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.

Emulation of Colonic Oxygen Gradients in a Microdevice.

Gut-on-a-chip in vitro modeling is an emerging field, as the human gut epithelium and gut microbiome have been recently identified as novel drug targets for a wide variety of diseases. Realistic in vitro gut models require a variety of precise environmental cues, such as chemical and gas gradients, in combination with substrates like mucus that support the growth of microbial communities. This technical brief describes a microfluidic architecture capable of developing a physiologically relevant oxygen gradient that emulates the oxygen profile proximal to the epithelial inner lining of the human colon. The device generates stable and repeatable defined oxygen gradients from 0% to 4 % partial pressure O2 over a length scale of hundreds of microns, and was applied to study the effects of oxygenation on the structure of native mucus that lines the colon wall. Using simulation as a design tool for hybrid gas-liquid microfluidic devices enables on-chip creation of defined, physiologically oxygen gradients. These microfluidic architectures have powerful potential applications for gut physiology, including providing optimal oxygenation conditions for the culture of mammalian epithelial cells in the gut lining, as well as creating a realistic mimic of the oxygen gradient found in the intestinal lumen for complex microbiome cultures.

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.

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.

3D Printed Multimaterial Microfluidic Valve.

We present a novel 3D printed multimaterial microfluidic proportional valve. The microfluidic valve is a fundamental primitive that enables the development of programmable, automated devices for controlling fluids in a precise manner. We discuss valve characterization results, as well as exploratory design variations in channel width, membrane thickness, and membrane stiffness. Compared to previous single material 3D printed valves that are stiff, these printed valves constrain fluidic deformation spatially, through combinations of stiff and flexible materials, to enable intricate geometries in an actuated, functionally graded device. Research presented marks a shift towards 3D printing multi-property programmable fluidic devices in a single step, in which integrated multimaterial valves can be used to control complex fluidic reactions for a variety of applications, including DNA assembly and analysis, continuous sampling and sensing, and soft robotics.

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.

Barriers to drug delivery in interventional oncology.

Although much attention has been paid to mechanisms of anticancer drug resistance that focus on intracellular processes that protect tumor cells, it has recently become increasingly evident that the unique features of the tumor microenvironment profoundly impact the efficacy of cancer therapies. The properties of this extracellular milieu, including increased interstitial pressure, decreased pH, hypoxia, and abnormal vascularity, result in limited drug efficacy; this finding is true not only for systemic chemotherapy but also for catheter-based therapies, including chemoembolization and radioembolization. The present review summarizes the barriers to drug delivery imposed by the tumor microenvironment and provides methods to overcome these hurdles.

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.