Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

DropSynth is a scalable, low-cost method to build thousands of defined gene-length constructs in a multiplexed manner. Briefly, this approach involves isolating oligos required to assemble each gene from a large oligo pool onto barcoded microbeads which are then emulsified into droplets where the assembly reaction takes place. We use DropSynth to successfully build >7000 synthetic genes that encode phylogenetically-diverse homologs of two essential genes in E. coli (folA encoding dihydrofolate reductase [DHFR] and coaD encoding phosphopantetheine adenylyltransferase [PPAT]). We tested the ability PPAT homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility.