Development of Quantitative Cell-Based Enzyme Assays in Microdroplets

Huebner, Ansgar; Olguín, Luis F.; Bratton, Daniel; Whyte, Graeme; Huck, Wilhelm T. S.; Mello, Andrew J. de; Edel, Joshua B.; Abell, Chris; Hollfelder, Florian

doi:10.1021/ac800338z

Cited by 197 publications

(200 citation statements)

References 43 publications

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“…By mixing aqueous samples together with a suitable surfactant-containing oil phase, millions of compartments are generated in minutes, not requiring any specialized equipment. Although monodisperse emulsion samples (uniform droplet size) produced with microfluidic droplet generators (40) allow for a high degree of control in encapsulating single cells in droplets (41), the additional technological complexity as well as the difficulty in handling a multitude of samples in parallel in our opinion outweighs the benefit of a likely only moderate acceleration of this passive selection process. However, future advances in microfluidic droplet sorting (42), for example, sorting based on optical density of droplet contents, could well result in experimental protocols capable of directly selecting cells with a high-yield phenotype and therefore greatly facilitate such evolution campaigns.…”

Section: Discussionmentioning

confidence: 99%

Availability of public goods shapes the evolution of competing metabolic strategies

Bachmann¹,

Fischlechner

Rabbers³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

173

220

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Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli, yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell-cell interactions, and the relationships between environmental and metabolic constraints.A lthough the existence of tradeoffs in evolution seems to be undisputable, experimental evidence obtained under controlled conditions is scarce. Several examples failed to show tradeoffs (1-4), whereas others could find them (5, 6) or found general but not universal tradeoffs (7,8). A tradeoff between growth rate and growth yield in microbes (9-11) has direct implications for experiments carried out in liquid cultures. This is especially of importance during prolonged cultivations such as laboratory evolution experiments. In suspension, fast-growing variants outcompete slower growing ones at the cost of biomass yield (5). The yield versus rate optimization is governed by a dilemma where fast growth is advantageous from the perspective of an individual cell, whereas slow growth, and therefore high yield, is advantageous from the perspective of a population. This dilemma is consistent with a concept termed the tragedy of the commons (12). It is well described that spatial structure is essential for the selection of high-yield strategies (13-15). The yield/rate tradeoff of microbial growth has been linked to metabolic strategies (11) such as the switch between respiration and fermentation in yeast (9). It is suggested that the underlying cause of this tradeoff is based on...

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Section: Discussionmentioning

confidence: 99%

Availability of public goods shapes the evolution of competing metabolic strategies

Bachmann¹,

Fischlechner

Rabbers³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

173

220

View full text Add to dashboard Cite

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“…Cell encapsulation into microfluidic droplets generally follows the Poisson distribution, 34 which implies that the cells need to be diluted to achieve predominately single cells in droplets. A cell to droplet ratio of 0.4 was used, which constitutes a tradeoff between higher throughput from having most droplets occupied by cells and the occurrence of adverse co-encapsulation events.…”

Section: Model Selection For α-Amylase Expressionmentioning

confidence: 99%

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

et al. 2014

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A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α-amylase production, close to the theoretical maximum enrichment. Furthermore, a 10 5 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.

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“…Other advantages of droplet-based [76] microfluidic system include precisely controllable reaction time inside a droplet by adjusting the length of channel and the flow rates of fluids. The availability of a wide range of technologies for flexible generation and manipulation of droplets has enabled the applications of droplet microfluidics in a wide variety of fields, from chemical reactions [9,79] and protein crystallization [10,11] to material synthesis [12,13,[80][81][82][83], single cell analysis [84][85][86][87][88][89][90][91], DNA amplification [16], protein engineering [62,92], and high-throughput screening technologies [17,93]. It is not possible to cover all application areas in this review.…”

Section: Applicationsmentioning

confidence: 99%

“…In this scene, droplet microfluidics is potentially a very competitive technical platform for high-throughput single cell analysis. Technologies for encapsulating individual cells into separated water in oil droplets [119][120][121], which allow for confining [122,123], content analysis [87,88,90,119,124], screening [36,57], and temporal changes tracing [63] of the cells, have become available recently, indicating promising applications for droplet microfluidics in this field.…”

Section: Single Cell Analysismentioning

confidence: 99%

Basic Technologies for Droplet Microfluidics

Zeng

Liu

Xie

et al. 2011

Topics in Current Chemistry

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In recent years droplet microfluidics has become a quickly evolving research field. The availability of a wide range of technologies for droplet generation and manipulation has enabled the applications of droplet microfluidics in a wide variety of fields, from single cell analysis to material synthesis. In this review we summarize the main technologies for droplet microfluidics and discuss the recent advances in technologies that enable droplets as microreactors with complete functions. Applications of microdroplets in chemical reactions, particle synthesis, and single cell analysis are also briefly reviewed.

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Development of Quantitative Cell-Based Enzyme Assays in Microdroplets

Cited by 197 publications

References 43 publications

Availability of public goods shapes the evolution of competing metabolic strategies

Availability of public goods shapes the evolution of competing metabolic strategies

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

Basic Technologies for Droplet Microfluidics

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