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Related Experiment Video

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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
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Dissecting enzyme function with microfluidic-based deep mutational scanning.

Philip A Romero1, Tuan M Tran1, Adam R Abate2

  • 1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158.

Proceedings of the National Academy of Sciences of the United States of America
|June 4, 2015
PubMed
Summary
This summary is machine-generated.

We developed a new method using droplet microfluidics and DNA sequencing to map millions of enzyme variants. This approach reveals crucial enzyme functions and identifies mutations for enhanced thermostability.

Keywords:
droplet-based microfluidicshigh-throughput DNA sequencingprotein engineering

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Enzyme engineering is complex due to the intricate relationship between sequence and function.
  • Developing new or improved enzyme functions requires advanced methods for mapping sequence-function landscapes.

Purpose of the Study:

  • To present an ultrahigh-throughput method for mapping enzyme sequence-function relationships.
  • To apply this method to glycosidase variants for comprehensive activity and thermostability mapping.

Main Methods:

  • Utilized droplet microfluidic screening combined with next-generation DNA sequencing.
  • Performed microfluidic-based deep mutational scanning on millions of glycosidase sequence variants.
  • Incorporated a high-temperature incubation step to assess enzyme thermotolerance.

Main Results:

  • Generated a comprehensive and unbiased map of the glycosidase enzyme function landscape.
  • Identified known patterns of mutational tolerance and correspondence with natural sequence variation.
  • Discovered previously unreported sites critical for glycosidase function.
  • Identified mutations that enhance enzyme thermostability.

Conclusions:

  • Droplet microfluidics coupled with DNA sequencing offers a powerful platform for high-throughput enzyme screening.
  • This method enables detailed mapping of enzyme sequence space, facilitating enzyme engineering.
  • The study revealed novel insights into glycosidase function and thermostability.