The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

The present invention provides a high throughput method for constructing and screening a compound library and a reaction device. Specifically, the method of the present invention comprises: (a) providing a reactor comprising n independent and addressable reaction chambers; (b) performing m independent synthesis reactions in said n reaction chambers, thereby constructing a compound library; and (c) performing activity tests in reaction chambers in which synthesis reactions are performed. In the present invention, the preparation and screening processes of a compound can be completed in the same reaction system. As the reactions of the present invention almost quantitatively generate products, the products can be directly used in enzymatic or even cytological activity test experiments without separation.

The present invention provides a high throughput method for constructing and screening a compound library and a reaction device. Specifically, the method of the present invention comprises: (a) providing a reactor comprising n independent and addressable reaction chambers; (b) performing m independent synthesis reactions in said n reaction chambers, thereby constructing a compound library; and (c) performing activity tests in reaction chambers in which synthesis reactions are performed. In the present invention, the preparation and screening processes of a compound can be completed in the same reaction system. As the reactions of the present invention almost quantitatively generate products, the products can be directly used in enzymatic or even cytological activity test experiments without separation.

Embodiments disclosed herein are directed to microfluidic devices that allow for scalable on-chip screening of combinatorial libraries and methods of use thereof. Droplets comprising individual molecular species to be screened are loaded onto the microfluidic device. The droplets are labeled by methods known in the art, including but not limited to barcoding, such that the molecular species in each droplet can be uniquely identified. The device randomly sorts the droplets into individual microwells of an array of microwells designed to hold a certain number of individual droplets in order to derive combinations of the various molecular species. The paired droplets are then merged in parallel to form merged droplets in each microwell, thereby avoiding issues associated with single stream merging. Each microwell is then scanned, e.g., using microscopy, such as high content imaging microscopy, to detect the optical labels, thereby identifying the combination of molecular species in each microwell.

Embodiments disclosed herein are directed to microfluidic devices that allow for scalable on-chip screening of combinatorial libraries and methods of use thereof. Droplets comprising individual molecular species to be screened are loaded onto the microfluidic device. The droplets are labeled by methods known in the art, including but not limited to barcoding, such that the molecular species in each droplet can be uniquely identified. The device randomly sorts the droplets into individual microwells of an array of microwells designed to hold a certain number of individual droplets in order to derive combinations of the various molecular species. The paired droplets are then merged in parallel to form merged droplets in each microwell, thereby avoiding issues associated with single stream merging. Each microwell is then scanned, e.g., using microscopy, such as high content imaging microscopy, to detect the optical labels, thereby identifying the combination of molecular species in each microwell.

A visual continuous spatial directed evolution method is disclosed. The host grows and moves in a solid culture space, the host carrying a foreign target gene to be evolved and containing a gene element that assists the evolution of the target gene, the target gene being correlated with the growth and movement of the host. Depending on different spatial distribution patterns formed in the solid culture space during the growth and movement of the host, screening is performed to obtain an evolved product. This method is carried out directly in the solid culture space. Depending on images of different spatial distribution morphologies visible to the naked eye that are locally formed, selection of evolved products is performed without the need for liquid fed-batch culture equipment. In addition, the evolution effect is visually observed through the infection spots formed during evolution, so that no real-time monitoring equipment is required.

A visual continuous spatial directed evolution method is disclosed. The host grows and moves in a solid culture space, the host carrying a foreign target gene to be evolved and containing a gene element that assists the evolution of the target gene, the target gene being correlated with the growth and movement of the host. Depending on different spatial distribution patterns formed in the solid culture space during the growth and movement of the host, screening is performed to obtain an evolved product. This method is carried out directly in the solid culture space. Depending on images of different spatial distribution morphologies visible to the naked eye that are locally formed, selection of evolved products is performed without the need for liquid fed-batch culture equipment. In addition, the evolution effect is visually observed through the infection spots formed during evolution, so that no real-time monitoring equipment is required.

Embodiments in accordance with the present disclosure are directed to apparatuses used for reaction screening and optimization purposes. An example apparatus includes a plurality of reaction vessels, a dispensing subsystem, at least one reactor module, an analysis subsystem, an automation subsystem, and control circuitry. The dispensing subsystem delivers reagents to the plurality of reaction vessels for a plurality of reaction mixtures having varied reaction conditions. The at least one reactor module drives a plurality of reactions within the plurality of reaction vessels. The analysis subsystem analyzes compositions contained in the plurality of reaction vessels. The automation subsystem selectively moves the plurality of reaction vessels from a location proximal to the dispensing subsystem to the at least one reactor module based on experimental design parameters. And, the control circuitry identifies optimum reaction conditions for a target end product based on the analysis.

The present disclosure provides methods for forming emulsions. A method for forming an emulsion comprises: bringing a first fluid phase in contact with a second fluid phase that is immiscible with the first fluid phase to generate the emulsion comprising the plurality of droplets. The plurality of droplets may comprise (i) the first fluid phase or the second fluid phase, (ii) a first surfactant at an interface between the first fluid phase and the second fluid phase, and (iii) a second surfactant that is different than the first surfactant. Subsequent to generating the emulsion, collect or direct the plurality of droplets along a channel. Subsequent to collecting or directing the plurality of droplets along the channel, at most 5% of the plurality of droplets coalesce.

The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. The invention provides for aqueous based emulsions containing uniquely labeled cells, enzymes, nucleic acids, etc., wherein the emulsions further comprise primers, labels, probes, and other reactants. An oil based carrier-fluid envelopes the emulsion library on a microfluidic device, such that a continuous channel provides for flow of the immiscible fluids, to accomplish pooling, coalescing, mixing, sorting, detection, etc., of the emulsion library.