Portable, On-Demand Biomolecular Manufacturing.

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP.

Paper-based synthetic gene networks.

Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides an alternate, versatile venue for synthetic biologists to operate and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze dried onto paper, enabling the inexpensive, sterile, and abiotic distribution of synthetic-biology-based technologies for the clinic, global health, industry, research, and education. For field use, we create circuits with colorimetric outputs for detection by eye and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small-molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

BACKGROUND: Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. METHODS: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-ß levels increase phosphorylation of ACTN4. RESULTS: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-ß stimulates phosphorylation of ACTN4 at S159 in podocytes. CONCLUSIONS: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

Efficient discovery of antibody binding pairs using a photobleaching strategy for bead encoding.

Dye-encoded bead-based assays are widely used for diagnostics. Multiple bead populations are required for multiplexing and can be produced using different dye colors, labeling levels, or combinations of dye ratios. Ready-to-use multiplex bead populations restrict users to specific targets, are costly, or require specialized instrumentation. In-house methods produce few bead plexes or require many fine-tuning steps. To expand bead encoding strategies, we present a simple, safe, and cost-effective bench-top system for generating bead populations using photobleaching. By photobleaching commercially available dye-encoded magnetic beads for different durations, we produce three times as many differentiable bead populations on flow cytometry from a single dye color. Our photobleaching system uses a high-power LED module connected to a light concentrator and a heat sink. The beads are photobleached in solution homogeneously by constant mixing. We demonstrate this photobleaching method can be utilized for cross-testing antibodies, which is the first step in developing immunoassays. The assay uses multiple photobleached encoded beads conjugated with capture antibodies to test many binding pairs simultaneously. To further expand the number of antibodies that can be tested at once, several antibodies were conjugated to the same bead, forming a pooled assay. Our assay predicts the performance of antibody pairs used in ultrasensitive Simoa assays, narrowing the number of cross-tested pairs that need to be tested by at least two-thirds and, therefore, providing a rapid alternative for an initial antibody pair screening. The photobleaching system can be utilized for other applications, such as multiplexing, and for photobleaching other particles in solution.

Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples.

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2-99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7-100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.

BioBits™ Explorer: A modular synthetic biology education kit.

Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.

Reactive oxygen species facilitate translocation of hormone sensitive lipase to the lipid droplet during lipolysis in human differentiated adipocytes.

In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies.