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One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution

One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution
One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution
Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1?000?000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at ?80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers
0003-2700
2526-2533
Zinchenko, Anastasia
e528bfdd-5ae9-4a0a-9ae3-b7a065b0c6b7
Devenish, Sean R.A.
c8e86e13-af5e-4654-86ac-f04237dbff47
Kintses, Balint
5c91d3b7-a827-429f-bcd3-386621561a47
Colin, Pierre-Yves
0f7a053a-735d-48f2-9fe1-e417f0bd99dd
Fischlechner, Martin
b3930129-0775-4c05-81c7-475934df97ee
Hollfelder, Florian
a9f01280-f05d-4057-b5d7-85eac249e477
Zinchenko, Anastasia
e528bfdd-5ae9-4a0a-9ae3-b7a065b0c6b7
Devenish, Sean R.A.
c8e86e13-af5e-4654-86ac-f04237dbff47
Kintses, Balint
5c91d3b7-a827-429f-bcd3-386621561a47
Colin, Pierre-Yves
0f7a053a-735d-48f2-9fe1-e417f0bd99dd
Fischlechner, Martin
b3930129-0775-4c05-81c7-475934df97ee
Hollfelder, Florian
a9f01280-f05d-4057-b5d7-85eac249e477

Zinchenko, Anastasia, Devenish, Sean R.A., Kintses, Balint, Colin, Pierre-Yves, Fischlechner, Martin and Hollfelder, Florian (2014) One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution. Analytical Chemistry, 86 (5), 2526-2533. (doi:10.1021/ac403585p). (PMID:24517505)

Record type: Article

Abstract

Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1?000?000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at ?80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers

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e-pub ahead of print date: 11 February 2014
Published date: 11 February 2014

Identifiers

Local EPrints ID: 363357
URI: http://eprints.soton.ac.uk/id/eprint/363357
ISSN: 0003-2700
PURE UUID: 973c1e1d-1d15-4d72-9a82-810880984ca8

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Date deposited: 24 Mar 2014 14:24
Last modified: 14 Mar 2024 16:23

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Contributors

Author: Anastasia Zinchenko
Author: Sean R.A. Devenish
Author: Balint Kintses
Author: Pierre-Yves Colin
Author: Martin Fischlechner
Author: Florian Hollfelder

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