scholarly journals Single-Round Remodeling of the Active Site of an Artificial Metalloenzyme using an Ultrahigh-Throughput Double Emulsion Screening Assay

2021 ◽  
Author(s):  
Jaicy Vallapurackal ◽  
Ariane Stucki ◽  
Alexandria Deliz Liang ◽  
Juliane Klehr ◽  
Petra S Dittrich ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Double Emulsion ◽  
Cell Encapsulation ◽  
Optimization Procedure ◽  
Droplet Microfluidics ◽  
Screening Assay ◽  
Fluorescent Intensity ◽  
Double Emulsions ◽  
Artificial Metalloenzyme

The potential of high-throughput compartmentalization renders droplet microfluidics an attractive tool for directed evolution of enzymes as it permits maintenance of the phenotype-genotype linkage throughout the entire optimization procedure. In particular, water-in-oil-in-water double emulsions droplets (DEs) produced by microfluidics enable the analysis of reaction compartments at ultra-high-throughput using commercially available fluorescence-activated cell sorting (FACS) devices. Here we report a streamlined method applicable for the ultrahigh-throughput screening of an artificial metalloenzyme (ArM), an artificial deallylase (ADAse), in double emulsions. The DE-protocol was validated by screening a four hundred member, double-mutant streptavidin library for the CpRu-catalyzed uncaging of aminocoumarin. The most active variants, identified by next-generation sequencing of the sorted DE droplets with highest fluorescent intensity, are in good agreement with 96-well plate screening hits. These findings, thus, pave the way towards the systematic implementation of commercially available FACS for the directed evolution of metalloenzymes making ultrahigh-throughput screening more broadly accessible. The use of microfluidics for the formation of uniform compartments with precise control over reagents and cell encapsulation further facilitates the establishment of highly reliable quantitative assays.

scholarly journals Synchronized Reagent Delivery in Double Emulsions for Triggering Chemical Reactions and Gene Expression

2021 ◽  
Author(s):  
Ariane Stucki ◽  
Petra Jusková ◽  
Nicola Nuti ◽  
Steven Schmitt ◽  
Petra Dittrich
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Double Emulsion ◽  
Lipid Vesicles ◽  
Enzyme Engineering ◽  
Droplet Microfluidics ◽  
Enzymatic Reactions ◽  
High Throughput Analysis ◽  
Double Emulsions

Microfluidic methods to form single emulsion and double emulsion (DE) droplets have greatly enhanced the toolbox for high throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed nanoliter compartments limit the applicability of droplet microfluidics. Here, we introduce a strategy for on-demand delivery of reactants in DEs. We use lipid vesicles as transport carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant SDS. The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the lipid bilayer. We demonstrate the versatility of the method with two critical applications, chosen as representative assays for high throughput screening. First, we trigger enzymatic reactions after releasing a reactant and second, we encapsulate bacteria and induce gene expression at a delayed time. The presented technique is compatible with the high throughput analysis of individual DE droplets using conventional flow cytometry as well as with microfluidic time-resolved studies. The possibility of delaying and controlling reagent delivery in current high throughput compartmentalization systems will significantly extend their range of applications e.g. for directed evolution, and further improve their compatibility with biological systems.

scholarly journals Synchronized Reagent Delivery in Double Emulsions for Triggering Chemical Reactions and Gene Expression

2021 ◽  
Author(s):  
Ariane Stucki ◽  
Petra Jusková ◽  
Nicola Nuti ◽  
Steven Schmitt ◽  
Petra Dittrich
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Double Emulsion ◽  
Lipid Vesicles ◽  
Enzyme Engineering ◽  
Droplet Microfluidics ◽  
Enzymatic Reactions ◽  
High Throughput Analysis ◽  
Double Emulsions

Microfluidic methods to form single emulsion and double emulsion (DE) droplets have greatly enhanced the toolbox for high throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed nanoliter compartments limit the applicability of droplet microfluidics. Here, we introduce a strategy for on-demand delivery of reactants in DEs. We use lipid vesicles as transport carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant SDS. The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the lipid bilayer. We demonstrate the versatility of the method with two critical applications, chosen as representative assays for high throughput screening. First, we trigger enzymatic reactions after releasing a reactant and second, we encapsulate bacteria and induce gene expression at a delayed time. The presented technique is compatible with the high throughput analysis of individual DE droplets using conventional flow cytometry as well as with microfluidic time-resolved studies. The possibility of delaying and controlling reagent delivery in current high throughput compartmentalization systems will significantly extend their range of applications e.g. for directed evolution, and further improve their compatibility with biological systems.

scholarly journals Towards the Directed Evolution of Artificial Metalloenzymes

2021 ◽  
Vol 75 (4) ◽  
pp. 257-260
Author(s):  
Jaicy Vallapurackal
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Droplet Microfluidics ◽  
Host Protein ◽  
Great Promise ◽  
Genetic Optimization ◽  
The Core ◽  
Technological Developments ◽  
Artificial Metalloenzymes ◽  
Tools And Methods

Artificial metalloenzymes (ArMs) are a class of enzymes holding great promise. In contrast to natural enzymes, the core of ArMs is a synthetic metallocofactor, with potential for bio-orthogonal reactivity, incorporated within a host protein. Next to chemical optimization of the metallocofactor, genetic optimization of the protein allows the further improvement of the ArM. Genetic optimization through directed evolution requires extensive screening of a large sequence-scape to enable the optimization of a desired phenotype. The process is however mostly limited by the throughput of the tools and methods available for screening. In recent years, versatile methods based on droplet microfluidics have been developed to address the need for higher throughput. This article aims to give an introduction into ArMs and the recent technological developments allowing high-throughput directed evolution of enzymes.

scholarly journals A Flow Cytometry–Based Screening System for Directed Evolution of Proteases

2011 ◽  
Vol 16 (3) ◽  
pp. 285-294 ◽  
Author(s):  
Ran Tu ◽  
Ronny Martinez ◽  
Radivoje Prodanovic ◽  
Mathias Klein ◽  
Ulrich Schwaneberg
Keyword(s):  
Flow Cytometry ◽  
Directed Evolution ◽  
High Throughput ◽  
Double Emulsion ◽  
Catalytic Efficiency ◽  
Small Population ◽  
Subtilis Strain ◽  
Screening System ◽  
Screening Technology

Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometry–based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry–based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110–containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance.

scholarly journals Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

2020 ◽  
Vol 92 (19) ◽  
pp. 13262-13270
Author(s):  
Kara K. Brower ◽  
Margarita Khariton ◽  
Peter H. Suzuki ◽  
Chris Still ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Double Emulsion ◽  
Cell Encapsulation ◽  
Single Cell Encapsulation

scholarly journals Droplet Microfluidics-Enabled High-Throughput Screening for Protein Engineering

Micromachines ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 734 ◽  
Author(s):  
Lindong Weng ◽  
James E. Spoonamore
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
High Throughput ◽  
Protein Design ◽  
High Throughput Screening ◽  
Fluid Phase ◽  
Droplet Microfluidics ◽  
New Paradigm ◽  
Wide Range ◽  
Challenges And Opportunities

Protein engineering—the process of developing useful or valuable proteins—has successfully created a wide range of proteins tailored to specific agricultural, industrial, and biomedical applications. Protein engineering may rely on rational techniques informed by structural models, phylogenic information, or computational methods or it may rely upon random techniques such as chemical mutation, DNA shuffling, error prone polymerase chain reaction (PCR), etc. The increasing capabilities of rational protein design coupled to the rapid production of large variant libraries have seriously challenged the capacity of traditional screening and selection techniques. Similarly, random approaches based on directed evolution, which relies on the Darwinian principles of mutation and selection to steer proteins toward desired traits, also requires the screening of very large libraries of mutants to be truly effective. For either rational or random approaches, the highest possible screening throughput facilitates efficient protein engineering strategies. In the last decade, high-throughput screening (HTS) for protein engineering has been leveraging the emerging technologies of droplet microfluidics. Droplet microfluidics, featuring controlled formation and manipulation of nano- to femtoliter droplets of one fluid phase in another, has presented a new paradigm for screening, providing increased throughput, reduced reagent volume, and scalability. We review here the recent droplet microfluidics-based HTS systems developed for protein engineering, particularly directed evolution. The current review can also serve as a tutorial guide for protein engineers and molecular biologists who need a droplet microfluidics-based HTS system for their specific applications but may not have prior knowledge about microfluidics. In the end, several challenges and opportunities are identified to motivate the continued innovation of microfluidics with implications for protein engineering.

scholarly journals Optimized double emulsion flow cytometry with high-throughput single droplet isolation

2019 ◽  
Author(s):  
Kara K. Brower ◽  
Catherine Carswell-Crumpton ◽  
Sandy Klemm ◽  
Bianca Cruz ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Double Emulsion ◽  
Droplet Microfluidics ◽  
Cell Analysis ◽  
Single Droplet ◽  
Emulsion Droplets ◽  
Droplet Sorting

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 μm nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

scholarly journals Identification of a Novel Laser Dye Substrate of Mammalian Cytochromes P450: Application in Rapid Kinetic Analysis, Inhibitor Screening, and Directed Evolution

2007 ◽  
Vol 12 (5) ◽  
pp. 677-682 ◽  
Author(s):  
Santosh Kumar
Keyword(s):  
Kinetic Analysis ◽  
Directed Evolution ◽  
High Throughput ◽  
Cumene Hydroperoxide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Significant Activity ◽  
Laser Dye ◽  
Screening Assay ◽  
Inhibitor Screening ◽  
Activity Screening

The author sought to develop a high-throughput activity screening assay to carry out rapid kinetic analysis, inhibitor screening, and directed evolution of cytochrome P450 2C enzymes. Initially, of the 9 fluorescent substrates and 10 P450 2C enzymes tested, several P450 2C enzymes showed > 1 nmol/min/nmol P450 activity in cumene hydroperoxide (CuOOH)—supported reaction with a laser dye, 7-dimethylamino-4-trifluoromethylcoumarin (C152). A high-throughput steady-state kinetic analysis of the human P450 2C8, 2C9, and 2C19 showed 1) kcat = 3 to 6 min—1, 2) Km, CuOOH = 100 to 200 µM, and 3) S50, C152 = 10 to 20 µM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high kcat (27 and 38 min—1, respectively) in the nicotinamide adenine dinucleotide phosphate (NADPH)—supported reaction. Subsequently, when mammalian P450s from the other subfamilies were tested, P450 2B1dH, 2B4dH, 2B5dH, 3A4, and 3A5 exhibited a significant activity in both CuOOH and NADPH systems. Furthermore, a high-throughput activity screening assay using whole-cell suspensions of the human P450 2C8, 2C9, and 2C19 was optimized. Overall, the data suggested that C152 can be used as a model substrate for mammalian P450s in CuOOH-supported reaction to perform rapid kinetic analysis, inhibitor screening, and directed evolution. ( Journal of Biomolecular Screening 2007:677-682)

scholarly journals High-Throughput Production of Micrometer Sized Double Emulsions and Microgel Capsules in Parallelized 3D Printed Microfluidic Devices

Polymers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1887 ◽  
Author(s):  
Alexander Jans ◽  
Jonas Lölsberg ◽  
Abdolrahman Omidinia-Anarkoli ◽  
Robin Viermann ◽  
Martin Möller ◽  
...  
Keyword(s):  
Surface Modification ◽  
High Throughput ◽  
Hollow Sphere ◽  
Double Emulsion ◽  
Continuous Phase ◽  
Flow Focusing ◽  
Double Emulsions ◽  
Emulsion Droplets ◽  
3D Printed ◽  
Double Emulsion Droplets

Double emulsions are useful geometries as templates for core-shell particles, hollow sphere capsules, and for the production of biomedical delivery vehicles. In microfluidics, two approaches are currently being pursued for the preparation of microfluidic double emulsion devices. The first approach utilizes soft lithography, where many identical double-flow-focusing channel geometries are produced in a hydrophobic silicone matrix. This technique requires selective surface modification of the respective channel sections to facilitate alternating wetting conditions of the channel walls to obtain monodisperse double emulsion droplets. The second technique relies on tapered glass capillaries, which are coaxially aligned, so that double emulsions are produced after flow focusing of two co-flowing streams. This technique does not require surface modification of the capillaries, as only the continuous phase is in contact with the emulsifying orifice; however, these devices cannot be fabricated in a reproducible manner, which results in polydisperse double emulsion droplets, if these capillary devices were to be parallelized. Here, we present 3D printing as a means to generate four identical and parallelized capillary device architectures, which produce monodisperse double emulsions with droplet diameters in the range of 500 µm. We demonstrate high throughput synthesis of W/O/W and O/W/O double emulsions, without the need for time-consuming surface treatment of the 3D printed microfluidic device architecture. Finally, we show that we can apply this device platform to generate hollow sphere microgels.

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