Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey

Stucki, Ariane; Vallapurackal, Jaicy; Ward, Thomas R.; Dittrich, Petra S.

doi:10.1002/anie.202016154

Cited by 82 publications

(60 citation statements)

References 168 publications

(188 reference statements)

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“…Cell-free water-in-oil droplets can be transformed into a double-emulsion using the same commercial microuidics setup. FACS instruments can then easily be employed for uorescent-activated droplet sorting [44,45].…”

Section: Discussionmentioning

confidence: 99%

Efficient multi-gene expression in cell-free droplet microreactors

Sierra

Arold

Grünberg

2021

Preprint

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Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

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Section: Discussionmentioning

confidence: 99%

Efficient multi-gene expression in cell-free droplet microreactors

Sierra

Arold

Grünberg

2021

Preprint

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“…There is a growing interest in the biochemical sciences for the compartmentalization or miniaturization of reactions [ 1 , 2 ]. When conducted in water-in-oil droplets, compartmentalization allows experimentalists to run millions of reactions in parallel, creating ample opportunity to analyze the vast diversity contained within a biochemical sample.…”

Section: Introductionmentioning

confidence: 99%

Fluorinated oil-surfactant mixtures with the density of water: Artificial cells for synthetic biology

Laos

Benner

2022

PLoS ONE

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There is a rising interest in biotechnology for the compartmentalization of biochemical reactions in water droplets. Several applications, such as the widely used digital PCR, seek to encapsulate a single molecule in a droplet to be amplified. Directed evolution, another technology with growing popularity, seeks to replicate what happens in nature by encapsulating a single gene and the protein encoded by this gene, linking genotype with phenotype. Compartmentalizing reactions in droplets also allows the experimentalist to run millions of different reactions in parallel. Compartmentalization requires a fluid that is immiscible with water and a surfactant to stabilize the droplets. While there are fluids and surfactants on the market that have been used to accomplish encapsulation, there are reported concerns with these. Span® 80, for example, a commonly used surfactant, has contaminants that interfere with various biochemical reactions. Similarly, synthetic fluids distributed by the cosmetic industry allow some researchers to produce experimental results that can be published, but then other researchers fail to reproduce some of these protocols due to the unreliable nature of these products, which are not manufactured with the intent of being used in biotechnology. The most reliable fluids, immiscible with water and suitable for biochemical reactions, are fluorinated fluids. Fluorinated compounds have the peculiar characteristic of being immiscible with water while at the same time not mixing with hydrophobic molecules. This peculiar characteristic has made fluorinated fluids attractive because it seems to be the basis of their being biologically inert. However, commercially available fluorinated fluids have densities between 1.4 to 1.6 g/mL. The higher-than-water density of fluorinated oils complicates handling of the droplets since these would float on the fluid since the water droplets would be less dense. This can cause aggregation and coalescence of the droplets. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities similar to the one of water at room temperature, and when mixed with non-ionic fluorinated surfactants, can produce droplets encapsulating biochemical reactions. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman® assays. Some of these use unnatural DNA built from an Artificially Expanded Genetic Information System (AEGIS) with six nucleotide "letters".

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“…Droplet microfluidics has established itself for studying biological and biochemical systems with high throughput and low cost. [1][2][3][4] By forming millions of near-identical water in oil droplets, samples are partitioned enabling in-drop directed evolution, high throughput screening, and single-molecule or single-cell analysis. [5][6][7] These foundational techniques have relied upon the efficient compartmentalization enabled by monodisperse SE droplets.…”

Section: Introductionmentioning

confidence: 99%

“…[22][23][24] Sorting by commercial flow cytometers eliminates the need for complex optical and electronic equipment and the associated expertise which is required to sort SEs. 4,7,25 This combination of factors, positions DEs as a promising modality in the proliferation of droplet-based high throughput assays and screening. In this paper, we test our hypothesis that DE drops effectively eliminate drop merging and content mixing and highlight the strength of DE-based approaches for single-cell analysis over traditional SE methods.…”

Section: Introductionmentioning

confidence: 99%

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis

Cowell

Dobria

Han

2021

Preprint

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Drop microfluidics has driven innovations for high throughput, low input analysis techniques such as single-cell RNA-seq. However, the instability of single emulsion (SE) drops occasionally causes significant merging during drop processing, limiting most applications to single-step reactions in drops. Here, we show that double emulsion (DE) drops address this critical limitation and completely prevent content mixing, which is essential for single entity analysis. DEs show excellent stability during thermal cycling. More importantly, DEs undergo rupture into the continuous phase instead of merging, preventing content mixing and eliminating unstable drops from the downstream analysis. Due to the lack of drop merging, the monodispersity of drops is maintained throughout a workflow, enabling the deterministic manipulation of drops downstream. We also developed a simple, one-layer fabrication method for DE drop makers. This design is powerful as it allows robust production of single-core DEs at a wide range of flow rates and better control over the shell thickness, both of which have been significant limitations of conventional two-layer devices. This approach makes the fabrication of DE devices much more accessible, facilitating its broader adoption. Finally, we show that DE droplets effectively maintain the compartmentalization of single virus genomes during PCR-based amplification and barcoding, while SEs mixed contents due to merging. With their resistance to content mixing, DE drops have key advantages for multistep reactions in drops, which is limited in SEs due to merging and content mixing.

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Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey

Abstract: From the Contents 1. Introduction 24369 2. Single Emulsions 24371 3. Double Emulsions 24378 4. Latest Developments and Label-Free Methods 24382

Cited by 82 publications

References 168 publications

Efficient multi-gene expression in cell-free droplet microreactors

Efficient multi-gene expression in cell-free droplet microreactors

Fluorinated oil-surfactant mixtures with the density of water: Artificial cells for synthetic biology

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis

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