Single-Cell Microencapsulation for Evolution and Discovery of Biocatalysts
Enzymes are responsible for many chemical reactions which support life and are used in numerous industries. Laboratory evolution experiments can mimic the Darwinian process of mutation and selection pressure, yielding newly evolved catalysts. Recently dev
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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrahigh-Throughput Enzyme Screens: Directed Evolution and Discovery . . . . . . . . . . . . . . . . . . . Single-Cell Encapsulation in Microfluidic Droplets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stochastic Encapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deterministic Single-Cell Encapsulation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microfluidic Workflow for Directed Evolution of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Construction of Multistep Workflows for Single-Cell Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Considerations for Setting Up Single Cell Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Operation of a Microdroplet Sorter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formats for Single-Cell Biocatalyst Functional Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single-Cell Lysate Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single-Cell Internal Expression and Surface Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single-Cell Secretion Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Metagenomics and Bioprospecting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro Workflows as Artificial Single Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations and Future Prospects of Single-Cell Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Enzymes are responsible for many chemical reactions which support life and are used in numerous industries. Laboratory evolution experiments can mimic the Darwinian process of mutation and selection pressure, yielding newly evolved catalysts. Recently developed single-cell microencapsulation techniques enable the screening of ultralarge enzyme-mutant pools, greatly increasing the odds o
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