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Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution

Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution
Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution
We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 107 monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable
1074-5521
1001-1009
Kintses, Balint
5c91d3b7-a827-429f-bcd3-386621561a47
Hein, Christopher
5fb45c30-b085-477d-909d-797dd1bd6425
Mohamed, Mark F.
8b6d6351-9403-41ec-aca5-2273974ed982
Fischlechner, Martin
b3930129-0775-4c05-81c7-475934df97ee
Courtois, Fabienne
96fbdf6b-bebc-442e-9701-f87be80c54c6
Lainé, Céline
94c4d85e-2666-4ce5-9543-20d18242605d
Hollfelder, Florian
a9f01280-f05d-4057-b5d7-85eac249e477
Kintses, Balint
5c91d3b7-a827-429f-bcd3-386621561a47
Hein, Christopher
5fb45c30-b085-477d-909d-797dd1bd6425
Mohamed, Mark F.
8b6d6351-9403-41ec-aca5-2273974ed982
Fischlechner, Martin
b3930129-0775-4c05-81c7-475934df97ee
Courtois, Fabienne
96fbdf6b-bebc-442e-9701-f87be80c54c6
Lainé, Céline
94c4d85e-2666-4ce5-9543-20d18242605d
Hollfelder, Florian
a9f01280-f05d-4057-b5d7-85eac249e477

Kintses, Balint, Hein, Christopher, Mohamed, Mark F., Fischlechner, Martin, Courtois, Fabienne, Lainé, Céline and Hollfelder, Florian (2012) Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chemistry & Biology, 19 (8), 1001-1009. (doi:10.1016/j.chembiol.2012.06.009). (PMID:22921067)

Record type: Article

Abstract

We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 107 monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable

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Published date: 24 August 2012
Organisations: Chemistry

Identifiers

Local EPrints ID: 342568
URI: http://eprints.soton.ac.uk/id/eprint/342568
ISSN: 1074-5521
PURE UUID: 280aacc8-8e93-4f6d-bc8d-862aca055c62

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Date deposited: 07 Sep 2012 13:47
Last modified: 16 Jul 2019 21:55

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Contributors

Author: Balint Kintses
Author: Christopher Hein
Author: Mark F. Mohamed
Author: Martin Fischlechner
Author: Fabienne Courtois
Author: Céline Lainé
Author: Florian Hollfelder

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