Multiplexed gene synthesis in emulsions for exploring protein functional landscapes

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 ba...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 2018-01, Vol.359 (6373), p.343-347
Main Authors: Plesa, Calin, Sidore, Angus M, Lubock, Nathan B, Zhang, Di, Kosuri, Sriram
Format: Article
Language:English
Subjects:
Beads
Biology
Construction costs
Deoxyribonucleic acid
DNA
Droplets
E coli
Emulsions
Escherichia coli
Escherichia coli - genetics
Functional testing
Gene Knockout Techniques
Gene sequencing
Genes
Genes, Essential
Genes, Synthetic
Genetic Complementation Test
Homology
Incompatibility
Multiplexing
Mutation
Nucleotide sequence
Oligonucleotides
Oligonucleotides - chemical synthesis
Oligonucleotides - chemistry
Oligonucleotides - genetics
Phosphopantetheine adenylyltransferase
Phylogeny
Proteins - genetics
Proteins - physiology
Synthesis
Synthetic Biology - methods
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Summary: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 We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout 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.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.aao5167