1. A general strategy for expanding polymerase function by droplet microfluidics.
- Author
-
Larsen, Andrew C, Dunn, Matthew R, Hatch, Andrew, Sau, Sujay P, Youngbull, Cody, and Chaput, John C
- Subjects
Cells ,Immobilized ,Escherichia coli ,DNA-Directed DNA Polymerase ,Monosaccharides ,Nucleic Acids ,Biological Assay ,Protein Engineering ,Microfluidics ,Base Pairing ,Biomimetic Materials ,Optical Devices ,Cells ,Immobilized ,Genetics ,Bioengineering ,Biotechnology ,Generic Health Relevance - Abstract
Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.
- Published
- 2016