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bioRxiv · 10.64898/2026.04.30.721591

A droplet microfluidic-based platform for enhanced DNA delivery in non-model organisms

Stibelman, A.·Tran, A.·Chappell, J.·Shamoo, Y.
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Abstract

Expanding genetic engineering beyond model microorganisms is critical to unlocking novel applications in biotechnology, yet the low efficiency of DNA delivery methods like conjugation, remains a major bottleneck in non-model and environmental microbes. Here, we present an automated, high-throughput droplet microfluidic platform that enhances conjugation by encapsulating donor and recipient microbes in picoliter-scale water-in-oil microdroplets, stabilizing cell-cell contact and DNA transfer. Optimization of incubation time, donor to recipient ratio, and plasmid type yielded over a 100-fold increase in conjugation efficiency compared to conventional methods and enabled delivery of complex DNA libraries in low reaction volumes, demonstrating scalability for pooled plasmid library delivery. We further utilized a synthetic biology circuit for donor removal within microdroplets without antibiotic selection, eliminating the need for host-specific selection markers or engineered auxotrophs. When applied to a soil microbial community, this platform improved community-level conjugation, preserving microbial diversity and enabling the identification of genetically accessible chassis. Collectively, this platform establishes a scalable, generalizable solution for high throughput DNA delivery in previously inaccessible microbial hosts. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/721591v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@2ce034org.highwire.dtl.DTLVardef@3399eaorg.highwire.dtl.DTLVardef@1bde851org.highwire.dtl.DTLVardef@df5727_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Stibelman, A., Tran, A., Chappell, J., Shamoo, Y.. 2026-05-03. A droplet microfluidic-based platform for enhanced DNA delivery in non-model organisms. https://doi.org/10.64898/2026.04.30.721591

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