CD BioGlyco addresses the challenges of traditional screening methods by offering chassis-based strain screening services using cutting-edge microfluidic systems. These challenges include the inability to capture the complexity of extracellular glycan secretion, limited throughput, and high reagent consumption. By leveraging the precision of microfluidics, we encapsulate individual cells within picoliter-sized droplets, individual "micro-reactors", allowing for the ultra-high-throughput evaluation of millions of variants. This technology is uniquely suited for the selection of strains optimized for glycan synthesis, enzyme secretion, and metabolic efficiency, ensuring that only the most productive candidates proceed to industrial scale-up.
Key Technologies
Droplet-based Microfluidics (DMF)
This technology generates monodisperse water-in-oil (W/O) droplets at kilohertz (kHz) frequencies. Each droplet acts as an isolated environment for a cell, preventing cross-contamination and allowing for the accumulation of secreted products (like glycans or enzymes) to detectable concentrations.
Fluorescence-Activated Droplet Sorting (FADS)
Unlike standard FACS, which primarily detects surface or intracellular signals, FADS allows us to sort droplets based on the activity or concentration of substances within the droplet. This is critical for screening chassis strains designed to secrete glyco-products into the medium.
Lab-on-a-Chip (LOC) Integration
We design custom microfluidic chips that integrate multiple steps, including cell encapsulation, incubation, reagent injection (e.g., fluorogenic substrates), and sorting, onto a single, automated platform.
Unlocking Microbial Potential through Picoliter-Scale Precision: Microfluidic System
At CD BioGlyco, our service scope is meticulously designed to support every phase of chassis strain discovery and optimization. We understand that a "one-size-fits-all" approach does not work in synthetic biology. Therefore, our microfluidic screening services are tailored to the specific metabolic requirements of your target molecule, whether it be a complex human-milk oligosaccharide (HMO), a therapeutic glycoprotein, or an industrial enzyme.
Custom design of screening assays
We develop specialized fluorogenic sensors or coupled enzymatic assays that translate your target's production level into a clear fluorescent signal within the microfluidic droplet. This allows us to screen massive libraries, often exceeding 107 to 108 variants, in a single day.
Library Screening and Isolation
Whether your library is generated via random mutagenesis, directed evolution, or genome editing, our microfluidic system handles the diversity. The service includes the encapsulation, controlled on-chip or off-chip incubation to allow for product accumulation, and high-speed sorting.
Environmentally Controlled Screening
Microfluidic systems allow us to precisely manipulate the chemical environment within the droplets. We screen for strains that maintain high productivity under specific stressors, such as varied pH levels, temperature fluctuations, or the presence of inhibitory byproducts. This ensures that the selected chassis is not only productive but also robust enough for the rigors of large-scale fermentation.
Workflow
Library Preparation and Assay Design
The process begins with the client's mutant library or a library generated by CD BioGlyco. Concurrently, we optimize the fluorescent detection chemistry to ensure a high signal-to-noise ratio within the picoliter environment.
Cell Encapsulation
Using flow-focusing microfluidic geometries, we encapsulate individual microbial cells (bacteria, yeast, or fungi) into droplets. We carefully control the cell density to follow a Poisson distribution, ensuring that the majority of occupied droplets contain exactly one cell.
Droplet Incubation and Product Accumulation
Droplets are incubated either in a specialized on-chip delay line or in an off-chip "emulsion storage" system. During this time, the encapsulated strain grows and secretes the target glycans or enzymes, which are trapped within the droplet.
Reagent Injection and Signal Development
If the assay requires secondary reagents, we utilize Pico-injection technology to add precise volumes of substrates or dyes into each pre-formed droplet without causing coalescence, triggering the fluorescent reaction.
High-Throughput Sorting (FADS)
The emulsion is reinjected into a sorting chip. As each droplet passes a laser detection point, the fluorescent signal is analyzed. Droplets meeting the pre-defined criteria are diverted into a "positive" collection channel using an integrated dielectrophoretic (DEP) force.
Strain Recovery and Validation
The sorted "winning" strains are recovered from the oil phase, plated, and subjected to secondary validation, including next-generation sequencing (NGS) and small-scale fermentation to confirm the improved phenotype.
Publication Data
DoI: 10.3390/mi11100943
Journal: Micromachines
IF: 3.0
Published: 2020
Results: This study presents a flexible rotary microfluidic dispensing system based on gap switch technology (GST) for high-speed, low-cost drug combination screening. The system uses aqueous-in-oil droplets to test drug combinations, with stainless steel wells, a brass dispensing shuttle, and polytetrafluoroethylene (PTFE) channels to avoid cross-contamination and enable reusability. It operates via circumferential (ring rotation) and radial (shuttle movement) motions, generating mixed droplets driven by Laplace pressure differences. A 48×4 commercial design is proposed, capable of 54 million combinations without dilutions—reduced to ~19.9 million by accounting for drug dilutions (avoiding redundant same-drug dilution combinations). Machine learning further cuts screening scale by scoring synergy (2 for synergy, 1 for additivity, 0 for antagonism) to focus on promising regions. This system addresses "combinatorial explosion," offering faster throughput and lower reagent use than traditional 96-well plates, supporting personalized medicine and organoid-based drug testing.
Applications
Therapeutic Glycoprotein Production
Screening for yeast or mammalian chassis strains with optimized glycosylation pathways to ensure human-like glycan structures on monoclonal antibodies and recombinant proteins.
Industrial Enzyme Discovery
Rapid identification of high-secreting strains for glycosidases, cellulases, and proteases used in the textile and biofuel industries, focusing on enhanced thermal and pH stability.
Metabolic Engineering for HMOs
Optimizing bacterial chassis (e.g., E. coli) for the high-yield synthesis of HMOs by screening libraries for improved precursor flux and reduced byproduct formation.
Biosurfactant and Glycolipid Optimization
Selecting strains with superior production of rhamnolipids or sophorolipids by monitoring interfacial tension or specific metabolic markers within micro-droplets.
Advantages
Ultra-High Throughput
Our systems process up to 107 variants per day, which is several orders of magnitude faster than conventional microtiter plate-based screening methods, shortening the cycle.
Minimal Reagent Consumption
By miniaturizing the reaction volume to picoliters, we reduce the consumption of expensive substrates, media, and reagents by up to 1,000-fold, providing a cost-effective solution for large-scale screens.
Detection of Extracellular Secretion
Unlike FACS, which is limited to intracellular or cell-surface targets, our droplet system traps secreted products, making it the ideal choice for glycan and enzyme secretion studies.
Single-Cell Resolution
We eliminate the "averaging effect" of bulk cultures, allowing for the identification of rare, high-performing individuals within a heterogeneous population that would otherwise be missed.
Frequently Asked Questions
Customer Review
"The microfluidic screening service at CD BioGlyco was a game-changer for our enzyme evolution project. We were able to screen a library of 107 mutants in a fraction of the time it would have taken using our in-house plate reader assays. The accuracy of the FADS system was impressive, and the recovered strains showed a 5-fold increase in secretion levels."
– Q.T., Lead Scientist
"Working with CD BioGlyco allowed us to identify a rare yeast variant with improved N-glycan site occupancy. Their expertise in both microfluidics and glycobiology is a unique combination that saved us months of trial and error."
– C.R., Director of R&D
"We were concerned about the viability of our filamentous fungi in droplets, but the team at CD BioGlyco optimized the encapsulation and incubation parameters perfectly. The results were robust, and the transition to 5L fermentation was seamless."
CD BioGlyco is your premier partner for high-throughput chassis strain screening. By combining advanced microfluidic technology with deep expertise in synthetic glycobiology, we help you overcome the most challenging hurdles in strain optimization. Please feel free to contact us to help you design the optimal screening strategy for your unique goals.
Reference
Davies, M.; et al. A flexible, microfluidic, dispensing system for screening drug combinations. Micromachines. 2020, 11: 943. (Open Access)
For research use only. Not intended for any clinical use.
