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Performance variability is a common challenge in cell-free protein production and hinders a wider adoption of these systems for both research and biomanufacturing. While the inherent stochasticity and complexity of biology likely contributes to variability, other systematic factors may also play a role, including the source and preparation of the cell extract, the composition of the supplemental reaction buffer, the facility at which experiments are conducted, and the human operator (Cole et al. ACS Synth Biol 8:2080-2091, 2019). Variability in protein production could also arise from differences in the DNA template-specifically the amount of functional DNA added to a cell-free reaction and the quality of the DNA preparation in terms of contaminants and strand breakage. Here, we present protocols and suggest best practices optimized for DNA template preparation and quantitation for cell-free systems toward reducing variability in cell-free protein production.

Engineered molecular circuits that process information in biological systems could address emerging human health and biomanufacturing needs. However, such circuits can be difficult to rationally design and scale. DNA-based strand displacement reactions have demonstrated the largest and most computationally powerful molecular circuits to date but are limited in biological systems due to the difficulty in genetically encoding components. Here, we develop scalable co-transcriptional RNA strand displacement (ctRSD) circuits that are rationally programmed via base pairing interactions. ctRSD addresses the limitations of DNA-based strand displacement circuits by isothermally producing circuit components via transcription. We demonstrate the programmability of ctRSD in vitro by implementing logic and amplification elements, and multi-layer signaling cascades. Further, we show ctRSD kinetics are accurately predicted by a simple model of coupled transcription and strand displacement, enabling model-driven design. We envision ctRSD will enable rational design of powerful molecular circuits that operate in biological systems, including living cells.

Genome-scale engineering holds great potential to impact science, industry, medicine, and society, and recent improvements in DNA synthesis have enabled the manipulation of megabase genomes. However, coordinating and integrating the workflows and large teams necessary for gigabase genome engineering remains a considerable challenge. We examine this issue and recommend a path forward by: 1) adopting and extending existing representations for designs, assembly plans, samples, data, and workflows; 2) developing new technologies for data curation and quality control; 3) conducting fundamental research on genome-scale modeling and design; and 4) developing new legal and contractual infrastructure to facilitate collaboration., Genome-scale engineering requires the integration of a wide range of in silico and in vivo technologies, as well data management procedures and legal infrastructure. Here the authors provide a list of recommendations to address these challenges.

This article reports a process for rapidly prototyping nanofluidic devices, particularly those comprising slits with microscale widths and nanoscale depths, in silicone. This process consists of designing a nanofluidic device, fabricating a photomask, fabricating a device mold in epoxy photoresist, molding a device in silicone, cutting and punching a molded silicone device, bonding a silicone device to a glass substrate, and filling the device with aqueous solution. By using a bilayer of hard and soft silicone, we have formed and filled nanofluidic slits with depths of less than 400 nm and aspect ratios of width to depth exceeding 250 without collapse of the slits. An important attribute of this article is that the description of this rapid prototyping process is very comprehensive, presenting context and details which are highly relevant to the rational implementation and reliable repetition of the process. Moreover, this process makes use of equipment commonly found in nanofabrication facilities and research laboratories, facilitating the broad adaptation and application of the process. Therefore, while this article specifically informs users of the Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST), we anticipate that this information will be generally useful for the nanofabrication and nanofluidics research communities at large, and particularly useful for neophyte nanofabricators and nanofluidicists.

There has been much interest in the dimensional properties of double-stranded DNA (dsDNA) confined to nanoscale environments as a problem of fundamental importance in both biological and technological fields. This has led to a series of measurements by fluorescence microscopy of single dsDNA molecules under confinement to nanofluidic slits. Despite the efforts expended on such experiments and the corresponding theory and simulations of confined polymers, a consistent description of changes of the radius of gyration of dsDNA under strong confinement has not yet emerged. Here, we perform molecular dynamics (MD) simulations to identify relevant factors that might account for this inconsistency. Our simulations indicate a significant amplification of excluded volume interactions under confinement at the nanoscale due to the reduction of the effective dimensionality of the system. Thus, any factor influencing the excluded volume interaction of dsDNA, such as ionic strength, solution chemistry, and even fluorescent labels, can greatly influence the dsDNA size under strong confinement. These factors, which are normally less important in bulk solutions of dsDNA at moderate ionic strengths because of the relative weakness of the excluded volume interaction, must therefore be under tight control to achieve reproducible measurements of dsDNA under conditions of dimensional reduction. By simulating semi-flexible polymers over a range of parameter values relevant to the experimental systems and exploiting past theoretical treatments of the dimensional variation of swelling exponents and prefactors, we have developed a novel predictive relationship for the in-plane radius of gyration of long semi-flexible polymers under slit-like confinement. Importantly, these analytic expressions allow us to estimate the properties of dsDNA for the experimentally and biologically relevant range of contour lengths that is not currently accessible by state-of-the-art MD simulations.

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.

We observed the free diffusion of individual 10 kbp, (1/2)λ (24.2 kbp), and λ (48.5 kbp) double-stranded deoxyribonucleic acid (DNA) molecules in fused-silica nanoslits with depths from 541 to 24 nm using epifluorescence video microscopy. Diffusivity, D, scaled with nanoslit depth, h, according to D ∝ hα, where α was 0.47 ± 0.05, 0.51 ± 0.06, and 0.47 ± 0.05 for 10 kbp, (1/2)λ, and λ DNA, respectively, in disagreement with the value of two-thirds predicted by blob theory. We observed no change in scaling behavior for h less than the persistence length of DNA, as predicted by reflecting rod theory. The scaling of D with DNA length, N, followed D ∝ N−1, indicating hydrodynamic screening in accordance with Rouse dynamics. Our results establish these scaling exponents for smaller h and greater DNA confinement than previous studies.

We have used the flow characteristics of an electrospinning jet to elongate and fix DNA molecules. We embedded and observed fluorescently labeled lambda bacteriophage DNA molecules in polyethylene oxide nanofibers. The embedded DNA molecules were imaged using fluorescence microscopy and found to be stretched to lengths approaching the full dyed contour length.

INTRODUCTION Cells respond to their environment, make decisions, build structures, and coordinate tasks. Underlying these processes are computational operations performed by networks of regulatory proteins that integrate signals and control the timing of gene expression. Harnessing this capability is critical for biotechnology projects that require decision-making, control, sensing, or spatial organization. It has been shown that cells can be programmed using synthetic genetic circuits composed of regulators organized to generate a desired operation. However, the construction of even simple circuits is time-intensive and unreliable. RATIONALE Electronic design automation (EDA) was developed to aid engineers in the design of semiconductor-based electronics. In an effort to accelerate genetic circuit design, we applied principles from EDA to enable increased circuit complexity and to simplify the incorporation of synthetic gene regulation into genetic engineering projects. We used the hardware description language Verilog to enable a user to describe a circuit function. The user also specifies the sensors, actuators, and “user constraints file” (UCF), which defines the organism, gate technology, and valid operating conditions. Cello (www.cellocad.org) uses this information to automatically design a DNA sequence encoding the desired circuit. This is done via a set of algorithms that parse the Verilog text, create the circuit diagram, assign gates, balance constraints to build the DNA, and simulate performance. RESULTS Cello designs circuits by drawing upon a library of Boolean logic gates. Here, the gate technology consists of NOT/NOR logic based on repressors. Gate connection is simplified by defining the input and output signals as RNA polymerase (RNAP) fluxes. We found that the gates need to be insulated from their genetic context to function reliably in the context of different circuits. Each gate is isolated using strong terminators to block RNAP leakage, and input interchangeability is improved using ribozymes and promoter spacers. These parts are varied for each gate to avoid breakage due to recombination. Measuring the load of each gate and incorporating this into the optimization algorithms further reduces evolutionary pressure. Cello was applied to the design of 60 circuits for Escherichia coli , where the circuit function was specified using Verilog code and transformed to a DNA sequence. The DNA sequences were built as specified with no additional tuning, requiring 880,000 base pairs of DNA assembly. Of these, 45 circuits performed correctly in every output state (up to 10 regulators and 55 parts). Across all circuits, 92% of the 412 output states functioned as predicted. CONCLUSION Our work constitutes a hardware description language for programming living cells. This required the co-development of design algorithms with gates that are sufficiently simple and robust to be connected by automated algorithms. We demonstrate that engineering principles can be applied to identify and suppress errors that complicate the compositions of larger systems. This approach leads to highly repetitive and modular genetics, in stark contrast to the encoding of natural regulatory networks. The use of a hardware-independent language and the creation of additional UCFs will allow a single design to be transformed into DNA for different organisms, genetic endpoints, operating conditions, and gate technologies.

A complex entropy gradient for confined DNA molecules was engineered for the first time. Following the second law of thermodynamics, this enabled the directed self-transport and self-concentration of DNA molecules. This new nanofluidic method is termed entropophoresis. As implemented in experiments, long DNA molecules were dyed with cyanine dimers, dispersed in a high ionic strength buffer, and confined by a nanofluidic channel with a depth profile approximated by a staircase function. The staircase step depths spanned the transition from strong to moderate confinement. The diffusion of DNA molecules across slitlike steps was ratcheted by entropic forces applied at step edges, so that DNA molecules descended and collected at the bottom of the staircase, as observed by fluorescence microscopy. Different DNA morphologies, lengths, and stoichiometric base pair to dye molecule ratios were tested and determined to influence the rate of transport by entropophoresis. A model of ratcheted diffusion was used to interpret a shifting balance of forces applied to linear DNA molecules of standard length in a complex free energy landscape. Related metrics for the overall and optimum performance of entropophoresis were developed. The device and method reported here transcend current limitations in nanofluidics and present new possibilities in polymer physics, biophysics, separation science, and lab-on-a-chip technology.

A nonequilibrium regime of size-based separation was observed experimentally for double-stranded deoxyribonucleic acid (DNA) molecules with lengths below 1 kbp moving electrokinetically through nanofluidic nanoslit arrays. The breakdown of Ogston sieving was supplanted at higher electric fields to recover rapid separation with a reversed elution order and elution times one to two orders of magnitude faster than with Ogston sieving at lower fields. A simple kinetic model describes the experimental results.

We have used the flow characteristics of an electrospinning jet to elongate and fix DNA molecules. We embedded and observed fluorescently labeled lambda bacteriophage DNA molecules in polyethylene oxide nanofibers. The embedded DNA molecules were imaged using fluorescence microscopy and found to be stretched to lengths approaching the full dyed contour length.

Nanofluidic slits are used to investigate surface interactions during electrophoresis between DNA molecules and channel walls. The channels have vertical dimensions of 19 and 70nm and contain no sieving matrix. Size-dependent mobility is observed for DNA in the 19nm channels. We present a model for double stranded DNA mobility in the nanochannels that accurately predicts the size dependence of the DNA mobility in the range of 2000–10000bp. Due to surface interactions, the DNA mobility in the nanochannels scales as N−1∕2. These results suggest that the notion of free solution DNA electrophoresis breaks down due to surface interactions in nanoscale environments.