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Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression
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Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression
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Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression
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Paper
Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression
2026
Overview
We present a microfluidic workflow that couples reconstituted in vitro transcription-translation (IVTT) with ultra-high throughput droplet screening to directly link genotype and phenotype within complex, heterogeneous DNA pools. The approach employs DNA nanoflowers as clonal, high-copy templates, enabling robust protein expression from single DNA molecules encapsulated in picoliter droplets. When integrated with fluorescence-assisted microdroplet sorting (FADS) and a DNA recovery pipeline that reconstituted selected libraries for subsequent iterative rounds, the platform achieves approximately 400-500 fold enrichment per selection cycle and supports functional discovery and directed evolution entirely independent of host cell expression. As a proof of principle, we demonstrate recovery of the recombinase RecA from an E. coli genomic library screened for single-stranded DNA binders, highlighting the platform's capability to identify DNA-interacting and DNA-modifying enzymes. By eliminating host-derived background activity and toxicity constraints that often complicate lysate or cell-based metagenomic screens, this method expands access to enzyme classes that have historically been difficult to assay.Competing Interest StatementThe authors have declared no competing interest.
Publisher
Cold Spring Harbor Laboratory Press
Subject
