Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions.

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.

A Multiplexed Cell-Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets.

The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analyzed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

Synchronized Reagent Delivery in Double Emulsions for Triggering Chemical Reactions and Gene Expression.

Microfluidic methods for the formation of single and double emulsion (DE) droplets allow for the encapsulation and isolation of reactants inside nanoliter compartments. Such methods have greatly enhanced the toolbox for high-throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed compartments limit the applicability of droplet microfluidics. Here, a strategy is introduced for on-demand delivery of reactants in DEs. Lipid vesicles are used as reactant carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant sodium dodecyl sulfate (SDS). The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the vesicles' lipid bilayer. The versatility of the method is demonstrated with two critical applications chosen as representative assays for high-throughput screening: the induction of gene expression in bacteria and the initiation of an enzymatic reaction. This method not only allows for the release of the lipid vesicle content inside DEs to be synchronized for all DEs but also for the release to be triggered at any desired time.

Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey.

Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronological overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes.

"Basicles": Microbial Growth and Production Monitoring in Giant Lipid Vesicles.

We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.