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Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.

Jeremy J Agresti1, Eugene Antipov, Adam R Abate

  • 1Harvard School of Engineering and Applied Sciences, Cambridge, MA 02138, USA.

Proceedings of the National Academy of Sciences of the United States of America
|February 10, 2010
PubMed
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We developed a novel microfluidic platform for ultrahigh-throughput screening of biochemical reactions. This technology accelerates enzyme evolution, identifying highly efficient mutants for biotechnological applications.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Genomic, proteomic, and metabolomic data fuel understanding of life's biochemistry and evolution.
  • Modern biotechnologies like synthetic biology offer solutions for drug development and sustainable energy.
  • High-throughput biochemical reaction measurements are crucial but often limited.

Purpose of the Study:

  • To present a general ultrahigh-throughput screening platform using drop-based microfluidics.
  • To overcome limitations in the scale and speed of biochemical reaction screening.
  • To demonstrate the platform's power in directed enzyme evolution.

Main Methods:

  • Utilized drop-based microfluidics with aqueous drops in oil as picoliter reaction vessels.
  • Screened reactions at rates of thousands per second.

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  • Applied the system to directed evolution of the enzyme horseradish peroxidase.

    • Main Results:

    • Identified horseradish peroxidase mutants with catalytic rates over 10 times faster than the parent enzyme.
    • Screened approximately 10^8 enzyme reactions in 10 hours using < 150 microL reagent volume.
    • Achieved a 1,000-fold increase in speed and a 1-million-fold cost reduction compared to robotic systems.

    Conclusions:

    • The drop-based microfluidic platform revolutionizes biochemical screening scale and speed.
    • Enables rapid identification of significantly improved enzyme variants.
    • Offers a powerful tool for advancing molecular engineering and synthetic biology.