Programming cells by multiplex genome engineering and accelerated evolution.

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection.

Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.

A Methodology for the Assessment and Prioritization of Genetic Biocontainment Technologies for Engineered Microbes.

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.

DNA construction: homemade or ordered out?

Automation and optimization of DNA construction results in the efficient production of large target sequences.

Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA.

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American's wildlife populations.

Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA.

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes ( Vulpes vulples and Urocyon cineroargentus , respectively), fishers ( Martes pennati ), river otters ( Lutra canadensis ), coyotes ( Canis lantrans ), bobcats ( Lynx rufus rufus ), black bears ( Ursus americanus ), and white-tailed deer ( Odocoileus virginianus ). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Our results indicate that no sampled wildlife were positive for SARS-CoV-2. This finding is surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American's wildlife populations.

Measurement of Transcription, Translation, and Other Enzymatic Processes During Cell-Free Expression Using PERSIA.

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.

Parallel gene synthesis in a microfluidic device.

The ability to synthesize custom de novo DNA constructs rapidly, accurately and inexpensively is highly desired by researchers, as synthetic genes and longer DNA constructs are enabling to numerous powerful applications in both traditional molecular biology and the emerging field of synthetic biology. However, the current cost of de novo synthesis-driven largely by reagent and handling costs-is a significant barrier to the widespread availability of such technology. In this work, we demonstrate, to our knowledge, the first gene synthesis in a microfluidic environment. The use of microfluidic technology greatly reduces reaction volumes and the corresponding reagent and handling costs. Additionally, microfluidic technology enables large numbers of complex reactions to be performed in parallel. Here, we report the fabrication of a multi-chamber microfluidic device and its use in carrying out the syntheses of several DNA constructs. Genes up to 1 kb in length were synthesized in parallel at minute starting oligonucleotide concentrations (10-25 nM) in four 500 nl reactors. Such volumes are one to two orders of magnitude lower than those utilized in conventional gene synthesis. The identity of all target genes was verified by sequencing, and the resultant error rate was determined to be 1 per 560 bases.

A Makerspace for Life Support Systems in Space.

Human space exploration and settlement will require leaps forward in life support for environmental management and healthcare. Life support systems must efficiently use nonrenewable resources packed from Earth while increasingly relying on resources available locally in space. On-demand production of components and materials (e.g., 3D printing and synthetic biology) holds promise to satisfy the evolving set of supplies necessary to outfit human missions to space. We consider here life support systems for missions planned in the 2020s, and discuss how the maker and 'do-it-yourself' (DIY) biology communities can develop rapid, on-demand manufacturing techniques and platforms to address these needs. This Opinion invites the diverse maker community into building the next generation of flight hardware for near-term space exploration.

PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems.

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.

Standardizing Automated DNA Assembly: Best Practices, Metrics, and Protocols Using Robots.

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.

Enabling Microfluidics: from Clean Rooms to Makerspaces.

The traditional requirement for clean rooms and specialized skills has inhibited many biologists from pursuing new microfluidic innovations. Makerspaces provide a growing alternative to clean rooms: they provide low-cost access to fabrication equipment such as laser cutters, plotter cutters, and 3D printers; use commercially available materials; and attract a diverse community of product designers. This Opinion discusses the materials, tools, and building methodologies particularly suited for developing novel microfluidic devices in these spaces, with insight into biological applications and leveraging the maker community. The lower barrier to access of makerspaces ameliorates the otherwise poor accessibility and scalability of microfluidic prototyping.

Open-source, community-driven microfluidics with Metafluidics.

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.

Protein-mediated error correction for de novo DNA synthesis.

The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10 000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.

Multiplexed Sequence Encoding: A Framework for DNA Communication.

Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication-data encoding, data transfer & data extraction-and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system-Multiplexed Sequence Encoding (MuSE)-that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA.

Direct Analysis of Gene Synthesis Reactions Using Solid-State Nanopores.

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.

In situ microfluidic SERS assay for monitoring enzymatic breakdown of organophosphates.

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.

DNA Assembly in 3D Printed Fluidics.

The process of connecting genetic parts-DNA assembly-is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to $5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

Genomically recoded organisms expand biological functions.

We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

Precise manipulation of chromosomes in vivo enables genome-wide codon replacement.

We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.