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Introduction: Organisms engineered with synthetic genes and genomes have the potential to play critical roles to address important issues in the environment, human health, and manufacturing. Engineered genetic biocontainment technologies are needed to manage the risks of unintended consequences when deploying these biological systems in consultation with the biosafety and biosecurity communities. Metrics measuring genetic biocontainment and a methodology to apply them are required to determine which genetic biocontainment technologies warrant further development for real-world applications. In this study, we develop and apply a systems analysis of the current technical landscape using expert opinion and a metric-based scoring system resulting in a semiquantitative comparative assessment of genetic biocontainment technologies in microorganisms. Methods: Genetic biocontainment technologies were evaluated according to multiple metrics, falling into two broad classes: feasibility and applicability. Specific genetic biocontainment example scenarios and generalized categories were scored with these metrics. Gap analysis was carried out, indicating particular areas where genetic biocontainment can be improved. Results: Metric analysis scoring of feasibility and applicability enabled prioritization of genetic biocontainment technologies for real-world applications. Gap analysis showed that technology readiness and containment stability scored low for a number of scenarios and categories, indicating a general need for further development before they can be ready for deployment. Conclusion: Developing an assessment framework with defined metrics produced a straightforward system for evaluating genetic biocontainment strategies intended for various real-world applications. Use of the methodology also provided insights into existing gaps in genetic biocontainment strategies, and by altering the metrics, can be applied to other biotechnologies.
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We developed the PERSIA technique with an interest in quantifying proteins as they are being produced during a cell-free synthesis reaction. A short 6-amino acid sequence added to a protein of interest reacts with a fluorogenic reagent (ReAsH), yielding a measure of protein concentration in close to real time. We combine this measurement with simultaneous fluorescent detection of mRNA production, quantifying both transcription and translation. Alternatively, we combine simultaneous measurement of protein synthesis and that protein's enzymatic activity. We have found these simple capabilities enabling for multiple applications, including sequence-structure-function studies and target-specific assessment of drug candidate compounds.
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Biossíntese de Proteínas , Pérsia , RNA Mensageiro/genéticaRESUMO
Quantification of biology's central dogma (transcription and translation) is pursued by a variety of methods. Direct, immediate, and ongoing quantification of these events is difficult to achieve. Common practice is to use fluorescent or luminescent proteins to report indirectly on prior cellular events, such as turning on a gene in a genetic circuit. We present an alternative approach, PURExpress-ReAsH-Spinach In-vitro Analysis (PERSIA). PERSIA provides information on the production of RNA and protein during cell-free reactions by employing short RNA and peptide tags. Upon synthesis, these tags yield quantifiable fluorescent signal without interfering with other biochemical events. We demonstrate the applicability of PERSIA in measuring cell-free transcription, translation, and other enzymatic activity in a variety of applications: from sequence-structure-function studies, to genetic code engineering, to testing antiviral drug resistance.
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Sistema Livre de Células , Biossíntese de Proteínas , Transcrição Gênica , Engenharia Genética/métodos , HIV/enzimologia , Protease de HIV/genética , Protease de HIV/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Fluorescência , Spinacia oleracea/genética , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
The advancement of synthetic biology requires the ability to create new DNA sequences to produce unique behaviors in biological systems. Automation is increasingly employed to carry out well-established assembly methods of DNA fragments in a multiplexed, high-throughput fashion, allowing many different configurations to be tested simultaneously. However, metrics are required to determine when automation is warranted based on factors such as assembly methodology, protocol details, and number of samples. The goal of our synthetic biology automation work is to develop and test protocols, hardware, and software to investigate and optimize DNA assembly through quantifiable metrics. We performed a parameter analysis of DNA assembly to develop a standardized, highly efficient, and reproducible MoClo protocol, suitable to be used both manually and with liquid-handling robots. We created a key DNA assembly metric (Q-metric) to characterize a given automation method's advantages over conventional manual manipulations with regard to researchers' highest-priority parameters: output, cost, and time. A software tool called Puppeteer was developed to formally capture these metrics, help define the assembly design, and provide human and robotic liquid-handling instructions. Altogether, we contribute to a growing foundation of standardizing practices, metrics, and protocols for automating DNA assembly.
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Automação Laboratorial/métodos , Clonagem Molecular/métodos , DNA/genética , Engenharia Genética/métodos , Guias de Prática Clínica como Assunto , Robótica/métodos , Biologia Sintética/métodos , Engenharia Genética/normasRESUMO
Microfluidic devices have the potential to automate and miniaturize biological experiments, but open-source sharing of device designs has lagged behind sharing of other resources such as software. Synthetic biologists have used microfluidics for DNA assembly, cell-free expression, and cell culture, but a combination of expense, device complexity, and reliance on custom set-ups hampers their widespread adoption. We present Metafluidics, an open-source, community-driven repository that hosts digital design files, assembly specifications, and open-source software to enable users to build, configure, and operate a microfluidic device. We use Metafluidics to share designs and fabrication instructions for both a microfluidic ring-mixer device and a 32-channel tabletop microfluidic controller. This device and controller are applied to build genetic circuits using standard DNA assembly methods including ligation, Gateway, Gibson, and Golden Gate. Metafluidics is intended to enable a broad community of engineers, DIY enthusiasts, and other nontraditional participants with limited fabrication skills to contribute to microfluidic research.
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DNA/genética , Redes Reguladoras de Genes/genética , Engenharia Genética/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Software , Biologia Sintética/instrumentação , Algoritmos , Bases de Dados FactuaisRESUMO
Synthetic nucleic acids offer rich potential to understand and engineer new cellular functions, yet an unresolved limitation in their production and usage is deleterious products, which restrict design complexity and add cost. Herein, we employ a solid-state nanopore to differentiate molecules of a gene synthesis reaction into categories of correct and incorrect assemblies. This new method offers a solution that provides information on gene synthesis reactions in near-real time with higher complexity and lower costs. This advance can permit insights into gene synthesis reactions such as kinetics monitoring, real-time tuning, and optimization of factors that drive reaction-to-reaction variations as well as open venues between nanopore-sensing, synthetic biology, and DNA nanotechnology.
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DNA/genética , Genes/genética , Nanoporos , Nanotecnologia/métodos , Análise de Sequência de DNA/métodos , Biologia Sintética/métodos , DNA/química , DNA/metabolismo , Protease de HIV/genética , Protease de HIV/metabolismo , CinéticaRESUMO
In this paper, we report on a method to probe the breakdown of the organophosphate (OP) simulants o,s-diethyl methyl phosphonothioate (OSDMP) and demeton S by the enzyme organophosphorous hydrolase (OPH) in a microfluidic device by surface enhanced Raman spectroscopy (SERS). SERS hotspots were formed on-demand inside the microfluidic device by laser-induced aggregation of injected Ag NPs suspensions. The Ag NP clusters, covering micron-sized areas, were formed within minutes using a conventional confocal Raman laser microscope. These Ag NP clusters were used to enhance the Raman spectra of the thiol products of OP breakdown in the microfluidic device: ethanethiol (EtSH) and (ethylsulfanyl) ethane-1-thiol (2-EET). When the OPH enzyme and its substrates OSDMP and demeton S were introduced, the thiolated breakdown products were generated, resulting in changes in the SERS spectra. With the ability to analyze reaction volumes as low as 20 nL, our approach demonstrates great potential for miniaturization of SERS analytical protocols.
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Arildialquilfosfatase/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Organofosfatos/análise , Análise Espectral Raman/métodos , Biotecnologia/instrumentação , Biotecnologia/métodos , Desenho de Equipamento , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Organofosfatos/química , Organofosfatos/metabolismo , Prata/químicaRESUMO
Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered.