Evolution in Microfluidic Droplet

Levin, Itay; Aharoni, Amir

doi:10.1016/j.chembiol.2012.08.004

Cited by 13 publications

(11 citation statements)

References 12 publications

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“…34,37,41 Polydisperse emulsions give rise to a situation in which droplets carrying genes encoding proteins with the same activity can exhibit dramatically different assay outcomes depending on their size, although selections in polydisperse droplets may still be successful if the activity difference between positive hits and the rest of the library is very large. Some researchers have addressed the polydispersity problem by introducing external markers, 42 such as coexpression of GFP, 41 but the inclusion of markers complicates the biological setup and does not fully remedy the problem of varying catalyst concentration and the volume dependence of fluorescence intensity.…”

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confidence: 99%

One in a Million: Flow Cytometric Sorting of Single Cell-Lysate Assays in Monodisperse Picolitre Double Emulsion Droplets for Directed Evolution

et al. 2014

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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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confidence: 99%

One in a Million: Flow Cytometric Sorting of Single Cell-Lysate Assays in Monodisperse Picolitre Double Emulsion Droplets for Directed Evolution

et al. 2014

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“…This principle finds application in microfluidic devices for batch processing of samples in e.g. pharmaceutical manufacturing or bio-engineering using droplets as chemical reactors or cell incubators so as to boost reaction rates, shield against contamination and/or provide protected environments [164,165,166,167,168,169]. • Digital microfluidic platforms Here discrete droplets are trapped (and displaced) on solid substrates.…”

Section: Industrial and Technological Relevancementioning

confidence: 99%

Lagrangian Transport and Chaotic Advection in Three-Dimensional Laminar Flows

Speetjens

Metcalfe

Rudman

2021

Applied Mechanics Reviews

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Transport and mixing of scalar quantities in fluid flows is ubiquitous in industry and Nature. Turbulent flows promote efficient transport and mixing by their inherent randomness. Laminar flows lack such a natural mixing mechanism and efficient transport is far more challenging. However, laminar flow is essential to many problems and insight into its transport characteristics of great importance. Laminar transport, arguably, is best described by the Lagrangian fluid motion ("advection") and the geometry, topology and coherence of fluid trajectories. Efficient laminar transport being equivalent to "chaotic advection" is a key finding of this approach. The Lagrangian framework enables systematic analysis and design of laminar flows. However, the gap between scientific insights into Lagrangian transport and technological applications is formidable primarily for two reasons. First, many studies concern two-dimensional (2D) flows yet the real world is three dimensional (3D). Second, Lagrangian transport is typically investigated for idealised flows yet practical relevance requires studies on realistic 3D flows. The present review aims to stimulate further development and utilisation of know-how on 3D Lagrangian transport and its dissemination to practice. To this end 3D practical flows are categorised into canonical problems. First, to expose the diversity of Lagrangian transport and create awareness of its broad relevance. Second, to enable knowledge transfer both within and between scientific disciplines. Third, to reconcile practical flows with fundamentals on Lagrangian transport and chaotic advection. This may be a first incentive to structurally integrate the "Lagrangian mindset" into the analysis and design of 3D practical flows.

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“…Recently, high‐throughput platforms for screening large populations of biological samples for DE of enzymes have been reported in the literature (See Section 3 and 4). Specifically, pL‐volume droplets (each containing a single cell) can be formed and sorted at kHz frequencies, which in turn yields a population of droplets containing the most active enzymes (Fig. ).…”

Section: Introductionmentioning

confidence: 99%

“…Subsequently, droplets are incubated and then sorted according to the fluorescence signal. Reprinted and reproduced from , with the permission of Elsevier Publishing.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

Chiu

Stavrakis

2019

Electrophoresis

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Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user-defined goal) coupled with droplet-based microfluidics allows the detailed analysis of millions of enzyme variants on ultra-short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet-based microfluidics, made in this context towards the high-throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell-based assays, as in the case of directed evolution experimentations.

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Evolution in Microfluidic Droplet

Cited by 13 publications

References 12 publications

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

Lagrangian Transport and Chaotic Advection in Three-Dimensional Laminar Flows

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

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