Engineering highly active nuclease enzymes with machine learning and high-throughput screening.

Optimizing enzymes to function in novel chemical environments is a central goal of synthetic biology, but optimization is often hindered by a rugged fitness landscape and costly experiments. In this work, we present TeleProt, a machine learning (ML) framework that blends evolutionary and experimental data to design diverse protein libraries, and employ it to improve the catalytic activity of a nuclease enzyme that degrades biofilms that accumulate on chronic wounds. After multiple rounds of high-throughput experiments, TeleProt found a significantly better top-performing enzyme than directed evolution (DE), had a better hit rate at finding diverse, high-activity variants, and was even able to design a high-performance initial library using no prior experimental data.

High-resolution dose-response screening using droplet-based microfluidics.

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound.

Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity.

We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s(-1). To validate the system, mixtures of E.

Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis.

We have developed a method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system. DNA amplification in droplets was analyzed using an intercalating fluorochrome, allowing fast and accurate "digital" quantification of the template DNA based on the Poisson distribution of DNA molecules in droplets. The clonal amplified DNA in each 2 pL droplet was further analyzed by measuring the enzymatic activity of the encoded proteins after fusion with a 15 pL droplet containing an in vitro translation system.

Reliable microfluidic on-chip incubation of droplets in delay-lines.

Together with droplet creation, fusion and sorting, the incubation of droplets is one of the most important and essential operations for droplet-based microfluidic assays. This manuscript concerns the development of delay-lines, which are necessary to allow incubation of reactions for precise time periods. We analyze the problems associated with creating delay-lines for incubation in the minute to hour time range, which arise from back-pressure and from the dispersion in the incubation time due to the unequal speeds with which droplets pass through the delay-line.

Microfluidic production of droplet pairs.

We study a microfluidic dual nozzle for the production of water-in-oil droplet pairs. Droplets are paired by the hydrodynamic coupling of two nozzles over a wide range of aqueous and oil flow rates provided that they are larger than the channel dimensions. The droplet production frequencies and volumes are related to the flow rates through a single, experimentally determined power-law.

Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms.

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil.