DropSynth-Gold: Golden Gate Assembly in Emulsions Extends Multiplexed Gene Libraries to Greater Lengths

The ability to synthesize longer genes at scale remains a central challenge in multiplexed gene synthesis. DropSynth is a pooled gene synthesis platform that enables highly multiplexed, compartmentalized assembly from microarray-derived oligonucleotides, but current implementations rely on polymerase cycling assembly (PCA), which constrains fragment number, construct length, and assembly fidelity. Here we present DropSynth-Gold, an evolution of the DropSynth platform that replaces PCA with Golden Gate assembly (GGA) performed within emulsion droplets. This modification preserves the core workflow, including bead-linked oligonucleotide capture and pooled processing, while altering only the assembly chemistry and computational oligo design strategy. Emulsion-based Golden Gate assembly enables directional, multi-fragment ligation within isolated droplets, followed by recovery and amplification of full-length constructs. As a proof of concept, we constructed six 384-member libraries spanning increasing construct lengths and fragment counts, including designs from 5x300-mer fragments to 12x350-mer architectures (~3 kb). DropSynth-Gold reliably assembled full-length constructs across all libraries. A direct comparison of a shared 5x300-mer library demonstrated comparable recovery and fidelity to PCA-based DropSynth, indicating that Golden Gate assembly can replace PCA without compromising assembly performance. These gains were achieved without increasing cost, workflow complexity, or turnaround time, expanding the accessible design space for multiplexed gene synthesis.