Darwinian Evolution on a Chip

Computer control of Darwinian evolution has been demonstrated by propagating a population of RNA enzymes in a microfluidic device. The RNA population was challenged to catalyze the ligation of an oligonucleotide substrate under conditions of progressively lower substrate concentrations. A microchip-based serial dilution circuit automated an exponential growth phase followed by a 10-fold dilution, which was repeated for 500 log-growth iterations. Evolution was observed in real time as the population adapted and achieved progressively faster growth rates over time. The final evolved enzyme contained a set of 11 mutations that conferred a 90-fold improvement in substrate utilization, coinciding with the applied selective pressure. This system reduces evolution to a microfluidic algorithm, allowing the experimenter to observe and manipulate adaptation

Multiplexed Enzyme Activity-Based Probe Display via Hybridization

Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types

Stepwise Synthesis of Giant Unilamellar Vesicles on a Microfluidic Assembly Line

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore

Stepwise Synthesis of Giant Unilamellar Vesicles on a Microfluidic Assembly Line

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore

Stepwise Synthesis of Giant Unilamellar Vesicles on a Microfluidic Assembly Line

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore

Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer