A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast library yielding improved production hosts of secreted industrial enzymes. The validated enriching strain 14 times based on its α-amylase production, close the theoretical maximum enrichment. Furthermore, 10(5) member screened clone with more than 2-fold increase in production. enzyme results from an improvement cellular functions host contrast previous droplet-based...

Significance Increasing demand for recombinant proteins leads to continuous attempts improving the protein secretion capacity of host cells. In this study, we show that by combining high-throughput microfluidic screening with whole-genome sequencing selected clones from yeast libraries can identify and map mutations associated significantly improved production. These identified be used as reverse metabolic engineering target genes in design efficient cell factories secretion. The will also...

We present a novel method for passive separation of microfluidic droplets by size at high throughput using deterministic lateral displacement (DLD). also show that containing Saccharomyces cerevisiae shrink significantly during incubation while only yeast media retain or slightly increase their size. demonstrate the DLD device sorting out shrunken yeast-cell from 31% larger diameter which were generated same time media, >40-fold excess. This demonstrates resolving power droplet and...

Abstract Cyanobacteria are model organisms for photosynthesis and attractive biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library Synechocystis sp. PCC 6803, where aim to target all genes repression. We track the growth members multiple conditions estimate fitness. The reveals several clones with increased rates, these have a common upregulation related cyclic electron flow. challenge...

Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts generate functional and biosensing the extracellular product concentration that accurately reflects effective production capacity. We describe effects different translation initiation rates on dynamic range p-coumaric acid biosensor based...

Abstract Proteine auf Zelloberflächen sind nützliche Biomarker für Krankheiten, allerdings konnten Hochdurchsatzmethoden bisher nur Zellen detektieren, die mindestens einige Hundert exprimieren. Enzymatische Amplifikation in Tröpfchen einem Mikrofluidikgerät eignet sich zur Messung und Analyse von Biomarkern, geringer Konzentration einzelnen menschlichen exprimiert werden. Durch Farbstoffmarkierung der können mehrere Proben gleichzeitig analysiert magnified image

Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine industrial bioprocess technology. We present novel microdroplet-based device extensive characterization the reaction enzyme substrate inhibitor systems in single experiment utilizing an integrated droplet picoinjector bioanalysis. This enables scanning multiple fluorescently-barcoded concentrations conditions single, highly time-resolved yielding Michaelis constant (Km), turnover...

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered production. Due to complexity such machinery, challenge in generating a superior factory is fully exploit production potential by finding beneficial targets for optimized strains, which ideally could be used improved other proteins. We focused on an approach yeast Saccharomyces cerevisiae allows attenuation gene expression, using RNAi combined with high-throughput microfluidic...

Abstract In this study, synthetic polymeric particles were effectively fabricated by combining modern technologies of artificial intelligence (AI) and microfluidics. Because size uniformity is a key factor that significantly influences the stability particles, therefore, work aimed to establish new AI application using machine learning technology for prediction poly( d , l -lactide-co-glycolide) (PLGA) microparticles produced diverse microfluidic systems either in form single or multiple...

Abstract Microfluidics has emerged rapidly over the past 20 years and been investigated for a variety of applications from life sciences to environmental monitoring. Although continuous-flow microfluidics is ubiquitous, segmented-flow or droplet offers several attractive features. Droplets can be independently manipulated analyzed with very high throughput. Typically, carried out within planar networks microchannels, namely, microfluidic chips. We propose that fibers offer an interesting...

We present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells over time. This high-throughput assay allows encapsulation in micro-droplets traps intact droplets arrays miniature wells on PDMS-glass chip. Automated time-lapse fluorescence imaging image analysis incubated chip determination viability individual In order containing cells, we developed simple method based circular Hough transform identify images quantify number live dead...

Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening mutant libraries using microtiter plates is not feasible.We present method high-throughput, single-cell analysis and sorting genetically engineered l-lactate-producing strains Synechocystis sp. PCC6803. A microfluidic device used encapsulate single cells in picoliter droplets, assay the droplets l-lactate production, sort with high...

In this paper, we utilize bulk acoustic waves to control the position of microparticles inside droplets in two-phase microfluidic systems and demonstrate a method enrich microparticles. droplet microfluidics, different unit operations are combined integrated on-chip miniaturize complex biochemical assays. We present operation capable controlling during trident shaped split. An standing wave field is generated microchannel, forces direct encapsulated center droplets. The generic, requires no...

The future of the life sciences is linked to automation and microfluidics. As robots start working side by with scientists, robotic microfluidics in general, droplet particular, will significantly extend accelerate sciences. Here, we demonstrate using an inexpensive liquid-handling robot produce human scaffold-free cell spheroids at high throughput. We use pipette actuation interface pipetting tip a droplet-generating microfluidic device. In this device, highly monodisperse droplets diameter...

Abstract Immune‐modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered dampen the immune reaction increase treatment efficacy. Mucin‐derived materials have gained attention for their ability modulate macrophage dendritic cell activity, trigger mild foreign body response when implanted in vivo. In this study, potential of mucin hydrogels (Muc‐gels) as cell‐encapsulating is investigated. When placed contact with blood, Muc‐gels significantly lower...

The health hazards of micro- and nanoplastic contaminants in drinking water has recently emerged as an area concern to policy makers industry. Plastic range size from (5 mm 1 μm) nanoplastics (<1 μm). Microfluidics provides many tools for particle manipulation at the microscale, particularly diagnostics biomedicine, but general a limited capacity process large volumes. Drinking environmental samples with low-level contamination microplastics require processing deciliter liter sample volumes...

We present the fabrication and characteristics of monolithically integrated ink dyed poly(dimethylsiloxane) (PDMS) filters for optical sensing in disposable lab-on-a-chip. This represents a migration auxillary functions onto chip with goal producing truly portable systems. Filters made from commercially available (Pelikan) directly mixed into PDMS oligomer without use any additional solvents were patterned standard soft lithography technologies. Furthermore, process based on capillary forces...

The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous division during cultivation droplets. Here, we report influence size on and viability Chinese Hamster Ovary (CHO) cells, most widely used host cells biopharmaceuticals production were encapsulated cultivated 33, 180 320 pL droplets 3 days. Periodic monitoring incubation showed that divisions 33 stopped after 24 h, whereas was observed 72 h. also...

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures different microfluidic formats. Control is critical for single cell/clone screening droplets, such as directed evolution yeast, directly affects production yields from factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites yeast glucose dissimilation to establish formats respiring well fermenting yeast....

Whole genome amplification (WGA) is a challenging, key step in metagenomic studies of samples containing minute amounts DNA, such as from low biomass environments. It well known that multiple displacement (MDA), the most commonly used WGA method for microbial samples, skews genomic representation sample. We have combined MDA with droplet microfluidics to perform reaction homogeneous emulsion. Each this emulsion can be considered an individual chamber, allowing partitioning into millions...

3D cell culture models are important tools in translational research but have been out of reach for high-throughput screening due to complexity, requirement large numbers and inadequate standardization. Microfluidics model miniaturization technologies could overcome these challenges. Here, we present a workflow produce characterize the formation miniaturized spheroids using deep learning. We train convolutional neural network (CNN) ensemble morphology classification droplet microfluidic...