Multiplexed gene synthesis in emulsions for exploring protein functional landscapes

Large-scale gene synthesis in tiny droplets Gene synthesis technology is important for functional characterization of DNA sequences and for the development of synthetic biology. However, current methods are limited by their low scalability and high cost. Plesa et al. developed a gene synthesis method, DropSynth, which uses barcoded beads to concentrate oligos and subsequently assemble them into synthetic genes within picoliter emulsion droplets. DropSynth allows generation of large libraries of thousands of genes and functional testing of all possible mutations of a particular sequence. Science, this issue p. 343 A gene synthesis method, DropSynth, allows for the synthesis and characterization of thousands of pooled genes. Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene’s assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in Escherichia coli. We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.

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