic study of Type II TA systems, to provide a comprehensive assessment of their distribution and relative abundance, to confirm activity of prevalent TA systems, and to understand putative roles these may play in gut associated bacteria and the gut microbiome. A comparative genomic and metagenomic analysis of 3919 bacterial chromosomes, 4580 plasmids, 711 bacteriophage genomes, and 781 metagenomes encompassing 16 distinct habitats was conducted using all known Type II TA systems present in the Toxin Antitoxin Database (~10,100 TA genes ~1:1 Tx:ATx). Of the 817 Type II TA system homologues found in human gut datasets, 686 were observed to have significantly higher relative abundance in the human gut microbiome over other microbial ecosystems. In parallel to these in silico findings, PCR and qPCR surveys of microbiomes from 65 stool samples obtained from healthy volunteers, as well as those with polyps or colorectal cancer, were undertaken. This demonstrated a higher ATx presence than Tx or complete module, however no differences in Tx copy number between health groups was seen. To confirm the activity of the most abundant TA system homologues identified in sequence surveys, ORFs were amplified from gut metagenomic DNA, and individual Tx or ATx cloned under the control of inducible promoters. Induction of Tx expression under normal growth conditions resulted in bacterial growth inhibition, while live dead staining showed entry into a viable but non-cultivatable state, commensurate with TA function. Experiments simulating environmental stresses encountered during colonisation of the GI tract (starvation, low pH, bile), indicated that expression of these TA systems could increase cell survival when carbon or nitrogen availability was limited (starvation). Since antibiotics are also commonly encountered by gut associated-bacteria (both as residents of the GI tract and during colonisation of other body sties) a role for gut associated TA systems in facilitating survival during antibiotic exposure was also explored. This revealed an increased number of cells surviving two hours post-treatment with β-lactams when Tx genes were expressed, and in keeping with an impact on cell growth. To test the hypothesis that TA systems may stabilize associated regions of DNA, the composition of gene neighbourhoods surrounding TA systems were also explored. ORFs surrounding TA system homologues identified in metagenomic and genomic datasets were identified using the Metagene annotator, and ORF functions predicted based on searches of the Clusters of Orthlogous Groups (COG) database. This revealed significant increases in ORFs with functions related to replication/recombination/repair and those with unknown functions. It also identified a decrease in the proportion of ORFs encoding functions such as carbohydrate and lipid transport and metabolism in regions surrounding TA systems, suggesting involvement with stabilization of mobile elements. Finally, we explored the potential for gut associated TA systems to modulate phage-microbe, and host-microbe interactions. In the case of phage-host interactions, TA systems have previously been shown to function as mediators of phage resistance at the population level, by directing cells towards a dormant state which prevents phage replication, and permits a sub-set of cells to survive phage attack. Our findings indicated the potential for gut associated TA systems to provide some degree of protection during particular host-phage interactions, but specific modules did not provide universal protection against phage. In the case of host-microbe interaction, some Type II TA system Tx components have been shown to be functional in cultured eukaryotic cells, promoting apoptosis when introduced and expressed in these cell types. However, no studies to date have examined the potential for bacterially expressed TA systems to influence eukaryotic cell health in co-culture models. To investigate this, we assessed the impact of bacterial TA system expression on the health of the intestinal epithelial cell line Caco-2 in co-culture systems specifically focusing on cell apoptosis and necrosis whilst in the presence of Escherichia coli expressing p22-RelBE

    Engineering cell-free systems for synthetic biologists

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    Synthetic biology (synbio) has emerged as a transformative scientific field with immense potential to address a wide-range of global problems. A specific sub-field of synbio utilizes cellular biomolecular machinery outside of a living cell. In theory, these “cell-free” systems offer a simpler approach and unique features compared to cell-based systems for biotechnology development. However, in practice limited accessibility and poor protein synthesis capacity hinder the overall scope and application of cell-free synbio. To address these challenges, it was our goal to create new engineering tools that will help expand the overall utility of cell-free expression systems. Data presented here provides: 1) detailed methods for the in-house preparation of a cost-effective in vitro reconstituted cell-free system, 2) an in-depth proteomic analysis of the system building blocks as a tool to characterize the composition and inform optimization, and 3) an improvement to protein synthesis capacity by modifying the ribosome composition. Furthermore, a critical assessment of the regulatory landscape is provided, promoting the safe and responsible use of cell-free synbio

    Genetics and Genomics of Forest Trees

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    Forest tree genetics and genomics are advancing at an accelerated rate, thanks to recent developments in high-throughput, next-generation sequencing capabilities, and novel biostatistical tools. Population and landscape genetics and genomics have seen the rise of new approaches implemented in large-scale studies that employ the use of genome-wide sampling. Such studies have started to discern the dynamics of neutral and adaptive variation in nature and the processes that underlie spatially explicit patterns of genetic and genomic variation in nature. The continuous development of genetic maps in forest trees and the expansion of QTL and association mapping approaches contribute to the unravelling of the genotype-phenotype relationship and lead to marker-assisted and genome-wide selection. However, major challenges lie ahead. Recent literature suggests that species demography and genetic diversity have been affected both by climatic oscillations and anthropogenically induced stresses in a way calls into question the possibility of future adaptation. Moreover, the pace of contemporary environmental change presents a great challenge to forest tree populations and their ability to adapt, taking into consideration their life history characteristics. Several questions emerge that include, but are not limited to, the interpretation of forest tree genome surveillance and their structural/functional properties, the adaptive and neutral processes that have shaped forest tree genomes, the analysis of phenotypic traits relevant to adaptation (especially adaptation under contemporary climate change), the link between epigenetics/epigenomics and phenotype/genotype, and the use of genetics/genomics as well as genetic monitoring to advance conservation priorities
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