Integrated microdroplet workflow for high-throughput cell-free transcription in double emulsion picoreactors

Single-molecule nucleic acid assays offer a powerful route to understand gene regulation. To enable rapid and precise characterization of regulatory sequences such as promoters within a pool of synthetic 5 ^′ regulatory sequences, we present an integrated microdroplet workflow for high-throughput cell-free transcription in double emulsion (DE) picoreactors. For this, we utilize the T7 promoter as a positive control and the Mango III RNA aptamer for fluorescent readout of transcription in an in vitro setup. First, DNA templates are compartmentalized into picolitre droplets along with rolling circle amplification reagents and incubated off-chip. The resulting emulsion is then reinjected into a second device that integrates three operations on a single chip : (i) emulsion reinjection, (ii) electric-field-mediated StepInjection of IVT reagents and (iii) downstream water-in-oil-in-water (W/O/W) double emulsification. Post IVT incubation, the fluorescent DE droplets are selectively isolated by FACS, enabling sorting as well as the classification of droplets on the basis of their fluorescent intensities. We characterize the workflow with proof-of-principle experiments with three input DNA concentrations that yield distinct fractions of fluorescent DEs and also demonstrate intensity-based binning and indexed sorting of droplets into defined wells of a microtiter plate. This workflow establishes a practical route to high-throughput, single-template transcription assays in FACS compatible double emulsions and provides a foundation for future screening of synthetic regulatory libraries and promoter variants with fluorescence encoded readouts.