Microbiome single cell atlases generated with a commercial instrument.

Single cell sequencing is useful for resolving complex systems into their composite cell types and computationally mining them for unique features that are masked in pooled sequencing. However, while commercial instruments have made single cell analysis widespread for mammalian cells, analogous tools for microbes are limited. Here, we present EASi-seq (Easily Accessible Single microbe sequencing).

Printhead on a chip: empowering droplet-based bioprinting with microfluidics.

Droplet-based bioprinting has long struggled with the manipulation and dispensation of individual cells from a printhead, hindering the fabrication of artificial cellular structures with high precision. The integration of modern microfluidic modules into the printhead of a bioprinter is emerging as one approach to overcome this bottleneck. This convergence allows for high-accuracy manipulation and spatial control over placement of cells during printing, and enables the fabrication of cell arrays and hierarchical heterogenous microtissues, opening new applications in bioanalysis and high-throughput screening.

Microbiome single cell atlases generated with a commercial instrument.

Single cell sequencing is useful for resolving complex systems into their composite cell types and computationally mining them for unique features that are masked in pooled sequencing. However, while commercial instruments have made single cell analysis widespread for mammalian cells, analogous tools for microbes are limited. Here, we present EASi-seq (Easily Accessible Single microbe sequencing).

Robotic automation of droplet microfluidics.

Droplet microfluidics enables powerful analytic capabilities but often requires workflows involving macro- and microfluidic processing steps that are cumbersome to perform manually. Here, we demonstrate the automation of droplet microfluidics with commercial fluid-handling robotics. The workflows incorporate common microfluidic devices including droplet generators, mergers, and sorters and utilize the robot's native capabilities for thermal control, incubation, and plate scanning.

Dual-layered hydrogels allow complete genome recovery with nucleic acid cytometry.

Targeting specific cells for sequencing is important for applications in cancer, microbiology, and infectious disease. Nucleic acid cytometry (NAC) is a powerful approach for accomplishing this because it allows specific cells to be isolated based on sequence biomarkers that are otherwise impossible to detect. However, existing methods require specialized microfluidic devices, limiting adoption.

Microbowls with Controlled Concavity for Accurate Microscale Mass Spectrometry.

Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces is described. The wells can also concentrate and localize cells and beads for cell-based assays.

Mapping enzyme catalysis with metabolic biosensing.

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry.

Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery.

Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment.

Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses.

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP.

CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window.

The capacity to diversify genetic codes advances our ability to understand and engineer biological systems. A method for continuously diversifying user-defined regions of a genome would enable forward genetic approaches in systems that are not amenable to efficient homology-directed oligonucleotide integration. It would also facilitate the rapid evolution of biotechnologically useful phenotypes through accelerated and parallelized rounds of mutagenesis and selection, as well as cell-lineage tracking through barcode mutagenesis.

Bioproduction of a betalain color palette in Saccharomyces cerevisiae.

Betalains are a family of natural pigments found exclusively in the plant order Caryophyllales. All members of this chemical family are biosynthesized through the common intermediate betalamic acid, which is capable of spontaneously condensing with various primary and secondary amines to produce betalains. Of particular interest is the red-violet betanin, most commonly obtained from Beta vulgaris (beet) as a natural food dye.

Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay.

Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies. In this paper, we demonstrate the versatility of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA) and its ability to support high-throughput data acquisition through next-generation sequencing (NGS).

Tracking transience: a method for dynamic monitoring of biological events in Arabidopsis thaliana biosensors.

The activation and level of expression of an endogenous, stress-responsive biosensor (bioreporter) can be visualized in real-time and non-destructively using highly accessible equipment (fluorometer). Biosensor output can be linked to computer-controlled systems to enable feedback-based control of a greenhouse environment. Today's agriculture requires an ability to precisely and rapidly assess the physiological stress status of plants in order to optimize crop yield.

DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification.