Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 64,995 bioRxiv papers from 288,040 authors.
Most downloaded bioRxiv papers, all time
in category synthetic biology
604 results found. For more information, click each entry to expand.
1,201 downloads synthetic biology
Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented timescales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) in V. natriegens by targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ~100 times more PHB than the parent isolate and ~3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.
1,195 downloads synthetic biology
Gene regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function in gene regulatory networks and can be tuned at the level of DNA, protein, and cooperative protein - protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity, specificity, and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription factor binding energy landscapes and mechanistic modeling. By integrating these methods we generated a library of well-characterized synthetic transcription factors and corresponding promoters, and used these standardized parts to build gene regulatory networks de novo in a cell-free environment. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene regulatory networks and for studying the biophysics of transcriptional regulation.
1,193 downloads synthetic biology
The dynamics of the bacterial population that comprises the gut microbiota plays key roles in overall mammalian health. However, a detailed understanding of bacterial growth within the gut is limited by the inherent complexity and inaccessibility of the gut environment. Here, we deploy an improved synthetic genetic oscillator to investigate dynamics of bacterial colonization and growth in the mammalian gut under both healthy and disease conditions. The synthetic oscillator, when introduced into both Escherichia coli and Salmonella Typhimurium maintains regular oscillations with a constant period in generations across growth conditions. We determine the phase of oscillation from individual bacteria using image analysis of resultant colonies and thereby infer the number of cell divisions elapsed. In doing so, we demonstrate robust functionality and controllability of the oscillator circuit's activity during bacterial growth in vitro, in a simulated murine gut microfluidic environment, and in vivo within the mouse gut. We determine different dynamics of bacterial colonization and growth in the gut under normal and inflammatory conditions. Our results show that a precise genetic oscillator can function in a complex environment and reveal single cell behavior under diverse conditions where disease may create otherwise impossible-to-quantify variability in growth across the population.
1,183 downloads synthetic biology
We employ a reporter assay and SHAPE-seq to study translational regulation by RNA-binding proteins, in bacteria. For the reporter assay, we designed 50 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on the position of the hairpin, and exhibits three-nucleotide periodicity. For hairpin positions within the initiation region, in the presence of cognate RNA-binding protein, we observe strong translational repression. In vivo SHAPE-seq results for a representative construct indicate that repression correlates with protection of both the hairpin and the ribosome binding site. Our data suggest that the RBP- hairpin complex entraps the 30S subunit, thereby stalling initiation. We utilize the repression phenomenon in a high-throughput assay for quantitative study of protein-RNA binding in vivo.
1,175 downloads synthetic biology
The construction of complex gene regulatory networks requires both inhibitory and up-regulatory modules. However, the vast majority of RNA-based regulatory parts are inhibitory. Using a synthetic biology approach combined with SHAPE-Seq, we explored the regulatory effect of RBP-RNA interactions in bacterial 5-UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in-vivo: in the absence of RBPs, the RNA molecules can be found either in a molten state that is amenable to translation, or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene regulatory circuits at the post-transcriptional level.
1,172 downloads synthetic biology
Colin JB Harvey, Mancheng Tang, Ulrich Schlecht, Joe Horecka, Curt R Fischer, Hsiao-ching Lin, Jian Li, Brian Naughton, James Cherry, Molly Miranda, Yong Fuga Li, Angela M Chu, James R Hennessy, Gergana A Vandova, Diane Inglis, Raeka Aiyar, Lars M Steinmetz, Ronald W Davis, Marnix H Medema, Elizabeth Sattely, Chaitan Khosla, Robert P St.Onge, Yi Tang, Maureen E Hillenmeyer
For decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.
1,168 downloads synthetic biology
Recording biological signals can be difficult in three-dimensional matrices, such as tissue. We present a DNA polymerase-based strategy that records temporal biosignals locally onto DNA to be read out later, which could obviate the need to extract information from tissue on the fly. We use a template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT) that probabilistically adds dNTPs to single-stranded DNA (ssDNA) substrates without a template. We show that in vitro, the dNTP-incorporation preference of TdT changes with the presence of Co2+, Ca2+, Zn2+ and temperature. Extracting the signal profile over time is possible by examining the dNTP incorporation preference along the length of synthesized ssDNA strands like a molecular ticker tape. We call this TdT-based untemplated recording of temporal local environmental signals (TURTLES). We show that we can determine the time of Co2+ addition to within two minutes over a 60-minute period. Further, TURTLES has the capability to record multiple fluctuations. We can estimate the rise and fall of an input Co2+ pulse to within three minutes. TURTLES has at least 200-fold better temporal resolution than all previous DNA-based recording techniques.
1,158 downloads synthetic biology
Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for cell programming with improved controllability leveraging the fast production and degradation time scales of RNA molecules.
1,157 downloads synthetic biology
Point mutations underlie many genetic diseases. In this regard, while programmable DNA nucleases have been used to repair mutations, their use for gene therapy poses multiple challenges: one, efficiency of homologous recombination is typically low in cells; two, an active nuclease presents a risk of introducing permanent off-target mutations; and three, prevalent programmable nucleases typically comprise elements of non-human origin raising the potential of in vivo immunogenicity. In light of these, approaches to instead directly target RNA, and use of molecular machinery native to the host would be highly desirable. Towards this, we engineered and optimized two complementary approaches, referred together hereon as tRiAD, based on the use of tRNAs in codon suppression and adenosine deaminases in RNA editing. Specifically, by delivering modified endogenous tRNAs and/or the RNA editing enzyme ADAR2 and an associated guiding RNA (adRNA) via adeno-associated viruses, we enabled premature stop codon read-through and correction in the mdx mouse model of muscular dystrophy that harbors a nonsense mutation in the dystrophin gene. We further demonstrated inducible restoration of dystrophin expression by pyrolysyl-tRNA mediated incorporation of unnatural amino acids (UAAs) at the stop codon. Additionally, we also engineered ADAR2 mediated correction of a point mutation in liver RNA of the spfash mouse model of ornithine transcarbamylase (OTC) deficiency. Taken together, our results establish the use of suppressor tRNAs and ADAR2 for in vivo RNA targeting, and this integrated tRiAD approach is robust, genomically scarless, and potentially non-immunogenic as it utilizes effector RNAs and human proteins.
1,148 downloads synthetic biology
The engineering of transcriptional networks presents many challenges due to the inherent uncertainty in the system structure, changing cellular context and stochasticity in the governing dynamics. One approach to address these problems is to design and build systems that can function across a range of conditions; that is they are robust to uncertainty in their constituent components. Here we examine the robustness landscape of transcriptional oscillators, which underlie many important processes such as circadian rhythms and the cell cycle, plus also serve as a model for the engineering of complex and emergent phenomena. The central questions that we address are: Can we build genetic oscillators that are more robust than those already constructed? Can we make genetic oscillators arbitrarily robust? These questions are technically challenging due to the large model and parameter spaces that must be efficiently explored. Here we use a measure of robustness that coincides with the Bayesian model evidence combined with an efficient Monte Carlo method to traverse model space and concentrate on regions of high robustness, which enables the accurate evaluation of the relative structural robustness of gene network models governed by stochastic dynamics. We report the most robust two and three gene oscillator systems, plus examine how the number of interactions, the presence of auto-regulation, and degradation of mRNA and protein affects the frequency, amplitude and robustness of transcriptional oscillators. We also find that there is a limit to parametric robustness, beyond which there is nothing to be gained by adding additional feedback. Importantly, we provide predictions on new oscillator systems that can be constructed to verify the theory and advance design and modelling approaches to systems and synthetic biology.
1,133 downloads synthetic biology
Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.
1,119 downloads synthetic biology
A true engineering framework for synthetic multicellular systems requires a programmable means of cell-cell communication. Such a communication system would enable complex behaviors, such as pattern formation, division of labor in synthetic microbial communities, and improved modularity in synthetic circuits. However, it remains challenging to build synthetic cellular communication systems in eukaryotes due to a lack of molecular modules that are orthogonal to the host machinery, easy to reconfigure, and scalable. Here, we present a novel cell-to-cell communication system in Saccharomyces cerevisiae (yeast) based on CRISPR transcription factors and the plant hormone auxin that exhibits several of these features. Specifically, we engineered a sender strain of yeast that converts indole-3-acetamide (IAM) into auxin via the enzyme iaaH from Agrobacterium tumefaciens. To sense auxin and regulate transcription in a receiver strain, we engineered a reconfigurable library of auxin degradable CRISPR transcription factors (ADCTFs). Auxin-induced degradation is achieved through fusion of an auxin sensitive degron (from IAA co-repressors) to the CRISPR TF and co-expression with an auxin F-box protein. Mirroring the tunability of auxin perception in plants, our family of ADCTFs exhibits a broad range of auxin sensitivities. We characterized the kinetics and steady state behavior of the sender and receiver independently, and in co-cultures where both cell types were exposed to IAM. In the presence of IAM, auxin is produced by the sender cell and triggers de-activation of reporter expression in the receiver cell. The result is an orthogonal, rewireable, tunable, and arguably scalable cell-cell communication system for yeast and other eukaryotic cells.
1,118 downloads synthetic biology
Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. By taking genes from a filamentous fungus and directing their efficient expression and subcellular localisation, we here engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide. Using synthetic biology tools combined with long-read DNA sequencing, we optimise productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.
1,111 downloads synthetic biology
Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a >10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.
1,110 downloads synthetic biology
Gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. Here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits. Using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, AND, OR and NOT biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of Boolean operations that include NIMPLY, XOR, half adder and half subtractor logics in single cell. Subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context dependent effects, including 5′ UTR interference and uncontrolled switch-on behaviour of σ54 promoter. Importantly, this work provides a representative case study to the debugging of genetic context dependent effects through principles elucidated herein, thereby providing a rational design framework to program single prokaryotic cell with diversified digital operations.
1,099 downloads synthetic biology
Negative feedback is known to endow biological and man-made systems with robust performance in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules which can inhibit translation of target messenger RNAs (mRNAs). In this paper, we designed, modelled and built two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA which negatively regulates the translation of the mRNA encoding this output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.
1,098 downloads synthetic biology
Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer synthetic quality control, termed Post-Translational Proofreading (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our work presents a new paradigm for molecular recognition of amino acids in target proteins.
1,088 downloads synthetic biology
CRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.
1,088 downloads synthetic biology
The fast growing bacterium Vibrio natriegens is an emerging microbial host for biotechnology. Harnessing its productive cellular components may offer a compelling platform for rapid protein production and prototyping of metabolic pathways or genetic circuits. Here, we report the development of a V. natriegens cell-free expression system. We devised a simplified crude extract preparation protocol and achieved >260 μg/mL of super-folder GFP in a small-scale batch reaction after three hours. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and protein yield of extracts. We observed maximal protein yield at incubation temperatures of 26°C or 30°C, and show improved yield by tuning ions crucial for ribosomal stability. This work establishes an initial V. natriegens cell-free expression system, enables probing of V. natriegens biology, and will serve as a platform to accelerate metabolic engineering and synthetic biology applications.
1,079 downloads synthetic biology
L. J. Rothschild, D. T. Greenberg, J. R. Takahashi, K. A. Thompson, A. J. Maheshwari, R. E. Kent, G. McCutcheon, J. D. Shih, C. Calvet, T. D. Devlin, T. Ju, D. Kunin, E. Lieberman, T Nguyen, F. G. Tran, D. Xiang, K. Fujishima
The CRISPR/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be applied to facilitate efficient plasmid selection for transformation as well as selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts after restriction enzyme digestion and ligation. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes unwanted ligation byproducts and increases transformation efficiency of desired inserts from 20% up to 97% ± 3%. This CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient method for obtaining specific cloning products.
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