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

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 a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-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

Selection platforms for directed evolution in synthetic biology

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules – gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function – be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code

Bottom-up construction of complex biomolecular systems with cell-free synthetic biology

Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner

Microdroplets as microreactors for biology and chemistry in microfluidic systems

La compartimentation de la soupe primitive dans des vésicules est pensée comme étant une des premières étapes dans l’apparition de la vie. Ces compartiments primitifs permettent de lier une information génétique (génotype) avec les produits de son expression (phénotype). C’est une des premières étapes offrant la possibilité d’évolution et d’hérédité. En laboratoire l’évolution dirigée est basée sur les mêmes mécanismes de mutation et de sélection utilisés par l’évolution naturelle. Pour ce faire, le génotype et le phénotype sont couplés par une variété de méthodes, parmi lesquelles la Compartimentation In Vitro (CIV). Cette technique implique la création d’une émulsion dans laquelle des molécules d’ADN ou d’ARN, portant l’information génétique, sont isolées dans des gouttelettes aqueuses dispersées dans une phase d’huile non miscible. Ce confinement des gènes dans les gouttelettes permet leur maintien avec leurs produits d’expression établissant ainsi un lien entre génotype et phénotype. La conservation d’un lien entre génotype et phénotype est un pré-requis à l’évolution dirigée de macromolécules biologiques. Ces gouttelettes d’eau dans l’huile permettent de réaliser des millions d’expériences dans des compartiments microscopiques ayant des volumes de 103 à 109 fois plus petites que le puits d’une plaque de microtitration (1-2 μL). L’utilisation de ces émulsions pour l’évolution dirigée a déjà permis la sélection d'un large éventail de protéines et d’ARN impliqués dans différentes réactions catalytiques [...]The compartmentalization of the primordial soup into vesicles is thought to be one of the first steps of the emergence of organized cells. These water-in-oil-in-water droplets provide a linkage between genotype and phenotype. Moreover, through division they provide the possibility for heredity and evolution. By using manmade compartments, in form of bulk water-in-oil emulsions, it is possible to perform directed evolution experiments within the laboratory. These experiments use Darwinian principles comprising iterative cycles of mutation and selection. The bulk emulsions are composed of millions of individual droplets containing one single gene with all the ingredients necessary for the in vitro expression of those genes. The specific phenotype can then be selected under strictly controlled conditions. However, the emulsions created in bulk are highly polydisperse and have specific limitations when used for directed evolution experiments. Due to differences in droplet volumes it is difficult to perform quantitative experiments. Droplet-based microfluidics and in vitro compartmentalization (IVC) can be combined to perform directed evolution experiments. Droplet-based microfluidics produces highly monodisperse emulsions that can be manipulated in a highly controlled manner. By using a set of specific microfluidic devices it is possible to amplify single genes in droplets, to measure their in vitro expression and, in combination with a sorting device isolate the most active variants form a genetic library. [...

Microgouttelettes pour la biologie et la chimie en systèmes microfluidics

La compartimentation de la soupe primitive dans des vésicules est pensée comme étant une des premières étapes dans l'apparition de la vie. Ces compartiments primitifs permettent de lier une information génétique (génotype) avec les produits de son expressThe compartmentalization of the primordial soup into vesicles is thought to be one of the first steps of the emergence of organized cells. These water-in-oil-in-water droplets provide a linkage between genotype and phenotype. Moreover, through division t