Ultrahigh-Throughput Enzyme Engineering and Discovery in Compartments.

Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function.

Investigating host-microbiome interactions by droplet based microfluidics.

Background: Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota.

Results: Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries.

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h.

Bioinspired genotype-phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins.

The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments-water-in-oil droplets and gel-shell beads-for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

Microfluidic droplets: new integrated workflows for biological experiments.

Miniaturization of the classical test tube to picoliter dimensions is possible in monodisperse water-in-oil droplets that are generated in microfluidic devices. The establishment of standard unit operations for droplet handling and the ability to carry out experiments with DNA, proteins, cells and organisms provides the basis for the design of more complex workflows to address biological challenges. The emerging experimental format makes possible a quantitative readout for large numbers of experiments with a precision comparable to the macroscopic scale.

Relating chemical and biological diversity space: a tunable system for efficient gene transfection.

Polyethyleneimine (PEI), a well-established nonviral transfection reagent, was combinatorially modified with varying proportions of methyl, benzyl, and n-dodecyl groups to create a library of 435 derivatized polymers. Screening of this library for transfection, DNA binding, and toxicity allows systematic correlation of the biological properties of our polymers to their derivatizations. Combinations of derivatizations bring about a 100-fold variation in transfection efficiency between library members.

Polyethylene imine derivatives ('synzymes') accelerate phosphate transfer in the absence of metal.

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.

Molecular recognition of DNA by rigid [N]-polynorbornane-derived bifunctional intercalators: synthesis and evaluation of their binding properties.

We have exploited the concept of multivalency in the context of DNA recognition, using novel chemistry to synthesize a new type of bis-intercalator with unusual sequence-selectivity. Bis-intercalation has been observed previously, but design principles for de novo construction of such molecules are not known. Our compounds feature two aromatic moieties projecting from a rigid, polynorbornane-based scaffold.