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35
A synthetic biology framework for programming eukaryotic transcription functions
- Cell
, 2012
"... SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found with ..."
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SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.
GS: DNA assembly for synthetic biology: from parts to pathways and beyond
- Integr Biol (Camb
"... The assembly of DNA from small fragments into large constructs has seen significant recent development, becoming a pivotal technology in the ability to implement the vision of synthetic biology. As the cost of whole gene synthesis is decreasing, whole genome synthesis at the other end of the spectru ..."
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Cited by 21 (1 self)
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The assembly of DNA from small fragments into large constructs has seen significant recent development, becoming a pivotal technology in the ability to implement the vision of synthetic biology. As the cost of whole gene synthesis is decreasing, whole genome synthesis at the other end of the spectrum has expanded our horizons to the prospect of fully engineered synthetic cells. However, the recently proven ability to synthesise genome-scale DNA is at odds with our ability to rationally engineer biological devices, which lags significantly behind. Most work in synthetic biology takes place on an intermediate scale with the combinatorial construction of networks and metabolic pathways from registries of modular biopart components. Implementation for rapid prototyping of engineered biological circuits requires quick and reliable DNA assembly according to specific architectures. It is apparent that DNA assembly is now a limiting technology in advancing synthetic biology. Current techniques employ standardised restriction enzyme assembly protocols such as BioBrickst, BglBricks and Golden Gate methods. Alternatively, sequence-independent overlap techniques, such as In-Fusiont, SLIC and Gibson isothermal assembly are becoming popular for larger assemblies, and in vivo DNA assembly in yeast and bacillus appears adept for chromosome fabrication. It is important to consider how the use of different technologies may impact the outcome of a construction, since the assembly technique can direct the architecture and diversity of systems that can be made. This review provides a critical examination of recent DNA assembly strategies and considers how this important facilitating aspect of synthetic biology may proceed in the future.
Automatic compilation from high-level biologically-oriented programming language to genetic regulatory networks
- PLoS ONE
, 2011
"... Background: The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic ..."
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Cited by 17 (7 self)
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Background: The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry. Methodology/Principal Findings: To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes (*50%) and latency of the optimized engineered gene networks.
Evolved orthogonal ribosome purification for in vitro characterization
- Nucleic Acids Res
, 2010
"... We developed orthogonal ribosomemRNA pairs in which the orthogonal ribosome (O-ribosome) spe-cifically translates the orthogonal mRNA and the orthogonal mRNA is not a substrate for cellular ribosomes. O-ribosomes have been used to create new cellular circuits to control gene expression in new ways, ..."
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Cited by 7 (0 self)
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We developed orthogonal ribosomemRNA pairs in which the orthogonal ribosome (O-ribosome) spe-cifically translates the orthogonal mRNA and the orthogonal mRNA is not a substrate for cellular ribosomes. O-ribosomes have been used to create new cellular circuits to control gene expression in new ways, they have been used to provide new information about the ribosome, and they form a crucial part of foundational technologies for genetic code expansion and encoded and evolvable polymer synthesis in cells. The production of O-ribosomes in the cell makes it challenging to study the properties of O-ribosomes in vitro, because no method exists to purify functional O-ribosomes from cellular ribosomes and other cellular components. Here we present a method for the affinity purification of O-ribosomes, via tagging of the orthogonal 16S ribosomal RNA. We demonstrate that the purified O-ribosomes are pure by primer extension assays, and structurally homogenous by gel electrophoresis and sucrose gradients. We demonstrate the utility of this purifi-cation method by providing a preliminary in vitro characterization of Ribo-X, an O-ribosome previously evolved for enhanced unnatural amino acid incorporation in response to amber codons. Our data suggest that the basis of Ribo-X’s in vivo activity is a decreased affinity for RF1.
Synthetic human cell fate regulation by protein-driven RNA switches
"... Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its bin ..."
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Cited by 7 (2 self)
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Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks. 1
One-pot DNA construction for synthetic biology: The Modular Overlap-Directed Assembly with Linkers (MODAL) strategy. Nucleic Acids Res
, 2013
"... Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key prin-ciple of engineering: mod ..."
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Cited by 6 (1 self)
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Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key prin-ciple of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modular-ity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of syn-thetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of con-struct libraries valuable for synthetic biology applications.
Sensors for micro bio robots via synthetic biology
- in Robotics and Automation (ICRA), 2014 IEEE Intl. Conf., pp.3783–3788, May 31 2014 – June 7 2014. doi: 10.1109/ICRA.2014.6907407
"... Abstract Microscale robots offer an unprecedented oppor-tunity to perform tasks at resolutions approaching 1 m, but the great majority of research to this point focuses on actuation and control. Potential applications for microrobots can be considerably expanded by integrating sensing, signal proce ..."
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Cited by 4 (2 self)
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Abstract Microscale robots offer an unprecedented oppor-tunity to perform tasks at resolutions approaching 1 m, but the great majority of research to this point focuses on actuation and control. Potential applications for microrobots can be considerably expanded by integrating sensing, signal processing and feedback into the system. In this work, we demonstrate that technologies from the eld of synthetic biology may be directly integrated into microrobotic systems to create cell-based programmable mobile sensors, with signal processors and memory units. Specically, we integrate genetically engineered, ultraviolet light-sensing bacteria with magnetic microrobots, creating the rst controllable biological microrobot that is capable of exploring, recording and reporting on the state of the microscale environment. We demonstrate two proof-of-concept prototypes: (a) an integrated microrobot platform that is able to sense biochemical signals, and (b) a microrobot platform that is able to deploy biosensor payloads to monitor biochemical signals, both in a biological environment. These results have important implications for integrated micro-bio-robotic systems for applications in biological engineering and research. I.
Tuning response curves for synthetic biology
- ACS Synth. Biol
, 2013
"... ABSTRACT: Synthetic biology may be viewed as an effort to establish, formalize, and develop an engineering discipline in the context of biological systems. The ability to tune the properties of individual components is central to the process of system design in all fields of engineering, and synthet ..."
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Cited by 3 (0 self)
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ABSTRACT: Synthetic biology may be viewed as an effort to establish, formalize, and develop an engineering discipline in the context of biological systems. The ability to tune the properties of individual components is central to the process of system design in all fields of engineering, and synthetic biology is no exception. A large and growing number of approaches have been developed for tuning the responses of cellular systems, and here we address specifically the issue of tuning the rate of response of a system: given a system where an input affects the rate of change of an output, how can the shape of the response curve be altered experimentally? This affects a system’s dynamics as well as its steady-state properties, both of which are critical in the design of systems in synthetic biology, particularly those with multiple components. We begin by reviewing a mathematical formulation that captures a broad class of biological response curves and use this to define a standard set of varieties of tuning: vertical shifting, horizontal scaling, and the like. We then survey the experimental literature, classifying the results into our defined categories, and organizing them by regulatory level: transcriptional, post-transcriptional, and post-translational.
GenoCAD: linguistic approaches to synthetic biology
, 2010
"... Synthetic biology is an emerging interdisciplinary research field, which leverages the mat-uration of DNA synthesis technologies. By introducing engineering principles to synthetic biological systems design, synthetic biology shows great potential to shed new lights on biol-ogy and benefit human bei ..."
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Synthetic biology is an emerging interdisciplinary research field, which leverages the mat-uration of DNA synthesis technologies. By introducing engineering principles to synthetic biological systems design, synthetic biology shows great potential to shed new lights on biol-ogy and benefit human beings. Computer assisted design (CAD) tools will play an important role in the rational design of synthetic genetic systems. This dissertation presents the first CAD tool for synthetic biology – GenoCAD, a linguistic-based web application. By viewing DNA sequences as a language, we developed the first syntactic model to design and verify synthetic genetic constructs. Then we conducted a careful curation of the terminal set in the grammar- the first comprehensive analysis of the Registry of standard biological parts. The implementation and major features of GenoCAD are discussed, and in particular we showed how to develop a domain-specific grammar for BioBrick-based construct design and make GenoCAD a useful tool for the iGEM students. Finally, we went beyond the syntactic level to explore the semantics of synthetic DNA sequences: by associating attributes with biologi-cal parts and coupling semantic actions with grammar rules, we developed the first semantic models to relate the genotype to the phenotype of synthetic genetic constructs. The theories and techniques presented in this dissertation, along with the informative results presented, will serve as a foundation for the future developments of GenoCAD.
Engineering genetic circuits that compute and remember
"... Memory and logic are central to complex state-dependent computing, and state-dependent behaviors are a feature of natural biological systems. recently, we created a platform for integrated logic and memory by using synthetic gene circuits, and we demonstrated the implementation of all two-input log ..."
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Memory and logic are central to complex state-dependent computing, and state-dependent behaviors are a feature of natural biological systems. recently, we created a platform for integrated logic and memory by using synthetic gene circuits, and we demonstrated the implementation of all two-input logic gates with memory in living cells. Here we provide a detailed protocol for the construction of two-input Boolean logic functions with concomitant Dna-based memory. this technology platform allows for straightforward assembly of integrated logic-and-memory circuits that implement desired behaviors within a couple of weeks. It should enable the encoding of advanced computational operations in living cells, including sequential-logic and biological-state machines, for a broad range of applications in biotechnology, basic science and biosensing.
