Multiplexed genes synthesis from oligos within microdroplets

Abstract

Abstract This study investigates the adoption of DropSynth methodology for in-house gene synthesis and the development of a droplet-based Golden Gate (DropGG) assembly technique to address the inherent limitations of DropSynth, such as low accuracy (20-30%) and limited gene length (<1 kb). Motivated by the aim to lower cost and turn-around times associated with gene synthesis by transitioning to in-house production, the study involved assembling single genes using both DropSynth and Golden Gate methods within droplets, followed by scaling up to multiplexed gene assembly in a single reaction. Through a detailed evaluation of the DropSynth pro tocol, highlighting its weaker points, a novel workflow for DropGG was introduced, adapted for 96-gene assembly. The DropGG approach successfully demonstrated the assembly of genes up to 750 bp within droplets using barcoded beads, while also simplifying downstream processing steps. Collectively, these findings provide valuable insights into the strengths and limitations of droplet-based gene assembly technologies.