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Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics

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Protein Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1685))

Abstract

The success of ultrahigh-throughput screening experiments in directed evolution or functional metagenomics strongly depends on the availability of efficient technologies for the quantitative testing of a large number of variants. With advanced robotics, libraries of up to 105 clones can be screened per day as colonies on agar plates or cell lysates in microwell plates, albeit at high cost of capital, manpower and consumables. These cost considerations and the general need for high-throughput make miniaturization of assay volumes attractive. To provide a general solution to maintain genotype–phenotype linkage, biochemical assays have been compartmentalized into water-in-oil droplets. This chapter presents a microfluidic workflow that translates a frequently used screening procedure consisting of cytoplasmic/periplasmic protein expression and cell lysis to the single cell level in water-in-oil droplet compartments. These droplets are sorted based on reaction progress by fluorescence measurements at the picoliter scale.

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Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK

    Fabrice Gielen, Pierre-Yves Colin, Philip Mair & Florian Hollfelder

  2. Living Systems Institute, University of Exeter, Stocker Road, EX4 4QD, Exeter, UK

    Fabrice Gielen

  3. Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK

    Pierre-Yves Colin

Authors
  1. Fabrice Gielen
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  2. Pierre-Yves Colin
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  3. Philip Mair
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  4. Florian Hollfelder
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Corresponding author

Correspondence to Florian Hollfelder .

Editor information

Editors and Affiliations

  1. Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Uwe T. Bornscheuer

  2. Protein Biochemistry Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Matthias Höhne

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Gielen, F., Colin, PY., Mair, P., Hollfelder, F. (2018). Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics. In: Bornscheuer, U., Höhne, M. (eds) Protein Engineering. Methods in Molecular Biology, vol 1685. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7366-8_18

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  • DOI: https://doi.org/10.1007/978-1-4939-7366-8_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7364-4

  • Online ISBN: 978-1-4939-7366-8

  • eBook Packages: Springer Protocols

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