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Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

View ORCID ProfileCalin Plesa, Angus M. Sidore, View ORCID ProfileNathan B. Lubock, Di Zhang, Sriram Kosuri
doi: https://doi.org/10.1101/163550
Calin Plesa
1Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA
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Angus M. Sidore
2Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, California, USA
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Nathan B. Lubock
1Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA
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Di Zhang
3Genomics and Computational Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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Sriram Kosuri
1Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA
4UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California, USA
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  • For correspondence: sri@ucla.edu
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Abstract

Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a gene’s synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized ~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.

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Posted July 14, 2017.
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Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes
Calin Plesa, Angus M. Sidore, Nathan B. Lubock, Di Zhang, Sriram Kosuri
bioRxiv 163550; doi: https://doi.org/10.1101/163550
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Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes
Calin Plesa, Angus M. Sidore, Nathan B. Lubock, Di Zhang, Sriram Kosuri
bioRxiv 163550; doi: https://doi.org/10.1101/163550

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