RT Journal Article
SR Electronic
T1 Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 163550
DO 10.1101/163550
A1 Calin Plesa
A1 Angus M. Sidore
A1 Nathan B. Lubock
A1 Di Zhang
A1 Sriram Kosuri
YR 2017
UL http://biorxiv.org/content/early/2017/07/14/163550.abstract
AB 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.