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Multiplexed gene synthesis in emulsions for exploring protein functional landscapes

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Science  04 Jan 2018:
eaao5167
DOI: 10.1126/science.aao5167

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Abstract

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 use DropSynth to successfully build >7000 synthetic genes that encode phylogenetically-diverse homologs of two essential genes in E. 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 allow us to rationally explore sequence-function relationships at unprecedented scale.

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