Download or read book Cell targeted Regulation of Gene Expression Through Synthetic RNA Devices written by James Vincent Vowles and published by . This book was released on 2014 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function. We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation. We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein. The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

Download or read book The Science and Applications of Synthetic and Systems Biology written by Institute of Medicine and published by National Academies Press. This book was released on 2011-12-30 with total page 570 pages. Available in PDF, EPUB and Kindle. Book excerpt: Many potential applications of synthetic and systems biology are relevant to the challenges associated with the detection, surveillance, and responses to emerging and re-emerging infectious diseases. On March 14 and 15, 2011, the Institute of Medicine's (IOM's) Forum on Microbial Threats convened a public workshop in Washington, DC, to explore the current state of the science of synthetic biology, including its dependency on systems biology; discussed the different approaches that scientists are taking to engineer, or reengineer, biological systems; and discussed how the tools and approaches of synthetic and systems biology were being applied to mitigate the risks associated with emerging infectious diseases. The Science and Applications of Synthetic and Systems Biology is organized into sections as a topic-by-topic distillation of the presentations and discussions that took place at the workshop. Its purpose is to present information from relevant experience, to delineate a range of pivotal issues and their respective challenges, and to offer differing perspectives on the topic as discussed and described by the workshop participants. This report also includes a collection of individually authored papers and commentary.

Download or read book Synthetic mRNA written by Robert E. Rhoads and published by Humana. This book was released on 2016-05-29 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.

Download or read book Advances in Synthetic Biology written by Vijai Singh and published by Springer Nature. This book was released on 2020-04-13 with total page 349 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book addresses the design of emerging conceptual tools, technologies and systems including novel synthetic parts, devices, circuits, oscillators, biological gates, and small regulatory RNAs (riboregulators and riboswitches), which serve as versatile control elements for regulating gene expression. Synthetic biology, a rapidly growing field that involves the application of engineering principles in biology, is now being used to develop novel systems for a wide range of applications including diagnostics, cell reprogramming, therapeutics, enzymes, vaccines, biomaterials, biofuels, fine chemicals and many more. The book subsequently summarizes recent developments in technologies for assembling synthetic genomes, minimal genomes, synthetic biology toolboxes, CRISPR-Cas systems, cell-free protein synthesis systems and microfluidics. Accordingly, it offers a valuable resource not only for beginners in synthetic biology, but also for researchers, students, scientists, clinicians, stakeholders and policymakers interested in the potential held by synthetic biology.

Download or read book Riboswitches as Targets and Tools written by and published by Academic Press. This book was released on 2015-01-12 with total page 468 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in riboswitches as targets and tools and contains sections on such topics as constructing and optimizing artificial riboswitches, live cell imaging and intracellular sensors with artificial riboswitches, conditional control of gene expression with artificial riboswitches, using artificial riboswitches for protein evolution and pathway optimization, and anti-riboswitch drug screens. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in riboswitches as targets and tools Contains sections on such topics as constructing and optimizing artificial riboswitches, synthetic biology: live cell imaging and intracellular sensors with artificial riboswitches, synthetic biology: conditional control of gene expression with artificial riboswitches, synthetic biology: using artificial riboswitches for protein evolution and pathway optimization, anti-riboswitches drug screens

Download or read book Engineering Synthetic Ligand responsive RNA Devices for Controlling the Cell Cycle written by Kathy Yufeng Wei and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Synthetic biology aims to program cells by applying engineering principles to the design of genetic circuits to control and monitor cellular behavior. Programmed cells can harness existing dynamically coordinated biologically processes to rescue cellular malfunctions, control existing cellular morphology, and introduce useful novel functionality. Recent advances in the field are leading to genetic circuits of higher-order complexity in mammalian cells, which may ultimately be used to study and reprogram phenotypes that play a key role in human health and disease, such as those linked to the cell cycle. RNA-based control devices can provide specific and modular control of gene expression in response to endogenous or exogenous inputs in living cells. Specifically, we show that for a ribozyme-based device platform, ribozyme switches previously prototyped in yeast are able to regulate gene expression in a predictable (R-squared = 0.63--0.97), ligand-responsive manner in human HEK 293, HeLa, and U2-OS cell lines without any change to device sequence nor further optimization. We also show that these simple, general-purpose components (an RNA ribozyme coupled to an RNA aptamer) are a compact (~200 nt) and effective way to control a complex phenomenon such as arresting up to 80% of a population of mammalian cells in the G2/M phase of the cell cycle. Current methods for altering cell cycle progression can present a number of limitations, including exhibiting specificity towards particular cell types, being broadly disruptive of cellular processes, and being limited in capacity to extend to different networks. A genetically encoded system supporting control over cell cycle progression has the potential to address limitations with existing chemical approaches. Further, these switches are modular and readily adapted to arrest cells in the G0/1 phase of the cell cycle and in response to at least two different small molecule inputs. This work describes the first example of the application of synthetic RNA-based gene-control devices to the regulation of cell cycle progression in human cells. We further show that siRNAs can induce the arrest of> 90% of a population of cells in the G0/1 phase of the cell cycle. This indicates the potential to build miRNA-based devices to control the cell cycle that complement the ribozyme-based approaches. Thus, ligand-responsive RNAs represent a class of synthetic biology tools that are both genetically encoded and capable of regulating intracellular protein levels in response to user-specified molecular signals, and can be adapted for sophisticated control of complex processes in human cells.

Download or read book Synthetic Biology written by Christina Smolke and published by John Wiley & Sons. This book was released on 2018-02-28 with total page 532 pages. Available in PDF, EPUB and Kindle. Book excerpt: A review of the interdisciplinary field of synthetic biology, from genome design to spatial engineering. Written by an international panel of experts, Synthetic Biology draws from various areas of research in biology and engineering and explores the current applications to provide an authoritative overview of this burgeoning field. The text reviews the synthesis of DNA and genome engineering and offers a discussion of the parts and devices that control protein expression and activity. The authors include information on the devices that support spatial engineering, RNA switches and explore the early applications of synthetic biology in protein synthesis, generation of pathway libraries, and immunotherapy. Filled with the most recent research, compelling discussions, and unique perspectives, Synthetic Biology offers an important resource for understanding how this new branch of science can improve on applications for industry or biological research.

Download or read book Synthetic Metabolic Pathways written by Michael Krogh Jensen and published by Humana Press. This book was released on 2017-11-24 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume outlines key steps associated with the design, building, and testing of synthetic metabolic pathways for optimal cell factory performance and robustness, and illustrates how data-driven learning from these steps can be used for rational cost-effective engineering of cell factories with improved performance. Chapters are divided into four sections focusing on the four steps of the iterative design-build-test-learn cycle related to modern cell factory engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Synthetic Metabolic Pathways: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Download or read book RNA level Controllers for Programmable Gene Expression in Mammalian Cells written by Katherine Ilia and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Synthetic biology is a burgeoning field that aims to design circuits using biomolecular components in order to equip cells with desired functionality for a variety of applications, such as medicine, biofuel production, and environmental health. As this field matures, engineers are taking on challenges that require the integration of regulatory logic in complex environments. While there has been significant progress in synthetic biology, there remains a need for genetic devices that allow for precise control over endogenously and exogenously expressed genes. Here, through two examples, we demonstrate that RNA is well-suited for engineering compact, programmable biomolecular tools with sense and actuate functionalities. In the first half of this thesis, we develop a microRNA-based strategy to precisely overwrite the expression level of an endogenous gene of interest, thereby insulating the expression levels of this gene from interference by the endogenous transcriptional unit. We incorporate this strategy into genetic controllers and leverage live cell imaging to develop a versatile strategy for probing the role of transcription factor dynamics on and enforcing the levels of transcription factors in cell fate transitions. In the second half of this thesis, we engineer programmable single-transcript RNA sensors in vivo, in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. This system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We anticipate that these approaches have extensive applications in cell- and RNA-therapeutics as well as for basic research, illustrating the potential of programmable RNA-based controllers.

Download or read book Cell Free Synthetic Biology written by Jian Li and published by Frontiers Media SA. This book was released on 2022-01-13 with total page 205 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Strategies and Technologies for Engineering Gene regulatory RNA Devices written by Andrew B. Kennedy and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Engineered biological systems will provide solutions to diverse global challenges, enabling new and enhanced products for application in chemical processing, materials synthesis, sustainable technologies, and human health. Ability to manipulate and probe biological systems is limited by our ability to noninvasively access, process, report on, and respond to information encoded in the properties of molecules in living systems. Developing genetically encoded information processing and control technologies is critical to addressing and overcoming fundamental challenges in basic and applied biomedical research. RNA poses a strong candidate for a substrate in which to build genetic control devices. Examples of functional RNA molecules playing key roles in controlling the behavior of natural biological systems have grown over the past decade. The relative ease in modeling RNA molecules has enabled design of synthetic counterparts, act with diverse function as components including sensors, regulators, controllers (ligand-responsive RNA regulators), and scaffolds. These synthetic regulatory RNAs are providing new tools for temporal and spatial control in biological systems. A modular platform was described for the construction of RNA devices composed of distinct domains that encode sensing, transmission and actuation functions. The sensor domain is composed of an RNA aptamer, a nucleic acid structure evolved in vitro to bind with high affinity to a given ligand. The actuator domain is composed of a hammerhead ribozyme, which self-cleaves at a specific sequence under proper secondary and tertiary folding. The transmitter domain couples the sensor and actuator domains and communicates the ligand-bound conformational state of the sensor domain to the actuator domain by affecting the folded state of the actuator into either its ribozyme-active or -inactive conformation. The devices are placed in the 3' untranslated region of a target transcript, where self-cleavage inactivates the transcript, thereby lowering gene expression. This framework has been extended to the assembly of devices exhibiting higher-order information processing operations, including logic gates, signal filters and programmed cooperativity. As a demonstration of the broader significance of this class of devices, they have been successfully implemented as biological control systems to regulate signaling pathways and clinically-relevant phenotypes. However, it is critical to develop an improved understanding of the underlying molecular mechanisms and parameters guiding the activities of these devices in vivo in order to develop improved design strategies and associated regulatory activity to extend the utility of these genetic devices for a broader range of applications. To advance the RNA device design, we have developed and implemented novel methods to measure important parameters. We characterized the causal relationship between the in vitro device cleavage rate constant device parameter and in vivo gene- regulatory activity, and utilized this for efficient device performance characterization and design. We described a have novel, two-color, in vivo fluorescent activated cell sorting-based approach to identify sequences that yielded improved catalytic activities within the device platform and enabled efficient tailoring of device regulatory activities. We further developed a quantitative assay based on surface plasmon resonance technology for rapid measurements of device cleavage and ligand binding, the important parameters governing the underlying device mechanism. We incorporate this assay into the RNA device design cycle, pre-filtering candidate devices by in vitro cleavage and binding activity for subsequent in vivo testing. By this method we efficiently developed of new protein-responsive RNA devices in both yeast and mammalian hosts. Finally, we devised a platform utilizing next generation sequencing and fluorescent activated cell sorting for simultaneous measurements of in vitro cleavage and in vivo gene-regulatory activities of large RNA device libraries. Enabled by the large screening capacity, we develop a new RNA device architecture, with modularity instilled at the design level. By this approach we generate RNA devices without a programmed conformational change that results in improved device gene- regulatory performance. Taken together, these new technologies for characterizing important device performance characters and resultant gene-regulatory activity provide a comprehensive framework for designing, testing and implementing RNA genetic controllers for engineering biological systems.

Download or read book Cell Biology by the Numbers written by Ron Milo and published by Garland Science. This book was released on 2015-12-07 with total page 400 pages. Available in PDF, EPUB and Kindle. Book excerpt: A Top 25 CHOICE 2016 Title, and recipient of the CHOICE Outstanding Academic Title (OAT) Award. How much energy is released in ATP hydrolysis? How many mRNAs are in a cell? How genetically similar are two random people? What is faster, transcription or translation?Cell Biology by the Numbers explores these questions and dozens of others provid

Download or read book Molecular Biology of The Cell written by Bruce Alberts and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Mammalian Synthetic Biology written by Jamie Davies and published by . This book was released on 2019-12-12 with total page 137 pages. Available in PDF, EPUB and Kindle. Book excerpt: Written primarily for mid-to-upper level undergraduates, this primer will introduce students to topics at the forefront of the subject that are being applied to probe biological problems, or to address the most pressing issues facing society. These topics will include those that form thecornerstone of contemporary research, helping students to make the transition to active researcher.This primer introduces the challenges and opportunities of applying synthetic biological techniques to mammalian cells, tissues, and organisms. It covers the special features that make engineering mammalian systems different from engineering bacteria, fungi, and plants, and provides an overview ofcurrent techniques. A variety of cutting-edge examples illustrate the different purposes of mammalian synthetic biology, including pure biomedical research, drug production, tissue engineering, and regenerative medicine.

Download or read book Scalable Genetic System Design Using Synthetic RNA Regulators written by Lei Qi and published by . This book was released on 2012 with total page 352 pages. Available in PDF, EPUB and Kindle. Book excerpt: Our ability to efficiently and predictably program cells is central to the fields of bioengineering and synthetic biology. Once thought to be a passive carrier of genetic information, RNA is now more appreciated as the main organizer of cellular networks. To harness the unique abilities of RNA molecules for programming cells, we show here how to rationally design novel synthetic RNA elements to recapitulate the functions of natural noncoding RNAs (ncRNAs), and how to assemble these synthetic elements into higher-order biological systems. To create synthetic RNA elements, we start with two primary types of ncRNA-mediated natural systems. Both modulate RNA-level regulatory signals encoded in the 5' untranslated region, and are mediated by ncRNAs. In the first system the ncRNA represses transcription elongation, whereas in the second system the ncRNA inhibits translation initiation. To create orthogonal RNA elements that work independently in the same cell, we systematically modify the RNA-RNA interaction in the natural systems. Our characterization results in families of orthogonally acting RNA elements for both transcription and translation controls. Furthermore, we develop mathematical thermodynamic models to predict new RNA elements in silico for translation controls. To engineer synthetic RNAs to sense and integrate cellular signals, we design allosteric RNA chimera molecules by fusing ncRNAs to RNA aptamers. We demonstrate the design principles for creating such chimeric RNA molecules that can sense proteins or small molecules and control transcription or translation. We show that the design strategy is modular, which allows us to reconfigure different ncRNA mutants and RNA aptamers to engineer orthogonal RNA chimeras that respond to different ligands and regulate different gene targets. We further show that multiple RNA chimeras allow logical integration of molecular signals in the same cell for cellular information processing. We assemble multiple synthetic RNA elements to create basic regulatory network motifs. These include independent control, logic control, and cascading control. We characterize the performance and properties of these engineered RNA circuits such as their time response, signal sensitivity, and noises across cell populations. We further explore a strategy that can effectively convert orthogonal translational regulators into orthogonal transcriptional regulators, which can be used to perform multi-input logic computation. In an effort to engineer feedback circuits, we demonstrate the use of translational repressor and activator based on RNA-binding proteins. The designed positive or negative feedback circuits form a basis for programming complex functions. To improve the predictability of engineered biological systems, we develop a synthetic RNA processing platform from the bacterial CRISPR genetic immune pathway. The synthetic RNA processing system can efficiently and specifically cleave desired precursor mRNAs at designed loci. Using this system, we show that transcript cleavage enables quantitative programming of gene expression by modular assembly of promoters, ribosome binding sites, cis regulatory elements, and riboregulators. These basic components can be grouped into multi-gene synthetic operons that behave predictably only after RNA processing. Physical separation of otherwise linked elements within biological assemblies allows design of sophisticated RNA-level regulatory systems that are not possible without it. Thus, our results exemplify a crucial design principle based on controllable RNA processing for improving the modularity and reliability of genetic systems. To sum, our work established bacterial ncRNAs as an intriguing engineering substrate for scalable genetic circuit design and for programming cells. We provide a set of engineering principles for designing synthetic RNA elements as well as using them to sense signals and form genetic circuits. Our RNA-based engineering platform provides a versatile and powerful strategy for designing higher-order cellular information processing and computation systems, which can be readily applied to practical applications including chemical production, environment remediation, and therapeutics.

Download or read book Synthetic Gene Networks written by Wilfried Weber and published by Humana Press. This book was released on 2011-11-17 with total page 393 pages. Available in PDF, EPUB and Kindle. Book excerpt: The rapid expansion of synthetic biology is due to the design and construction of synthetic gene networks that have opened many new avenues in fundamental and applied research. Synthetic Gene Networks: Methods and Protocols provides the necessary information to design and construct synthetic gene networks in different host backgrounds. Divided into four convenient sections, this volume focuses on design concepts to devise synthetic gene networks and how mathematical models can be applied to the predictable engineering of desired network features. The volume continues by highlighting the construction and validation of biologic tools, describing strategies to optimize and streamline the host cell for optimized network performance, and covering how optimally designed gene networks can be implemented in a large variety of host cells ranging from bacteria over yeast and insect cells to plant and mammalian cell culture. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Synthetic Gene Networks: Methods and Protocols serves as an invaluable resource for established biologists, engineers, and computer scientists or novices just entering into the rapidly growing field of synthetic biology

Download or read book High throughput Strategies for the Scalable Generation of RNA Component Functions written by Joe Chih Yao Liang and published by . This book was released on 2012 with total page 424 pages. Available in PDF, EPUB and Kindle. Book excerpt: Engineered biological systems hold great promise in providing solutions to many global challenges, including environmental remediation, sustainability, scalable manufacturing, and health and medicine. Synthetic biology is an emerging research field with a primary goal of making the engineering of biology more streamlined and reliable. Recent advances in synthetic RNA biology have led to design of RNA-based gene-regulatory devices from assembly of functional RNA components that encode more basic functions, including sensing, information transmission, and actuation functions. These synthetic RNA control devices allow access and control information on cellular state, thereby advancing our ability to interact with and program biology. A modular ribozyme device platform was recently described to link an aptamer (sensor) to a hammerhead ribozyme (actuator) through a distinct sequence (information transmitter) capable of a strand-displacement event. The utilization of ribozyme as the actuator in the platform, whose mechanism of action is independent of cell-specific machinery, allows transport of the resultant devices to in vitro or different cellular environments. The broad implementation of these devices requires enabling technologies to support efficient generation of new functional RNA components and quantitative tailoring of device regulatory performance for specific cellular applications. Current component generation and device tailoring strategies are limited in their throughputs and efficiencies, and thus have hampered our ability to generate new ribozyme devices for cellular engineering applications. To support scalable generation and tailoring of ribozyme devices, we have described high-throughput in vitro selection and in vivo screening strategies based on the modular ribozyme device platform. We proposed a high-throughput solution-based in vitro selection strategy to generate new sensing functions within the device platform. A high-throughput and quantitative two-color FACS-based screening strategy was developed to complement the in vitro selection strategy by allowing efficient tailoring of device regulatory activities in the cellular environments. We further developed quantitative assays based on the surface plasmon resonance (SPR) technology to allow rapid measurements of the device and component activities. Together, these enabling strategies will offer a scalable and integrated process for the construction and programming of RNA control devices for broad cellular engineering applications, thus laying an important foundation for engineering more complex biological systems.