WO2016048994A2

WO2016048994A2 - Systems and methods of cell-free protein synthesis in droplets and other compartments

Info

Publication number: WO2016048994A2
Application number: PCT/US2015/051401
Authority: WO
Inventors: David A. Weitz; Shaorong Chong; Huidan ZHANG; Nai Wen CUI; Ye Tao
Original assignee: President And Fellows Of Harvard College; New England Biolabs, Inc.
Priority date: 2014-09-23
Filing date: 2015-09-22
Publication date: 2016-03-31
Also published as: WO2016048994A3

Abstract

The present invention generally relates to cell-free protein synthesis in microfluidic droplets. Droplets may be used to encapsulate genetic information (DNA/RNA), and through cell-free protein synthesis, provide a linkage of the genetic information with the functional information, e.g., the activity of the expressed protein, and used to convert the functional information into a detectable signal, e.g., to allow sorting of the droplets and retrieve genetic information associated with the drops. In one set of embodiments, microfluidic droplets containing a cell-free protein synthesis system designed to detect protein-protein interaction (e.g., in vitro two-hybrid systems or IVT2H) can be used for high-throughput screening, e.g., of protein binders. This drop-based two-hybrid system may include two (or more) fusion proteins that can bind to each other such that their binding produces a complex that is able to produce a nucleic acid. The nucleic acid may be expressed to produce a protein. In certain embodiments, the protein may be produced within a cell-free system. The protein may be fluorescent or otherwise determinable, such that determination of the protein may be used to allow assays to occur within the droplets, to allow sorting of the droplets to occur, or the like, e.g., as discussed below, for instance, for screening or other applications.

Description

SYSTEMS AND METHODS OF CELL-FREE PROTEIN SYNTHESIS IN DROPLETS

AND OTHER COMPARTMENTS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No.

62/054,263, filed September 23, 2014, entitled "Two-Hybrid Systems and Methods in Droplets and Other Compartments," by Weitz, et ah, incorporated herein by reference in its entirety.

FIELD

The present invention generally relates to cell-free protein synthesis in microfluidic droplets.

BACKGROUND

Cell function is regulated by protein-protein binding affinities that underlie the interactome network. Between 40,000 and 200,000 protein-protein interactions (PPIs) have been predicted to exist within the human interactome, and their malfunction is one of the fundamental causes of human diseases. One promising therapeutic strategy involves the targeted introduction of protein binders with high binding affinity to up or down regulate certain PPIs. To further develop such drugs, fast, low-cost and high-throughput methods for screening binding affinity are required due to the limitless number of candidates and large size of the interactome network.

SUMMARY

A cell can be thought of as a compartment in which DNA/RNA is expressed as proteins, which allows the genetic information to be linked to the functional information. Microfluidic droplets encapsulating DNA/RNA and a cell-free protein synthesis system can serve as a celllike compartment in which DNA is expressed into protein, and provide a stable linkage between the genetic information and the functional information.

A cell-free protein synthesis system can be engineered to convert genetic information

(DNA/RNA) encapsulated in droplets into the functional information that is a detectable signal. Such signal can be used in drop-based microfluidics to sort droplets (or other compartments) containing the genetic information linked to the signal. This may allow fast, low-cost and ultra- high-throughput screening of large amounts of genetic information, such as a protein-encoding DNA library, for desired functions, such as high affinity binding or enzymatic activity.

The present invention generally relates to two-hybrid systems, e.g., in in vitro systems or microfluidic droplets. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the present invention is a method, for example, a method of protein production.

The method, in one set of embodiments, includes producing a first fusion protein, wherein the first fusion protein comprises a first portion and a second portion comprising a binding domain; producing a second fusion protein, wherein the second fusion protein comprises a first portion able to bind to the first portion of the first fusion protein and a second portion comprising an activation domain; binding the first portion of the first fusion protein and the first portion of the second fusion protein within a compartment having a volume of less than about 1 microliter; binding an RNA polymerase to the activation domain; binding the binding domain to a nucleic acid, wherein the RNA polymerase is able to express at least a portion of the nucleic acid to produce a protein; and expressing the protein.

In another set of embodiments, the method includes providing a plurality of droplets, at least some of which droplets comprise a nonconstant DNA sequence and a constant DNA sequence, wherein the nonconstant DNA sequences within the plurality of droplets together form a library of DNA sequences having at least 20 distinguishable members and at least about 50% homology, and wherein the constant DNA sequence is substantially identical in the at least some droplets; in at least some of the droplets, producing a first protein from the nonconstant DNA sequence and a second protein from the constant DNA sequence; forming a complex comprising the first protein, the second protein, and an RNA polymerase; binding the complex to a nucleic acid to express a protein encoded by the nucleic acid; and expressing the protein.

In yet another set of embodiments, the method comprises producing a first fusion protein; producing a second fusion protein; forming a complex comprising the first fusion protein, the second fusion protein, and an RNA polymerase within a compartment having a volume of less than about 1 microliter; binding the complex to a nucleic acid to express a protein encoded by the nucleic acid; and expressing the protein.

According to still another set of embodiments, the method includes producing a first fusion protein, wherein the first fusion protein comprises a first portion comprising MDM2 and a second portion comprising a binding domain; producing a second fusion protein, wherein the second fusion protein comprises a first portion comprising a peptide inhibitor of MDM2 and a second portion comprising an activation domain; binding the first portion of the first fusion protein and the first portion of the second fusion protein within a compartment having a volume of less than about 1 microliter; binding an RNA polymerase to the activation domain; and binding the binding domain to a nucleic acid, wherein the RNA polymerase is able to express at least a portion of the nucleic acid to produce a protein.

In accordance with yet another set of embodiments, the method includes providing a first protein; providing a second protein; binding at least a portion of the first protein to at least a portion of the second protein to produce a complex within a compartment having a volume of less than about 1 microliter, the compartment being free of cells; producing a nucleic acid using the complex; and expressing the nucleic acid as a third protein.

The method, in yet another set of embodiments, comprises producing a first protein and a second protein within a microfluidic droplet, the droplet being free of cells; binding at least a portion of the first protein to at least a portion of the second protein to produce a complex;

producing a nucleic acid using the complex; and expressing the nucleic acid as a third protein.

In another set of embodiments, the method comprises acts of producing a first fusion protein; producing a second fusion protein; forming a complex comprising the first fusion protein, the second fusion protein, and an RNA polymerase within a compartment having a volume of less than about 1 microliter; binding the complex to a nucleic acid to express a protein encoded by the nucleic acid; and expressing of the protein.

The method, in another set of embodiments, includes acts of producing a first fusion protein within a microfluidic droplet, wherein the first fusion protein comprises MDM2 and a second portion comprising a binding domain; producing a second fusion protein within the microfluidic droplet, wherein the second fusion protein comprises a first portion able to bind the MDM2 and a second portion comprising an activation domain; binding the first portion of the first fusion protein to the the first portion of the second fusion protein; binding an RNA polymerase to the activation domain; binding the binding domain to a nucleic acid, wherein the RNA polymerase is able to express at least a portion of the nucleic acid to produce a protein; determining expression of the protein; and sorting the microfluidic droplet based on the expression of the protein.

In yet another set of embodiments, the method is a method of protein production, comprising providing a first protein and a second protein within a microfluidic droplet, binding at least a portion of the first protein to at least a portion of the second protein to produce a complex, producing a nucleic acid using the complex, and expressing the nucleic acid as a third protein. In some cases, the microfluidic droplet is free of cells.

In another aspect, the present invention encompasses methods of making one or more of the embodiments described herein. In still another aspect, the present invention encompasses methods of using one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

Fig. 1A is a schematic diagram of a cell-free system for converting genetic information (e.g., protein-protein interactions) into a determinable fluorescent signal, in one embodiment of the invention; (SEP ID NO: 16 is TSxAEYxNLxSP):

Fig. IB illustrate microfluidic equipment that encapsulates DNA in a cell-free system in accordance with another embodiment of the invention;

Fig. 1C illustrates drop-based microfluidics for drop sorting and retrieval of genetic information from drops, in accordance with another embodiment of the invention;

Fig. 2A illustrates that genetic information encoding a higher affinity binder results in brighter fluorescent drops, while genetic information encoding a lower affinity binder, or no genetic information, results in no (or negligible) brighter drops, according to certain embodiments of the invention;

Fig. 2B illustrates that RNA from isolated drops can be amplified by RT-PCR, in some embodiments of the invention;

Fig. 2C illustrates that genetic information can be retrieved by sequencing amplified

DNA (SEQ ID NO: 25 is TSFAEYWNLLS, and SEQ ID NO: 26 is

ACCTCTTTCGCGGAATACTGGAACCTGCTGTCTCC);

Fig. 3A illustrates fluorescence-activated sorting of drops encapsulating a random DNA library and brighter drops containing potential high-affinity binders, in some embodiment of the inventions;

Fig. 3B illustrates RT-PCR amplification of RNA from isolated bright drops, in one embodiment of the invention;

Fig. 3C-3E illustrate retrieval of genetic information of high- affinity binders by deep sequencing, showing DNA encoding the known high-affinity binder (FWL) is enriched in droplets, in another embodiment of the invention;

Figs. 4A-4B illustrate interaction of fusion proteins, in another embodiment of the invention;

Fig. 5 shows a method of high throughput screening with drop-based synthetic cell-free protein synthesis, in yet another embodiment of the invention;

Fig. 6 shows another method of high throughput screening with drop-based synthetic cell-free protein synthesis, in still another embodiment of the invention; and

Fig. 7 shows the structure of SEQ ID NO: 9;

Fig. 8 shows the structure of SEQ ID NO: 21; and

Fig. 9 shows the structure of SEQ ID NO: 23.

DETAILED DESCRIPTION

The present invention generally relates to cell-free protein synthesis in microfluidic droplets. Droplets may be used to encapsulate genetic information (DNA/RNA), and through cell-free protein synthesis, provide a linkage of the genetic information with the functional information, e.g., the activity of the expressed protein, and used to convert the functional information into a detectable signal, e.g., to allow sorting of the droplets and retrieve genetic information associated with the drops. In one set of embodiments, microfluidic droplets containing a cell-free protein synthesis system designed to detect protein-protein interaction (e.g., in vitro two-hybrid systems or IVT2H) can be used for high-throughput screening, e.g., of protein binders. This drop-based two-hybrid system may include two (or more) fusion proteins that can bind to each other such that their binding produces a complex that is able to produce a nucleic acid. The nucleic acid may be expressed to produce a protein. In certain embodiments, the protein may be produced within a cell-free system. The protein may be fluorescent or otherwise determinable, such that determination of the protein may be used , to allow assays to occur within the droplets, to allow sorting of the droplets to occur, or the like, e.g., as discussed below, for instance, for screening or other applications.

In one embodiment, two (or more) fusion proteins may be provided that, when assembled, are able to cause a nucleic acid to be produced or expressed, e.g., to produce a reporter protein, such as a fluorescent protein, that is relatively easily determined. More than two fusion proteins may also be used in some instances. See, e.g., U.S. Prov. Pat. Apl. Ser. No. 62/008,341, filed June 5, 2014, entitled "Protein Analysis Assay System," by Weitz, et al, incorporated herein by reference in its entirety.

In addition, in some cases, systems such as these may be used to study systems where one of the fusion proteins is varied in some fashion (for example, systematically or randomly, mutated, selected from a library of different potential proteins, etc.). For example, in one set of embodiments, a series of microfluidic droplets may be provided that contain the two (or more) fusion proteins, where one of the fusion proteins is varied between the droplets. By determining the reporter protein within the droplets (e.g., through fluorescence of the droplets), the droplets may be accordingly screened or sorted.

An example embodiment is now described with respect to Fig. 4. However, other systems and methods may be used as well. In this figure, a reporter protein 60 is to be determined in some fashion, e.g., which can be used to assess an interaction between a first fusion protein 10 and a second fusion protein 20. Thus, for example, the amount of reporter protein that is present may be a function of the interaction between the fusion proteins.

In this figure, first fusion protein 10 comprises a first portion 11 and a second portion 12 comprising a binding domain ("BD"). Second fusion protein 20 comprises a first portion 21 able to bind to the first portion of the first fusion protein and a second portion 22 comprising an activation domain ("AD"). The first portions of the first fusion protein and the second fusion protein may bind to or interact with each other in some fashion. For example, the first portion of the second fusion protein may include a MDM2 protein (or fragment thereof), while the first portion of the first fusion protein may include a peptide inhibitor ("PMI") that is able to bind to or otherwise interact with MDM2. It should be noted that MDM2 and PMI are provided here by way of example only; other examples are discussed in more detail below.

The activation domain 22 of the second fusion protein may be selected to bind to (or recruit) a polymerase, such as RNA polymerase 40, as is discussed below. The activation domain need not be directly related to first portion 21, e.g., where both are part of fusion protein 20. Thus, for example, the regions may come from different chromosomes, different cells, different organisms, or even different species, and fusion protein 20 may comprise each of these regions (and optionally, other regions as well). As an example, activation domain 22 may include a protein such as TFIIIB 150, which is able to recruit RNA polymerase.

Similarly, fusion protein 10 may also have a second portion that is a binding domain ("DB"), or region 12. This region may be selected to be able to bind to nucleic acid 50, e.g., as discussed below. For instance, nucleic acid 50 may be reporter nucleic acid that can be used to produce a nucleic acid, such as RNA. In some cases, the RNA may also be expressed as a protein. In some cases, the protein may be fluorescent, and/or the protein may have other characteristics that allow for easy determination of the protein. Binding domain 12 need not be related to portion 11, e.g., where both are part of fusion protein 10. For instance, these regions may come from different chromosomes, different cells, different organisms, or even different species, and fusion protein 10 may comprise each of these regions (and optionally, other regions as well). As a non-limiting example, in one embodiment, binding domain 12 may comprise a sequence able to recognize a UAS (upstream activating sequence) region 51, or other region of nucleic acid 50 that can be specifically recognized by domain 12. For instance, domain 12 may comprise zinc finger DNA binding proteins, Cro protein, GAL4 protein, DAP I or DAP II

(derepression activating protein), or portions thereof, or fusion protein 10 may comprise a SH2 fusion protein which contains a binding domain.

In some embodiments, as mentioned, activation domain 22 may be selected to bind to (or recruit) a polymerase, such as RNA polymerase 40. RNA polymerase 40 may be any suitable polymerase that can be recruited, e.g., RNA polymerase I, II, or III. Recruitment of RNA polymerase 40 to activation domain 22 may occur before, during, or after binding or other interactions of portions 11 and 21 with each other. The polymerase may also interact with nucleic acid 50 in some fashion. For example, RNA polymerase 40 may bind to or recognize a promoter site 52 on nucleic acid 50.

Similarly, binding domain 32 may interact with a suitable nucleic acid 50 before, during, or after binding/interaction of portions 11 and 12 to each other. In some cases, nucleic acid 50 may include a UAS or upstream activating sequence, or other sequence that can be specifically recognized by binding domain 12.

The above-described interactions may occur in any suitable order, and in some cases, may result in the formation of a complex that is able to express at least a portion of nucleic acid 50. The presence or concentration of the target may affect the expression of at least a portion of nucleic acid 50, e.g., to produce a protein, which can be indicative of the binding or interaction of portions 11 and 12, qualitatively and/or quantitatively. For instance, as is shown in Fig. 7, RNA polymerase 40 is able to interact with nucleic acid 50 to produce RNA 45. The RNA may be directly detected, and/or the RNA may be subsequently expressed, e.g., to produce a reporter protein 60 that can be determined. For instance, the protein may be a fluorescent protein, such as GFP (green fluorescent protein).

In certain cases, the above-described system may include components able to cause in situ expression of proteins, e.g., where nucleic acids can be expressed to form proteins in situ or in vitro, rather than inside of living cells. Techniques for in vitro expression of proteins from nucleic acids are known, e.g., by using suitable transcription and translation proteins and other components in vitro. Kits for producing such in vitro expression of proteins are available commercially. In some cases, for example, nucleic acids encoding fusion proteins 10 and/or 20 may initially be present, such that fusion proteins 10 and 20 are produced in vitro in such an environment. Fusion proteins 10 and 20 can then react or proceed as discussed above. In some cases, RNA 45 may also be produced as discussed above, and then expressed in vitro into reporter protein 60. In some cases, the same molecular "machinery" may be involved in producing both fusion protein 10, fusion protein 20, and/or expressed protein 60. Once expressed, protein 60 can be detected using any suitable technique, and may be directly or indirectly determined, i.e., through its interactions with other proteins or species. In one set of embodiments, reporter protein 60 is fluorescent. Thus, by determining fluorescence in a sample, the presence or concentration of a target in the sample can be determined. In addition, in certain instances, systems such as those described herein may be used within a plurality of microfluidic droplets, e.g., for purposes such as screening or sorting. For example, as is discussed in more detail, fusion proteins 10 and 20 may be composed of interacting portions, one or both of which may be varied or mutated in some fashion, and the amount of interaction between the interacting portions, and thus fusion proteins 10 and 20, may be assessed within each droplet, e.g., by determining fluorescence within each droplet, or other assays to determine the reporter protein.

Another embodiment of the invention is discussed with reference to Fig. 5. In this figure, a technique involving a synthetic in vitro transcription and translation (sIVTT) system is shown. In this figure, DNA1 and 2 produce protein A and B, respectively. The presence or activity of protein A and B leads to the production of protein C from DNA 3, which can be a fluorescent protein, resulting in a detectable signal. In this case, sIVTT can be used as a simple synthetic gene "circuit," which correlates the functions or products of DNA 1 and 2 to a detectable signal. sIVTT can be designed to be a more complex gene circuit with more DNA templates and cascaded events, e.g., protein C leads to the production of another protein that is a fluorescent protein. Encapsulation of sIVTT in drops allows a DNA library containing a large number of different DNA templates to be distributed into different drops. The detectable signal generated by sIVTT allows drops to be sorted. As a result, the genetic information associated with the signals is enriched, and can be retrieved by subsequent RT-PCR of DNA/RNA from isolated drops and deep sequencing. This genetic information can be used to designed new functions based on known information such as crystal structure of a protein, and/or create a second set of genetic information for another round of screening. Iterative rounds of screening, which may be used in some embodiments such as is shown in the example of Fig. 5, can lead to significant improvements of gene functions or discovery of new functions from large pools of genetic materials. A similar example is shown in Fig. 6.

The above discussion is a non-limiting example of certain embodiments of the present invention. However, this is by way of explanation only, and other embodiments are also possible. Accordingly, more generally, various aspects of the invention are directed to various systems and methods for systems and methods for determining proteins, antibodies, or other targets. For instance, in some aspects, the present invention is generally directed to making proteins and other materials in droplets or other compartments, e.g., using systems and methods such as discussed herein. In some cases, this may be performed cell-free, e.g., proteins may be synthesized in droplets, without the presence of cells such as bacteria, yeast, or mammalian cells to assist in the production of the proteins. For instance, in one set of embodiments, one, two or more fusion proteins may be provided that can interact in a cell-free system to produce a protein, e.g., a reporter protein, upon suitable interactions of the fusion proteins. In some cases, the production of protein within the droplets may be controlled by the controlling the fusion proteins within the droplets. In addition, in some cases, synthetic in vitro transcription and translation (sIVTT) may occur within droplets (or other compartments, e.g., as discussed herein). For instance, in certain cases, two or more proteins may be provided or produced within a droplet or other compartment, where the proteins are able to interact, directly or indirectly, to produce a complex that is able to interact with a nucleic acid to cause expression of the nucleic acid as another protein. In addition, other components may be present as well in some embodiments, e.g., cofactors or the like, which may also become part of the complex. In some embodiments, the droplets or other compartments are free of cells, although in other embodiments, cells may be present.

In some cases, one, two, or more fusion proteins may be used. In some cases, the fusion proteins may exhibit an interaction with each other, e.g., such that the fusion proteins can assemble into a complex that is able to cause expression of another protein, such as a reporter protein to occur. In some cases, the protein may be used for detection, e.g., as a reporter. For instance, a droplet (or other compartment) may be sorted or screened by determining the reporter, e.g., qualitatively and/or quantitatively. For instance, in one set of embodiments, the protein may be fluorescent. As an example, two fusion proteins may be selected to have portions that can bind or otherwise interact, and one or both of the portions may be varied to find increased or decreased binding affinities. For instance, one or both of the portions may be systematically or randomly varied. Thus, for instance, one fusion protein may comprise a constant DNA sequence while another fusion protein may comprises a nonconstant DNA sequence that is allowed to vary in some fashion. When expressed, the fusion proteins that are produced may exhibit different amounts of binding or other interaction, which may result in different amounts of protein expression. The protein expression can be determined and sequences exhibiting desired characteristics (e.g., greater or lesser interactions) may be selected or screened using techniques such as those discussed herein.

In some cases, the nonconstant DNA sequence may be varied systematically. For instance, one or more locations may be varied systematically, e.g., by encoding each of the 20 amino acids in a location, e.g., in different droplets or compartments. More than one location may also be varied systematically, e.g., with 2, 3, 4, or more locations. In other cases, however, the nonconstant DNA sequence may be varied randomly, e.g., by introducing random mutations into the nonconstant DNA sequence. In some cases, the nonconstant DNA sequences may vary at 1, 2, 3, 4, or 5 locations. In some cases, the nonconstant DNA sequences may have a homology of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

However, in other embodiments, protein may be produced with the droplets or other compartments, e.g., for other uses. For example, the proteins may be allowed to collect within the droplets or compartments, then isolated from the droplets or compartments, e.g., for subsequent uses. In some cases, relatively large amounts of proteins may be produced in the droplets or other compartments. For instance, a protein may be produced within the droplet or compartment to a concentration of at least about 10 -^"8 M, at least about 10 -^"7 M, at least about 10 -^"6 M, at least about 10^"5 M, at least about 10^"5 M, or at least about 10^"4 M. In some cases, the amount of protein may be produced may be limited by the amount or concentration of free amino acids present within the droplets that are available for synthesis into a protein.

In certain embodiments, the fusion proteins may include two or more portions that are able to bind to or otherwise interact with each other. The binding may be specific or nonspecific, and typically is non-covalent binding. For instance, the two or more portions may have binding affinity to each other such that the K_<j (dissociation constant) is less than about 100 nM, less than about 50 nM, less than about 30 nM, less than about 10 nM, less than about 5 nM, less than about 3 nM, or less than about 1 nM. In some cases, the ¾ may be at least about 0.1 nM, about 1 nM, or about 10 nM. Combinations of any of these are also possible, e.g., the K_<j may be between about 1 nM and 10 nM.

In one set of embodiments, the fusion proteins may comprise a MDM2 protein (or fragment thereof) and a peptide inhibitor ("PMI") that is able to bind to or otherwise interact with MDM2. Other non-limiting examples of fusion proteins able to interact with each other include FKBP (FK506 binding protein)/FRB (FKBP12-Rapamycin Binding protein), Leucine zippers, or SspB/ssrA.

In some cases, other portions may also be present in a fusion protein as well. In one set of embodiments, a fusion protein may include a portion that is able to participate in a complex able to produce a nucleic acid or express a protein, and/or the portion may be able to recruit a component that is able to participate in a complex able to produce a nucleic acid or express a protein. If more than one fusion protein is present, such portions may independently be the same or different. In an assay, the fusion proteins may be introduced as proteins, and/or introduced as nucleic acids which can be expressed (e.g., in vitro) to produce such proteins, using in vitro or in situ techniques known to those of ordinary skill in the art.

A fusion protein may include regions such as activation domains or domains of binding such as those described herein. In some cases, a region may be able to specifically bind a nucleic acid, or a specific region of the nucleic acid, such as a UAS (upstream activating sequence), an operon sequence, a promoter sequence, etc. As another example, a region may include a region able to recruit a component such as a polymerase. Specific examples include, but are not limited to, Protein A and/or Protein G, TFIIIB 150, YEEI peptide (SEQ ID NO.: 13), FEEI peptide (SEQ ID NO.: 14), GAL4, DAP I, DAP II, SH2, Cro protein, zinc finger DNA binding proteins or the like.

If a polymerase is present, then the polymerase may be any suitable polymerase. For example, the polymerase may be a DNA polymerase (which produces DNA) or an RNA polymerase (which produces RNA). The polymerase may arise from any suitable organism (e.g., human, E. coli, etc.). One or more than one polymerase may be present. In some cases, the polymerase may be obtained commercially. Examples of DNA polymerases include, but are not limited to, DNA Polymerases beta, lambda, sigma, mu, alpha, delta, epsilon, I, II, III, IV, eta, iota, kappa, Revl, zeta, gamma, theta, etc. Non-limiting examples of RNA polymerases include RNA polymerase I, II, III, IV, V, bacterial RNA polymerase, etc.

If the target is present, a complex can be formed that is able to produce or express a nucleic acid, e.g., using a reporter nucleic acid. The nucleic acid may include DNA and/or RNA. In some cases, the complex may produce DNA or RNA from the reporter nucleic acid (which, in some cases, may then be expressed as a protein), or the complex may itself be able to express the nucleic acid to produce a protein. However, if the target is not present (or if the target is not present in a sufficient amount or quantity), then the complex is not formed (or is formed, but not in a sufficient amount or quantity), and thus, little or no production or expression of the nucleic acid occurs.

In one set of embodiments, the nucleic acid encodes a protein that can be expressed, e.g., directly or indirectly. For example, the protein may be a fluorescent protein, or the protein may be an enzyme. In one set of embodiments, for instance, the protein is an inherently fluorescent protein, such as GFP, or other similar proteins (e.g., blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet), yellow fluorescent protein (YFP, Citrine, Venus, YPet), etc. In another set of embodiments, the protein is an enzyme, such as horseradish peroxidase, whose activity can be readily determined, e.g., using spectrophotometric methods. In yet another set of embodiments, the protein is one that exhibits specific binding that may be assayable using other systems (e.g., streptavidin in a biotin- streptavidin assay).

The nucleic acid may also contain other regions, e.g., to facilitate binding by the complex, and/or production or expression. For example, in one set of embodiments, the nucleic acid may contain one or more promoter regions, operator regions, transcription start sites, upstream activating sequences, TATA elements, or the like. For instance, the nucleic acid may include one or more binding sites for RNA polymerase.

In one set of embodiments, RNA polymerase interacts with the nucleic acid to produce RNA. The RNA can itself be determined, and or the RNA may be expressed as protein, which is then determined, e.g., enzymatically or fluorescently, etc. In some cases, RNA expression may occur in vitro or in situ, e.g., using in situ expression techniques. Many such kits may be readily obtained commercially. In some cases, the same systems may also be used to produce fusion proteins, for example, as discussed above.

In addition, as mentioned, in some cases, such reactions may occur in compartments, such as droplets. In some cases, the compartments are isolated from each other, such that target remains within the compartments and cannot move into adjacent compartments or diffuse away from the compartment. In one embodiment, the compartments are droplets. Systems and methods of manipulating droplets, e.g., for sorting purposes, are discussed in more detail below. In another embodiment, the compartments may be wells of a microwell plate (e.g., a 96-well, a 384-well, a 1536-well, a 3456-well microwell plate, etc.). In yet other embodiments, the compartments may be individual tubes or containers, test tubes, microfuge tubes, glass vials, bottles, petri dishes, or the like. In some cases, the compartments may have relatively small volumes (e.g., less than about 1 microliter, less than about 300 nl, less than about 100 nl, less than about 30 nl, less than about 10 nl, less than about 3 nl, less than about 1 nl, etc.), such that the target may be present at a relatively high concentration within the compartment. In some cases, the compartments may be individually accessible.

In some cases, the compartments may be analyzed, e.g., to determine a target. For example, compartments containing a target (or a suitable concentration of target) may be identified or distinguished from other compartments that do not. Thus, for example, if a target results in the expression of a fluorescent protein, then compartments that are fluorescent (or sufficiently fluorescent) may be identified or distinguished from other compartments. In addition, in some cases, sorting of compartments may occur. Thus, for example, a first group of compartments may be identified for subsequent processing or analysis, while a second group of compartments is not. For instance, if the compartments are droplets, then a first group of droplets may be retained for subsequent processing or analysis, while a second group of droplets is sent to waste (or, in some cases, retained for different processing or analysis, etc.). Systems and methods for manipulating droplets in such fashion are discussed in further detail below.

For example, as mentioned, various aspects of the invention relates to systems and methods for producing droplets of fluid surrounded by a liquid. Any technique may be used to make a droplet, including those described herein. The fluid and the liquid may be essentially immiscible in many cases, i.e., immiscible on a time scale of interest (e.g., the time it takes a fluidic droplet to be transported through a particular system or device). In certain cases, the droplets may each be substantially the same shape or size, as described herein. The fluid may also contain other species, for example, certain molecular species (e.g., as discussed herein), cells, particles, etc.

In one set of embodiments, for example, electric charge may be created on a fluid surrounded by a liquid, which may cause the fluid to separate into individual droplets within the liquid. In some embodiments, the fluid and the liquid may be present in a channel, e.g., a microfluidic channel, or other constricted space that facilitates application of an electric field to the fluid (which may be "AC" or alternating current, "DC" or direct current etc.), for example, by limiting movement of the fluid with respect to the liquid. Thus, the fluid can be present as a series of individual charged and/or electrically inducible droplets within the liquid. In one embodiment, the electric force exerted on the fluidic droplet may be large enough to cause the droplet to move within the liquid. In some cases, the electric force exerted on the fluidic droplet may be used to direct a desired motion of the droplet within the liquid, for example, to or within a channel or a microfluidic channel (e.g., as further described herein), etc.

Electric charge may be created in the fluid within the liquid using any suitable technique, for example, by placing the fluid within an electric field (which may be AC, DC, etc.), and/or causing a reaction to occur that causes the fluid to have an electric charge, for example, a chemical reaction, an ionic reaction, a photocatalyzed reaction, etc. In one embodiment, the fluid is an electrical conductor. As used herein, a "conductor" is a material having a conductivity of at least about the conductivity of 18 megohm (MOhm or ΜΩ) water. The liquid surrounding the fluid may have a conductivity less than that of the fluid. For instance, the liquid may be an insulator, relative to the fluid, or at least a "leaky insulator," i.e., the liquid is able to at least partially electrically insulate the fluid for at least a short period of time. Those of ordinary skill in the art will be able to identify the conductivity of fluids. In one non-limiting embodiment, the fluid may be substantially hydrophilic, and the liquid surrounding the fluid may be substantially hydrophobic.

The electric field, in some embodiments, is generated from an electric field generator, i.e., a device or system able to create an electric field that can be applied to the fluid. The electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc. The electric field generator may be constructed and arranged to create an electric field within a fluid contained within a channel or a microfluidic channel. The electric field generator may be integral to or separate from the fluidic system containing the channel or microfluidic channel, according to some embodiments. As used herein, "integral" means that portions of the components integral to each other are joined in such a way that the components cannot be manually separated from each other without cutting or breaking at least one of the components.

In another set of embodiments, droplets of fluid can be created from a fluid surrounded by a liquid within a channel by altering the channel dimensions in a manner that is able to induce the fluid to form individual droplets. The channel may, for example, be a channel that expands relative to the direction of flow, e.g., such that the fluid does not adhere to the channel walls and forms individual droplets instead, or a channel that narrows relative to the direction of flow, e.g., such that the fluid is forced to coalesce into individual droplets. In other embodiments, internal obstructions may also be used to cause droplet formation to occur. For instance, baffles, ridges, posts, or the like may be used to disrupt liquid flow in a manner that causes the fluid to coalesce into fluidic droplets.

In some cases, the channel dimensions may be altered with respect to time (for example, mechanically or electromechanically, pneumatically, etc.) in such a manner as to cause the formation of individual fluidic droplets to occur. For example, the channel may be mechanically contracted ("squeezed") to cause droplet formation, or a fluid stream may be mechanically disrupted to cause droplet formation, for example, through the use of moving baffles, rotating blades, or the like. Other examples of methods for creating droplets include those disclosed in Int. Pat. Apl. No. PCT/US2003/020542, filed June 30, 2003, entitled "Method and Apparatus for Fluid Dispersion," by Stone, et al, published as WO 2004/002627 on January 8, 2004.

In some instances, the droplets may be created at relatively high rates. For instance, at least about 1 droplet per second may be created in some cases, and in other cases, at least about 10 droplets per second, at least about 20 droplets per second, at least about 30 droplets per second, at least about 100 droplets per second, at least about 200 droplets per second, at least about 300 droplets per second, at least about 500 droplets per second, at least about 750 droplets per second, at least about 1000 droplets per second, at least about 1500 droplets per second, at least about 2000 droplets per second, at least about 3000 droplets per second, at least about 5000 droplets per second, at least about 7500 droplets per second, at least about 10,000 droplets per second, at least about 15,000 droplets per second, at least about 20,000 droplets per second, at least about 30,000 droplets per second, at least about 50,000 droplets per second, at least about 75,000 droplets per second, at least about 100,000 droplets per second, at least about 150,000 droplets per second, at least about 200,000 droplets per second, at least about 300,000 droplets per second, at least about 500,000 droplets per second, at least about 750,000 droplets per second, at least about 1,000,000 droplets per second, at least about 1,500,000 droplets per second, at least about 2,000,000 or more droplets per second, or at least about 3,000,000 or more droplets per second may be created. Other examples of the production of droplets of fluid surrounded by a liquid are described in International Patent Application Serial No. PCT/US2004/010903, filed April 9, 2004 by Link, et al, and International Patent Application Serial No. PCT/US03/20542, filed June 30, 2003 by Stone, et al, published as WO 2004/002627 on January 8, 2004, each incorporated herein by reference.

In some embodiments, a species may be contained within the droplet, e.g., before or after formation. In some cases, more than one species may be present. Thus, for example, a precise quantity of a drug, pharmaceutical, or other agent can be contained within a droplet. Other species that can be contained within a droplet include, for example, biochemical species such as nucleic acids such as siRNA, mRNA, RNAi and DNA, proteins, peptides, or enzymes, or the like. Additional species that can be contained within a droplet include, but are not limited to, nanoparticles, quantum dots, proteins, indicators, dyes, fluorescent species, chemicals, amphiphilic compounds, detergents, drugs, or the like. Further examples of species that can be contained within a droplet include, but are not limited to, growth regulators, vitamins, hormones, or microbicides.

In certain instances, the invention provides for the production of droplets consisting essentially of a substantially uniform number of entities of a species therein (i.e., molecules, cells, particles, etc.). For example, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99%, or more of a plurality or series of droplets may each contain the same number of entities of a particular species. For instance, a substantial number of fluidic droplets produced, e.g., as described above, may each contain 1 entity, 2 entities, 3 entities, 4 entities, 5 entities, 7 entities, 10 entities, 15 entities, 20 entities, 25 entities, 30 entities, 40 entities, 50 entities, 60 entities, 70 entities, 80 entities, 90 entities, 100 entities, etc., where the entities are molecules or macromolecules, cells, particles, etc. In some cases, the droplets may each independently contain a range of entities, for example, less than 20 entities, less than 15 entities, less than 10 entities, less than 7 entities, less than 5 entities, or less than 3 entities in some cases.

As discussed, in some aspects, fluidic droplets may be screened and/or sorted, and in some cases, at relatively high rates. For example, a characteristic of a droplet may be sensed and/or determined in some fashion (e.g., as herein described), then the droplet may be directed towards a particular region of the device, for example, for sorting or screening purposes. For example, the fluidic droplets may be sorted into two or more than two channels, e.g., based on reactions present within the droplets. In some embodiments, a characteristic of a fluidic droplet may be sensed and/or determined in some fashion, for example, as described herein (e.g., fluorescence of the fluidic droplet may be determined), and, in response, an electric field may be applied or removed from the fluidic droplet to direct the fluidic droplet to a particular region (e.g. a channel). Other techniques for sensing and/or for sorting droplets that are known to those of ordinary skill in the art may also be used, in some embodiments of the invention.

In some cases, high sorting speeds may be achievable using certain systems and methods of the invention. For instance, at least about 1 droplet per second may be determined and/or sorted in some cases, and in other cases, at least about 10 droplets per second, at least about 20 droplets per second, at least about 30 droplets per second, at least about 100 droplets per second, at least about 200 droplets per second, at least about 300 droplets per second, at least about 500 droplets per second, at least about 750 droplets per second, at least about 1000 droplets per second, at least about 1500 droplets per second, at least about 2000 droplets per second, at least about 3000 droplets per second, at least about 5000 droplets per second, at least about 7500 droplets per second, at least about 10,000 droplets per second, at least about 15,000 droplets per second, at least about 20,000 droplets per second, at least about 30,000 droplets per second, at least about 50,000 droplets per second, at least about 75,000 droplets per second, at least about 100,000 droplets per second, at least about 150,000 droplets per second, at least about 200,000 droplets per second, at least about 300,000 droplets per second, at least about 500,000 droplets per second, at least about 750,000 droplets per second, at least about 1,000,000 droplets per second, at least about 1,500,000 droplets per second, at least about 2,000,000 or more droplets per second, or at least about 3,000,000 or more droplets per second may be determined and/or sorted in such a fashion.

In one set of embodiments, a fluidic droplet may be directed by creating an electric charge (e.g., as previously described) on the droplet, and steering the droplet using an applied electric field, which may be an AC field, a DC field, etc. As an example, an electric field may be selectively applied and removed (or a different electric field may be applied, e.g., a reversed electric field) as needed to direct the fluidic droplet to a particular region. The electric field may be selectively applied and removed as needed, in some embodiments, without substantially altering the flow of the liquid containing the fluidic droplet. For example, a liquid may flow on a substantially steady-state basis (i.e., the average flowrate of the liquid containing the fluidic droplet deviates by less than 20% or less than 15% of the steady-state flow or the expected value of the flow of liquid with respect to time, and in some cases, the average flowrate may deviate less than 10% or less than 5%) or other predetermined basis through a fluidic system of the invention (e.g., through a channel or a microchannel), and fluidic droplets contained within the liquid may be directed to various regions, e.g., using an electric field, without substantially altering the flow of the liquid through the fluidic system.

In another set of embodiments, a fluidic droplet may be sorted or steered by inducing a dipole in the fluidic droplet (which may be initially charged or uncharged), and sorting or steering the droplet using an applied electric field. The electric field may be an AC field, a DC field, etc.

In other embodiments, however, the fluidic droplets may be screened or sorted within a fluidic system of the invention by altering the flow of the liquid containing the droplets. For instance, in one set of embodiments, a fluidic droplet may be steered or sorted by directing the liquid surrounding the fluidic droplet into a first channel, a second channel, etc.

In another set of embodiments, pressure within a fluidic system, for example, within different channels or within different portions of a channel, can be controlled to direct the flow of fluidic droplets. For example, a droplet can be directed toward a channel junction including multiple options for further direction of flow (e.g., directed toward a branch, or fork, in a channel defining optional downstream flow channels). Pressure within one or more of the optional downstream flow channels can be controlled to direct the droplet selectively into one of the channels, and changes in pressure can be effected on the order of the time required for successive droplets to reach the junction, such that the downstream flow path of each successive droplet can be independently controlled. In one arrangement, the expansion and/or contraction of liquid reservoirs may be used to steer or sort a fluidic droplet into a channel, e.g., by causing directed movement of the liquid containing the fluidic droplet. The liquid reservoirs may be positioned such that, when activated, the movement of liquid caused by the activated reservoirs causes the liquid to flow in a preferred direction, carrying the fluidic droplet in that preferred direction. For instance, the expansion of a liquid reservoir may cause a flow of liquid towards the reservoir, while the contraction of a liquid reservoir may cause a flow of liquid away from the reservoir. In some cases, the expansion and/or contraction of the liquid reservoir may be combined with other flow-controlling devices and methods, e.g., as described herein. Non-limiting examples of devices able to cause the expansion and/or contraction of a liquid reservoir include pistons and piezoelectric components. In some cases, piezoelectric components may be particularly useful due to their relatively rapid response times, e.g., in response to an electrical signal.

In certain aspects of the invention, sensors are provided that can sense and/or determine one or more characteristics of the fluidic droplets, and/or a characteristic of a portion of the fluidic system containing the fluidic droplet (e.g., the liquid surrounding the fluidic droplet) in such a manner as to allow the determination of one or more characteristics of the fluidic droplets. Characteristics determinable with respect to the droplet and usable in the invention can be identified by those of ordinary skill in the art. Non-limiting examples of such characteristics include fluorescence, spectroscopy (e.g., optical, infrared, ultraviolet, etc.), radioactivity, mass, volume, density, temperature, viscosity, pH, concentration of a substance, such as a biological substance (e.g., a protein, a nucleic acid, etc.), or the like.

In some cases, the sensor may be connected to a processor, which in turn, cause an operation to be performed on the fluidic droplet, for example, by sorting the droplet, adding or removing electric charge from the droplet, fusing the droplet with another droplet, etc. One or more sensors and/or processors may be positioned to be in sensing communication with the fluidic droplet. "Sensing communication," as used herein, means that the sensor may be positioned anywhere such that the fluidic droplet within the fluidic system (e.g., within a channel), and/or a portion of the fluidic system containing the fluidic droplet may be sensed and/or determined in some fashion. For example, the sensor may be in sensing communication with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet fluidly, optically or visually, thermally, pneumatically, electronically, or the like. The sensor can be positioned proximate the fluidic system, for example, embedded within or integrally connected to a wall of a channel, or positioned separately from the fluidic system but with physical, electrical, and/or optical communication with the fluidic system so as to be able to sense and/or determine the fluidic droplet and/or a portion of the fluidic system containing the fluidic droplet (e.g., a channel or a microchannel, a liquid containing the fluidic droplet, etc.). For example, a sensor may be free of any physical connection with a channel containing a droplet, but may be positioned so as to detect electromagnetic radiation arising from the droplet or the fluidic system, such as infrared, ultraviolet, or visible light. The electromagnetic radiation may be produced by the droplet, and/or may arise from other portions of the fluidic system (or externally of the fluidic system) and interact with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet in such as a manner as to indicate one or more characteristics of the fluidic droplet, for example, through absorption, reflection, diffraction, refraction, fluorescence, phosphorescence, changes in polarity, phase changes, changes with respect to time, etc. As an example, a laser may be directed towards the fluidic droplet and/or the liquid surrounding the fluidic droplet, and the fluorescence of the fluidic droplet and/or the surrounding liquid may be determined. "Sensing communication," as used herein may also be direct or indirect. As an example, light from the fluidic droplet may be directed to a sensor, or directed first through a fiber optic system, a waveguide, etc., before being directed to a sensor.

Non-limiting examples of sensors useful in the invention include optical or

electromagnetically-based systems. For example, the sensor may be a fluorescence sensor (e.g., stimulated by a laser), a microscopy system (which may include a camera or other recording device), or the like. As another example, the sensor may be an electronic sensor, e.g., a sensor able to determine an electric field or other electrical characteristic. For example, the sensor may detect capacitance, inductance, etc., of a fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet.

As used herein, a "processor" or a "microprocessor" is any component or device able to receive a signal from one or more sensors, store the signal, and/or direct one or more responses (e.g., as described above), for example, by using a mathematical formula or an electronic or computational circuit. The signal may be any suitable signal indicative of the environmental factor determined by the sensor, for example a pneumatic signal, an electronic signal, an optical signal, a mechanical signal, etc.

As a particular non-limiting example, a device of the invention may contain fluidic droplets containing one or more signaling entities, such as a fluorescent signal marker that binds if a certain condition is present, for example, the marker may bind to a first species but not a second species , the marker may bind to an expressed protein, and the droplets may be directed through a fluidic system of the invention based on the presence/absence, and/or magnitude of the fluorescent signal marker. For instance, determination of the fluorescent signal marker may cause the droplets to be directed to one region of the device (e.g., a collection chamber), while the absence of the fluorescent signal marker may cause the droplets to be directed to another region of the device (e.g., a waste chamber). Thus, in this example, a population of droplets may be screened and/or sorted on the basis of one or more determinable or targetable characteristics of the droplets.

As mentioned, certain aspects of the invention are directed to the production of droplets using apparatuses and devices such as those described herein, for example, within microfluidic channels or other microfluidic systems. In some cases, e.g., relatively large droplet production rates may be achieved. For instance, in some cases, greater than about 1,000 droplets/s, greater than or equal to 5,000 droplets/s, greater than about 10,000 droplets/s, greater than about 50,000 droplets/s, greater than about 100,000 droplets/s, greater than about 300,000 droplets/s, greater than about 500,000 droplets/s, or greater than about 1,000,000 droplets/s, etc. may be produced. In some cases, such high droplet production may be used to produce a relatively large amount of protein, or to screen a relatively large number of droplets in a relatively short period of time, or other applications disclosed herein.

In addition, in some cases, a plurality of droplets may be produced that are substantially monodisperse, in some embodiments. In some cases, the plurality of droplets may have a distribution of characteristic dimensions such that no more than about 20%, no more than about 18%, no more than about 16%, no more than about 15%, no more than about 14%, no more than about 13%, no more than about 12%, no more than about 11%, no more than about 10%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or less, of the droplets have a characteristic dimension greater than or less than about 20%, less than about 30%, less than about 50%, less than about 75%, less than about 80%, less than about 90%, less than about 95%, less than about 99%, or more, of the average characteristic dimension of all of the droplets. Those of ordinary skill in the art will be able to determine the average characteristic dimension of a population of droplets, for example, using laser light scattering, microscopic examination, or other known techniques. In one set of embodiments, the plurality of droplets may have a distribution of characteristic dimension such that no more than about 20%, no more than about 10%, or no more than about 5% of the droplets may have a characteristic dimension greater than about 120% or less than about 80%, greater than about 115% or less than about 85%, or greater than about 110% or less than about 90% of the average of the characteristic dimension of the plurality of droplets. The "characteristic dimension" of a droplet, as used herein, is the diameter of a perfect sphere having the same volume as the droplet. In addition, in some instances, the coefficient of variation of the characteristic dimension of the exiting droplets may be less than or equal to about 20%, less than or equal to about 15%, or less than or equal to about 10%.

The average characteristic dimension or diameter of the plurality of droplets, in some embodiments, may be less than about 1 mm, less than about 500 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 25 micrometers, less than about 10 micrometers, or less than about 5 micrometers in some cases. The average characteristic dimension of a droplet (or plurality of droplets) may also be greater than or equal to about 1 micrometer, greater than or equal to about 2 micrometers, greater than or equal to about 3 micrometers, greater than or equal to about 5 micrometers, greater than or equal to about 10 micrometers, greater than or equal to about 15 micrometers, or greater than or equal to about 20 micrometers in certain cases.

In some embodiments, the fluidic droplets may each be substantially the same shape and/or size. The shape and/or size can be determined, for example, by measuring the average diameter or other characteristic dimension of the droplets. The term "determining," as used herein, generally refers to the analysis or measurement of a species, for example, quantitatively or qualitatively, and/or the detection of the presence or absence of the species. "Determining" may also refer to the analysis or measurement of an interaction between two or more species, for example, quantitatively or qualitatively, or by detecting the presence or absence of the interaction.

In some embodiments, a droplet may undergo additional processes. For example, as discussed, a droplet may be sorted and/or detected. For example, a species within a droplet may be determined, and the droplet may be sorted based on that determination. In general, a droplet may undergo any suitable process known to those of ordinary skill in the art. See, e.g., Int. Pat. Apl. No. PCT/US2004/010903, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link, et al, published as WO 2004/091763 on October 28, 2004; Int. Pat. Apl. No. PCT/US2003/020542, filed June 30, 2003, entitled "Method and Apparatus for Fluid

Dispersion," by Stone, et al, published as WO 2004/002627 on January 8, 2004; Int. Pat. Apl. No. PCT/US2006/007772, filed March 3, 2006, entitled "Method and Apparatus for Forming Multiple Emulsions," by Weitz, et al, published as WO 2006/096571 on September 14, 2006; Int. Pat. Apl. No. PCT/US2004/027912, filed August 27, 2004, entitled "Electronic Control of Fluidic Species," by Link, et al, published as WO 2005/021151 on March 10, 2005, each of which is incorporated herein by reference in their entireties. As a non-limiting example, droplets may be created, manipulated, sorted, etc. using electric fields or surface acoustic waves. See, e.g., Int. Pat. Apl. No. PCT/US2004/027912, filed August 27, 2004, entitled "Electronic Control of Fluidic Species," by Link, et al, published as WO 2005/021151 on March 10, 2005;

International Patent Application No. PCT/US2011/048804, filed August 23, 2011 , entitled

"Acoustic Waves in Microfluidics," by Weitz, et al., published as WO 2012/027366 on March 1, 2012; International Patent Application No. PCT/US2013/047829, filed June 26, 2013, entitled "Control of Entities Such as Droplets and Cells Using Acoustic Waves," by Weitz, et al., published as WO 2014/004630 on January 3, 2014; International Patent Application No.

PCT/US2013/066591, filed October 24, 2013, entitled "Systems and Methods for Droplet Production and Manipulation Using Acoustic Waves," by Weitz, et al., published as WO

2014/066624 on May 1, 2014; or U.S. Ser. No. 62/017,301, filed June 26, 2014, entitled "Fluid Injection Using Acoustic Waves," by Weitz, et al.; each incorporated herein by reference in their entireties.

Certain aspects of the invention are generally directed to devices containing channels such as those described above. In some cases, some of the channels may be microfluidic channels, but in certain instances, not all of the channels are microfluidic. There can be any number of channels, including microfluidic channels, within the device, and the channels may be arranged in any suitable configuration. The channels may be all interconnected, or there can be more than one network of channels present. The channels may independently be straight, curved, bent, etc. In some cases, there may be a relatively large number and/or a relatively large length of channels present in the device. For example, in some embodiments, the channels within a device, when added together, can have a total length of at least about 100 micrometers, at least about 300 micrometers, at least about 500 micrometers, at least about 1 mm, at least about 3 mm, at least about 5 mm, at least about 10 mm, at least about 30 mm, at least 50 mm, at least about 100 mm, at least about 300 mm, at least about 500 mm, at least about 1 m, at least about 2 m, or at least about 3 m in some cases. As another example, a device can have at least 1 channel, at least 3 channels, at least 5 channels, at least 10 channels, at least 20 channels, at least 30 channels, at least 40 channels, at least 50 channels, at least 70 channels, at least 100 channels, etc. In some embodiments, at least some of the channels within the device are microfhiidic channels. "Microfhiidic," as used herein, refers to a device, article, or system including at least one fluid channel having a cross-sectional dimension of less than about 1 mm. The "cross- sectional dimension" of the channel is measured perpendicular to the direction of net fluid flow within the channel. Thus, for example, some or all of the fluid channels in a device can have a maximum cross- sectional dimension less than about 2 mm, and in certain cases, less than about 1 mm. In one set of embodiments, all fluid channels in a device are microfhiidic and/or have a largest cross sectional dimension of no more than about 2 mm or about 1 mm. In certain embodiments, the fluid channels may be formed in part by a single component (e.g. an etched substrate or molded unit). Of course, larger channels, tubes, chambers, reservoirs, etc. can be used to store fluids and/or deliver fluids to various elements or systems in other embodiments of the invention, for example, as previously discussed. In one set of embodiments, the maximum cross- sectional dimension of the channels in a device is less than 500 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers, less than about 10 micrometers, less than about 5 micrometers, or less than about 1 micrometer.

A "channel," as used herein, means a feature on or in a device or substrate that at least partially directs flow of a fluid. The channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlets and/or outlets or openings. A channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2: 1, more typically at least about 3: 1, at least about 4: 1, at least about 5: 1, at least about 6: 1, at least about 8: 1, at least about 10: 1, at least about 15: 1, at least about 20: 1, at least about 30: 1, at least about 40: 1, at least about 50: 1, at least about 60: 1, at least about 70: 1, at least about 80: 1, at least about 90: 1, at least about 100: 1 or more. An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid. Non-limiting examples of force actuators that can produce suitable forces include piezo actuators, pressure valves, electrodes to apply AC electric fields, and the like. The fluid within the channel may partially or completely fill the channel. In some cases where an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).

The channel may be of any size, for example, having a largest dimension perpendicular to net fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. In some cases, the dimensions of the channel are chosen such that fluid is able to freely flow through the device or substrate. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel may be used. For example, two or more channels may be used, where they are positioned adjacent or proximate to each other, positioned to intersect with each other, etc.

In certain embodiments, one or more of the channels within the device may have an average cross- sectional dimension of less than about 10 cm. In certain instances, the average cross- sectional dimension of the channel is less than about 5 cm, less than about 3 cm, less than about 1 cm, less than about 5 mm, less than about 3 mm, less than about 1 mm, less than 500 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers. The "average cross-sectional dimension" is measured in a plane perpendicular to net fluid flow within the channel. If the channel is non-circular, the average cross- sectional dimension may be taken as the diameter of a circle having the same area as the cross- sectional area of the channel. Thus, the channel may have any suitable cross-sectional shape, for example, circular, oval, triangular, irregular, square, rectangular, quadrilateral, or the like. In some embodiments, the channels are sized so as to allow laminar flow of one or more fluids contained within the channel to occur.

The channel may also have any suitable cross-sectional aspect ratio. The "cross- sectional aspect ratio" is, for the cross- sectional shape of a channel, the largest possible ratio (large to small) of two measurements made orthogonal to each other on the cross-sectional shape. For example, the channel may have a cross- sectional aspect ratio of less than about 2: 1, less than about 1.5: 1 , or in some cases about 1: 1 (e.g., for a circular or a square cross- sectional shape). In other embodiments, the cross-sectional aspect ratio may be relatively large. For example, the cross- sectional aspect ratio may be at least about 2: 1, at least about 3: 1, at least about 4: 1, at least about 5: 1, at least about 6: 1, at least about 7: 1, at least about 8: 1, at least about 10: 1, at least about 12: 1, at least about 15: 1, or at least about 20: 1.

As mentioned, the channels can be arranged in any suitable configuration within the device. Different channel arrangements may be used, for example, to manipulate fluids, droplets, and/or other species within the channels. For example, channels within the device can be arranged to create droplets (e.g., discrete droplets, single emulsions, double emulsions or other multiple emulsions, etc.), to mix fluids and/or droplets or other species contained therein, to screen or sort fluids and/or droplets or other species contained therein, to split or divide fluids and/or droplets, to cause a reaction to occur (e.g., between two fluids, between a species carried by a first fluid and a second fluid, or between two species carried by two fluids to occur), or the like.

Non-limiting examples of systems for manipulating fluids, droplets, and/or other species are discussed below. Additional examples of suitable manipulation systems can also be seen in U.S. Patent Application Serial No. 11/246,911, filed October 7, 2005, entitled "Formation and Control of Fluidic Species," by Link, et ah, published as U.S. Patent Application Publication No. 2006/0163385 on July 27, 2006; U.S. Patent Application Serial No. 11/024,228, filed December 28, 2004, entitled "Method and Apparatus for Fluid Dispersion," by Stone, et ah, now U.S.

Patent No. 7,708,949, issued May 4, 2010; U.S. Patent Application Serial No. 11/885,306, filed August 29, 2007, entitled "Method and Apparatus for Forming Multiple Emulsions," by Weitz, et al, published as U.S. Patent Application Publication No. 2009/0131543 on May 21, 2009; and U.S. Patent Application Serial No. 11/360,845, filed February 23, 2006, entitled "Electronic Control of Fluidic Species," by Link, et al., published as U.S. Patent Application Publication No. 2007/0003442 on January 4, 2007; each of which is incorporated herein by reference in its entirety.

Fluids may be delivered into channels within a device via one or more fluid sources. Any suitable source of fluid can be used, and in some cases, more than one source of fluid is used. For example, a pump, gravity, capillary action, surface tension, electroosmosis, centrifugal forces, etc. may be used to deliver a fluid from a fluid source into one or more channels in the device. A vacuum (e.g., from a vacuum pump or other suitable vacuum source) can also be used in some embodiments. Non-limiting examples of pumps include syringe pumps, peristaltic pumps, pressurized fluid sources, or the like. The device can have any number of fluid sources associated with it, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., or more fluid sources. The fluid sources need not be used to deliver fluid into the same channel, e.g., a first fluid source can deliver a first fluid to a first channel while a second fluid source can deliver a second fluid to a second channel, etc. In some cases, two or more channels are arranged to intersect at one or more intersections. There may be any number of fluidic channel intersections within the device, for example, 2, 3, 4, 5, 6, etc., or more intersections.

A variety of materials and methods, according to certain aspects of the invention, can be used to form devices or components such as those described herein, e.g., channels such as microfluidic channels, chambers, etc. For example, various devices or components can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, physical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, electrodeposition, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al).

In one set of embodiments, various structures or components of the devices described herein can be formed of a suitable material, such as glass, metal, polymers, etc., for example, an elastomeric polymer such as polydimethylsiloxane ("PDMS"), polytetrafluoroethylene ("PTFE" or Teflon^®), or the like. For instance, according to one embodiment, a channel such as a microfluidic channel may be implemented by fabricating the fluidic system separately using PDMS or other soft lithography techniques (details of soft lithography techniques suitable for this embodiment are discussed in the references entitled "Soft Lithography," by Younan Xia and George M. Whitesides, published in the Annual Review of Material Science, 1998, Vol. 28, pages 153-184, and "Soft Lithography in Biology and Biochemistry," by George M. Whitesides, Emanuele Ostuni, Shuichi Takayama, Xingyu Jiang and Donald E. Ingber, published in the Annual Review of Biomedical Engineering, 2001, Vol. 3, pages 335-373; each of these references is incorporated herein by reference).

Other examples of potentially suitable polymers include, but are not limited to, polyethylene terephthalate (PET), polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyethylene, polypropylene, polyvinylchloride, cyclic olefin copolymer (COC),

polytetrafhioroethylene, a fluorinated polymer, a silicone such as polydimethylsiloxane, polyvinylidene chloride, bis-benzocyclobutene ("BCB"), a polyimide, a fluorinated derivative of a polyimide, or the like. Combinations, copolymers, or blends involving polymers including those described above are also envisioned. The device may also be formed from composite materials, for example, a composite of a polymer and a semiconductor material.

In some embodiments, various structures or components of the device are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer").

Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, waxes, metals, or mixtures or composites thereof heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well- known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc. Silicone polymers are used in certain embodiments, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of various structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 °C to about 75 °C for exposure times of, for example, at least about an hour. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the invention. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures or channels from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials. Thus, structures can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). Oxidation and sealing methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled "Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy et al), incorporated herein by reference.

Another advantage to forming channels or other structures (or interior, fluid-contacting surfaces) from oxidized silicone polymers is that these surfaces can be much more hydrophilic than the surfaces of typical elastomeric polymers (where a hydrophilic interior surface is desired). Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions than can structures comprised of typical, unoxidized elastomeric polymers or other hydrophobic materials.

In some aspects, such devices may be produced using more than one layer or substrate, e.g., more than one layer of PDMS. For instance, devices having channels with multiple heights and/or devices having interfaces positioned such as described herein may be produced using more than one layer or substrate, which may then be assembled or bonded together, e.g., e.g., using plasma bonding, to produce the final device. As a specific example, a device as discussed herein may be molded from masters comprising two or more layers of photoresists, e.g., where two PDMS molds are then bonded together by activating the PDMS surfaces using 0₂ plasma or other suitable techniques. For example, in some cases, the masters from which the PDMS device is cast may contain one or multiple layers of photoresist, e.g., to form a 3D device. In some embodiments, one or more of the layers may have one or more mating protrusions and/or indentations which are aligned to properly align the layers, e.g., in a lock-and-key fashion. For example, a first layer may have a protrusion (having any suitable shape) and a second layer may have a corresponding indentation which can receive the protrusion, thereby causing the two layers to become properly aligned with respect to each other.

In some aspects, one or more walls or portions of a channel may be coated, e.g., with a coating material, including photoactive coating materials. For example, in some embodiments, each of the microfluidic channels at the common junction may have substantially the same hydrophobicity, although in other embodiments, various channels may have different hydrophobicities. For example a first channel (or set of channels) at a common junction may exhibit a first hydrophobicity, while the other channels may exhibit a second hydrophobicity different from the first hydrophobicity, e.g., exhibiting a hydrophobicity that is greater or less than the first hydrophobicity. Non-limiting examples of systems and methods for coating microfluidic channels, for example, with sol-gel coatings, may be seen in International Patent Application No. PCT/US2009/000850, filed February 11, 2009, entitled "Surfaces, Including Microfluidic Channels, With Controlled Wetting Properties," by Abate, et ah, published as WO 2009/120254 on October 1, 2009, and International Patent Application No.

PCT/US2008/009477, filed August 7, 2008, entitled "Metal Oxide Coating on Surfaces," by Weitz, et ah, published as WO 2009/020633 on February 12, 2009, each incorporated herein by reference in its entirety. Other examples of coatings include polymers, metals, or ceramic coatings, e.g., using techniques known to those of ordinary skill in the art.

As mentioned, in some cases, some or all of the channels may be coated, or otherwise treated such that some or all of the channels, including the inlet and daughter channels, each have substantially the same hydrophilicity. The coating materials can be used in certain instances to control and/or alter the hydrophobicity of the wall of a channel. In some embodiments, a sol-gel is provided that can be formed as a coating on a substrate such as the wall of a channel such as a microfluidic channel. One or more portions of the sol-gel can be reacted to alter its

hydrophobicity, in some cases. For example, a portion of the sol-gel may be exposed to light, such as ultraviolet light, which can be used to induce a chemical reaction in the sol-gel that alters its hydrophobicity. The sol-gel may include a photoinitiator which, upon exposure to light, produces radicals. Optionally, the photoinitiator is conjugated to a silane or other material within the sol-gel. The radicals so produced may be used to cause a condensation or polymerization reaction to occur on the surface of the sol-gel, thus altering the hydrophobicity of the surface. In some cases, various portions may be reacted or left unreacted, e.g., by controlling exposure to light (for instance, using a mask).

A variety of definitions are now provided which will aid in understanding various aspects of the invention. Following, and interspersed with these definitions, is further disclosure that will more fully describe the invention.

A "droplet," as used herein, is an isolated portion of a first fluid that is completely surrounded by a second fluid. In some cases, the first fluid and the second fluid are substantially immiscible. It is to be noted that a droplet is not necessarily spherical, but may assume other shapes as well, for example, depending on the external environment. The diameter of a droplet, in a non-spherical droplet, is the diameter of a perfect mathematical sphere having the same volume as the non-spherical droplet. The droplets may be created using any suitable technique, as previously discussed. As used herein, a "fluid" is given its ordinary meaning, i.e., a liquid or a gas. A fluid cannot maintain a defined shape and will flow during an observable time frame to fill the container in which it is put. Thus, the fluid may have any suitable viscosity that permits flow. If two or more fluids are present, each fluid may be independently selected among essentially any fluids (liquids, gases, and the like) by those of ordinary skill in the art.

Certain embodiments of the present invention provide a plurality of droplets. In some embodiments, the plurality of droplets is formed from a first fluid, and may be substantially surrounded by a second fluid. As used herein, a droplet is "surrounded" by a fluid if a closed loop can be drawn around the droplet through only the fluid. A droplet is "completely surrounded" if closed loops going through only the fluid can be drawn around the droplet regardless of direction. A droplet is "substantially surrounded" if the loops going through only the fluid can be drawn around the droplet depending on the direction (e.g., in some cases, a loop around the droplet will comprise mostly of the fluid by may also comprise a second fluid, or a second droplet, etc.).

In most, but not all embodiments, the droplets and the fluid containing the droplets are substantially immiscible. In some cases, however, they may be miscible. In some cases, a hydrophilic liquid may be suspended in a hydrophobic liquid, a hydrophobic liquid may be suspended in a hydrophilic liquid, a gas bubble may be suspended in a liquid, etc. Typically, a hydrophobic liquid and a hydrophilic liquid are substantially immiscible with respect to each other, where the hydrophilic liquid has a greater affinity to water than does the hydrophobic liquid. Examples of hydrophilic liquids include, but are not limited to, water and other aqueous solutions comprising water, such as cell or biological media, ethanol, salt solutions, etc.

Examples of hydrophobic liquids include, but are not limited to, oils such as hydrocarbons, silicon oils, fluorocarbon oils, organic solvents etc. In some cases, two fluids can be selected to be substantially immiscible within the time frame of formation of a stream of fluids. Those of ordinary skill in the art can select suitable substantially miscible or substantially immiscible fluids, using contact angle measurements or the like, to carry out the techniques of the invention.

The following documents are incorporated herein by reference in their entireties: U.S. Prov. Pat. Apl. Ser. No. 62/008,341, filed June 5, 2014, entitled "Protein Analysis Assay System," by Weitz, et al. ; International Patent Application No. PCT/US04/10903, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link, et al., published as WO 2004/091763 on October 28, 2004; International Patent Application No. PCT/US03/20542, filed June 30, 2003, entitled "Method and Apparatus for Fluid Dispersion," by Stone, et al., published as WO 2004/002627 on January 8, 2004; International Patent Application No. PCT/US04/27912, filed August 27, 2004, entitled "Electronic Control of Fluidic Species," by Link, et al., published as WO 2005/021151 on March 10, 2005; and U.S. Pat. No. 8,337,778. Also incorporated herein by reference in their entireties are International Patent Application No. PCT/US2008/008563, filed July 11, 2008, entitled "Droplet-Based Selection," by Weitz, et al, published as WO 2009/011808 on January 22, 2009; and International Patent Application No.

PCT/US2009/004037, filed July 10, 2009, entitled "Systems and Methods of Droplet-Based Selection," by Weitz, et al, published as WO 2010/005593 on July 10, 2009. Also incorporated herein by reference in its entirety is U.S. Provisional Patent Application Serial No. 62/054,263, filed September 23, 2014, entitled "Two-Hybrid Systems and Methods in Droplets and Other Compartments," by Weitz, et al.

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1

In vitro directed evolution of proteins generally involves screening/selection of a protein- encoding DNA library, and often requires a stable linkage between the DNA sequence and the protein function. Drop-based microfluidics can provide such a stable genotype-phenotype linkage by encapsulating single DNA templates with a cell-free protein synthesis system and assaying in vitro-expressed proteins in the same drops. This example shows that microfluidic drops encapsulating protein-encoding DNA templates with a cell-free protein synthesis system can provide a stable genotype-phenotype linkage for in vitro directed protein evolution and avoid manipulation of cells in drops for protein expression. However, use of a drop-based

microfluidics with a cell-free protein synthesis system for screening a random DNA library has not been demonstrated.

In particular, this example shows a cell-free drop-based in vitro two-hybrid (dIVT2H) method for high-throughput screening of a DNA library for high affinity protein binders. Single DNA templates from a random DNA library are co-encapsulated with the IVT2H system based on Poisson distribution in drops. The in-drop IVT2H system expresses both potential protein binders and the binding target protein, and produces fluorescent signals correlated to the high affinity of the protein binders. Fluorescence-activated sorting is then used to isolate the drops containing potential high-affinity binders. This method is applied to a random DNA library derived from an inhibitor peptide that binds the MDM2 protein.

It was shown that the DNA sequence that encodes the known high-affinity binder of MDM2 from isolated fluorescent drops could be retrieved and verified. This approach involved streamlined steps (e.g., encapsulation, incubation, sorting) and did not require in-drop amplification, drop-fusion, micro-injection or transformation and encapsulation of cells.

Therefore, dIVT2H may simplify and accelerate drop-based microfluidics workflow for high throughput screening. Compared to other in vitro selection methods such as ribosome- and mRNA-display, dIVT2H avoids multi-step affinity panning and purification and immobilization of the binding target protein, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

This example shows an in vitro two-hybrid system (IVT2H) that is a mix-and-read assay for detection of protein-protein interactions. By simply incubating with IVT2H, DNA templates encoding a high-affinity binder can generate a significant fluorescent signal. In this example, drop-based microfluidics are used to encapsulate a random DNA library with IVT2H in picoliter drops. As a model system, a random DNA library was constructed based on the sequence of a peptide inhibitor (PMI) known to bind MDM2 with a high affinity and inhibit the interaction between p53 and MDM2. The PMI DNA library was encapsulated with IVT2H expressing MDM2 in drops. After incubation and fluorescence-activated sorting, the high-affinity binder sequence was retrieved from 8000 random PMI variants. This approach involved encapsulation, incubation, sorting of droplets. It was demonstrated that drop-based IVT2H (dIVT2H) may simplify and accelerate drop-based microfluidics workflow for high throughput screening, and can be used for in vitro directed evolution of proteins.

Detection and fluorescence-activated sorting of the high-affinity interaction of PMI-

Mdm2 using drop-based IVT2H (dIVT2H). PMI interacts with the full-length MDM2 in bulk solutions. The protein binder PMI is fused to the DNA binding domain (DB) (SEQ ID NOs.: 20- 21 and Fig. 8), and the binding target protein MDM2 is fused to the activation domain (AD) (SEQ ID NOs.: 22-23 and Fig. 9). The binding of PMI to MDM2 recruits AD to the promoter- bound RNA polymerase, thereby activating the expression of a GFP reporter (Fig. 1A and SEQ ID NO: 9). In bulk IVT2H reactions, PMI resulted in a significantly higher fluorescence (GFP expression) than the wild-type p53 peptide (p53p), consistent with the higher affinity of PMI compared to that of p53p. In these experiments, the optimal concentration of the PMI or p53p DNA template was 60 pM. However, screening a random DNA library with dIVT2H requires the distribution of single-DNA templates in drops, with the mean DNA concentration (λ, lambda) of 0.1 DNA per drops based on the Poisson distribution. This DNA template concentration corresponds to 60 fM in bulk solution, well below the detection range of the bulk IVT2H.

To determine if dIVT2H can detect the high- affinity interaction of PMI-MDM2 and distinguish this high affinity interaction from the low-affinity interaction of p53p-MDM2 at the single DNA template level, this example uses a drop-based microfluidics device to encapsulate an IVT2H expressing MDM2 with the PMI or p53p DNA template at λ = 0.1 DNA per drop. The drop-based microfluidics setup and workflow are shown in Figs. IB and C (see below for details).

After incubation, a significant number of drops were observed from the PMI DNA template exhibiting higher fluorescence than the background fluorescence, whereas the drops from the p53p DNA template exhibited only the background fluorescence (Fig. 2A). The histogram of fluorescence from the p53p DNA template (Fig. 2A) showed that all drops exhibit background fluorescence and appear to follow a Gaussian distribution, suggesting that the drops with the p53p DNA template have the same fluorescence as those without the DNA template. In contrast, the histogram of fluorescence from the PMI DNA template (Fig. 2A) showed that a number of drops exhibit significantly higher fluorescence than the background fluorescence, suggesting that IVT2H allowed the detection of the PMI-MDM2 interaction in these drops containing 1 or more PMI DNA templates.

Fluorescence-activated sorting was used to isolate five bright drops and performed RT- PCR on individual drops to amplify a 350 bp region of the mRNA encoding PMI. PCR fragments from three drops with the correct size were obtained (Fig. 2B). Subsequent Sanger sequencing confirmed that all three PCR fragments contain the PMI sequence (Fig. 2C).

Drop-based IVT2H (dIVT2H). The drop-based in vitro transcription and translation two- hybrid (dIVT2H) screening method combined a cell-free equivalent of genetic two-hybrid systems and microfluidics platform. A well-controlled cell-free system, IVT2H, was utilized where binding of prey and bait fusion proteins transcriptionally activate o⁵⁴-RNA polymerase holoenzyme (o⁵⁴-RNAP), resulting in elevated expression of the gfp reporter gene. To adapt the system for high-throughput screening of prey libraries, the IVT2H system was

compartmentalized in picoliter water-in-oil drops (Fig. IB). Monoclonal compartments containing at most a single prey plasmid are generated by employing sufficiently dilute concentrations of prey plasmids (vide infra). While compartmentalized in drops, single copies of prey plasmid together with multiple copies of the bait plasmid were constitutively expressed from a T7 promoter resulting in a large number of prey and bait fusion proteins. Previous work employing 4h incubation times has shown that the PURExpress system can generate around 10⁴- 10⁵ active proteins per copy of template, corresponding to nM to micromolar concentrations of bait and prey proteins in picoliter drops. At these concentrations, sufficiently strong binding between prey and bait fusion proteins on the reporter template results in a ternary complex that transcriptionally activates the vGFP gene. Upon in-vitro translation of vGFP mRNA in drops, the accumulated fluorescence of each drop could be assessed and used as a measure of binding affinity between the prey and bait protein inside the compartments.

For proof-of-principle experiments, the binding affinity was screened between the oncoprotein MDM2 (SEQ ID NO: 19) and peptides derived from the MDM2 inhibitor PMI, a dodecameric peptide with a binding affinity two orders higher (K^ = 3.3 nM) than that of the ^(17~ ²⁸⁾p53 peptide of comparable length. Compared to ^{(17 28)}p53 (ETFSDLWKLLPE, SEQ ID NO: 18, K_d = 300 nM), the sequence of the PMI peptide (TSFAEYWNLLSP, SEQ ID NO: 15) conserves the three most critical residues involved in p53-MDM2 binding (Phe-3, Trp-7, Leu- 10, residues emphasized) while binding affinity towards MDM2 is enhanced by an increased alpha-helicity and tightening of the intramolecular hydrogen-bonding interactions. In the <iIVT2H proof-of-principle experiments, the bait fusion protein (MDM2-AD) included MDM2 fused to the N-terminal activation domain of PspF while the prey library contains three-residue randomized variants of PMI (TSxAEYxNLxSP, SEQ ID NO: 16, DB-PMI_m) fused to the DNA binding domain of lambda repressor protein Cro. Mutations in the hydrophobic triad Phe-3, Trp- 7, Leu- 10 are expected to result in a dramatic decrease in binding affinity, hence, the successful recovery of PMI from this library would establish <iIVT2H as a novel screening method for high- affinity protein binders.

Monodisperse 8 pi w/o drops containing IVTT mixture and templates encoding for prey, bait and reporter genes were produced using a flow-focussing microfluidic device using low temperatures (0-5 °C) to prevent transcription of templates in solution prior to encapsulation (Fig. IB). The encapsulation of templates in drops is described by Poisson statistics. The concentration of prey template in solution was adjusted to give an average occupancy of λ = 0.1, resulting in a drop population in which less than 1% contains two or more prey templates. After an eight hour off-chip incubation period at 37 °C, drops are reinjected into a microfluidic sorter and are sorted based on their fluorescence emission (Fig. 1C).

The capability of this platform to screen binders for the case of a library of Mdm2 binders was also demonstrated. The library contained 8000 variants of PMI, where three of the eleven amino acids are randomized, each to one of the 20 natural amino acids (20 = 8000). SEQ ID NO. 16 (TSxAEYxNLxSP) shows PMI peptide and variations; each x can independently be one of the 20 natural amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalaine, proline, serine, threonine, tryptophan, tyrosine, and valine), for a total of 8000 sequences (20 ).

Next-Generation Sequencing of the library shows that within 10% fluctuations, each variant was equally represented (Fig. 3D). The histogram of fluorescence intensities of drops encapsulating the DNA library showed a major peak corresponding to drops without a prey template followed by a tail corresponding to drops containing potential strong binders to Mdm2, as shown in Fig. 3A. Additionally, outliers were observed similar to those for PMI.

13 of the brightest drops from a collection of 1,048,576 drops were collected for identifying the strongest binders. The DNA/RNA of these drops was amplified by RT-PCR (Fig. 3B), which shows a band with the expected size. To enable high throughput identification, Next Generation Sequencing of the collection of these bright drops was used. For each of the three positions, the relative frequency of each amino acid was determined. The most common amino acids in positions 1, 2, and 3 are F, W, and L, which have a relative frequency of 27%, 25%, and 29% respectively; this sequence corresponds to PMI, as shown in Fig. 3C. Next, the ten most frequent variants were identified, which are shown in Fig. 3D. The most common variant was FWL (PMI, i.e., TSFAEYWNLLSP, SEQ ID NO: 15)

EXAMPLE 2

This example illustrates various materials and methods used in the previous examples. Construction of screening library. The peptide PMI, which binds to MDM2 with a dissociation constant K_D(PMI) = 490 pM, was chosen as a basis for our screening library of MDM2 binders. This peptide had twelve residues, TSFAEYWNLLSP (SEQ ID NO: 15), where the three residues that are emphasized are binding- anchors; replacement of F or W abolishes binding and replacement of L significantly reduces binding. Accordingly, a library with three-residue randomized variants of PMI was craeted, TSxAEYxNLxSP (SEQ ID NO: 16), that contained 8000 (20 ) different peptides, by sysetmatically and independently varying each positoin with one of the twenty amino acids, thereby creating 8000 different sequences in all.

The encoding DNA library was genetically engineered by replacing the three native codons corresponding to the three variant residues with randomized nucleotide distributions NNK (N: A,G,C,T; K: G,T). The resulting 32 oligonucleotides (Integrated DNA

Technologies, Coralville, IA) encoded all 20 amino acids and exclude stop codons apart from the amber stop-codon UAG. The oligonucleotides were flanked with constant regions creating overlaps with the 5'-situated region of transcriptional and translational control and a 3'-positioned linker sequence (GGGS, SEQ ID NO: 24). To create a gene library appropriate for the IVT2H system from the oligonucleotides, a library was prepared that contained the 8000 peptide sequences, fused to a DNA binding domain for the GFP reporter gene.

Microfmidic device fabrication. Polydimethylsiloxane (PDMS) microfmidic devices were fabricated using standard soft lithographic methods. Briefly, SU8 photoresist

(MicroChem, Newton, MA) is spin-coated onto a silicon wafer (University Wafer, Boston, MA), patterned by UV exposure (OAI, San Jose, CA) through a photolithography mask, and developed. Then Sylgard 184 silicone elastomer mixture (Dow Corning, Midland, MI) at a weight ratio of 10 Base: 1 Curing agent was poured onto the SU8 mold and degassed under vacuum. After curing for two hours at 65 °C, the PDMS was peeled from the mold and input/output ports are punched into the PDMS with a 0.75 mm diameter Harris Uni-Core biopsy punch (Ted-Pella, Redding, CA). The PDMS and a glass slide were plasma treated and then bonded to each other. Finally, the microfmidic channel walls were rendered hydrophobic by treating them with Aquapel (PPG, Pittsburgh, PA). In the PDMS device for the sorting experiments, the electrodes were designed as channels. These channels were filled with Indalloy 19 (51In, 32.5 Bi, 16.5 Sn; 0.020 inch diameter, 1 inch = 2.54 cm), a low melting point metal alloy (Indium, Clinton, NY), by pushing the wire into the punched holes on a 80 °C hot plate. Electrical connections were made using eight-pin terminal blocks (Phoenix Contact, Middletown, PA). Drop formation and incubation. Reagents A and B of the IVT2H system were mixed, and the diluted DNA screening library was added to obtain at most one DNA template per drop. The solution was kept on ice to minimize transcription and translation before encapsulation into drops which served as reaction vessels. A microfmidic chip containing a flow-focusing junction with a cross section of 15x15 micrometers was used to encapsulate this solution into 7.2 pi monodisperse drops with diameter of 24 micrometers in HFE-7500 fluorinated oil (3M, Saint Paul, MN, U.S. A), containing 1% (w/w) Krytox-PEG diblock copolymer surfactant (32-34). Rather than driving the flow using syringe pumps, a house vacuum at -0.4 psi (1 psi = 6894.757 Pa) was applied at the outlet to suck the reagents that are placed directly into the inlets through the microfmidic channels. The benefits of using a vacuum include 1) no initial transients in drop size, 2) no dead volume of reagents remaining inside the device, and 3) single parameter control versus coordinating two or more syringe pumps. Typical production rates were about four thousand drops per second. The water-in- oil emulsion generated in the microfmidic devices was collected in a PCR tube, as shown in Fig. 1C, and then covered with mineral oil for a 6 h incubation at 37 °C.

Drop detection and sorting. To detect and isolate bright drops containing high- affinity MDM2 binders, a microfmidic drop sorter was used. The incubated drops were reinjected into the sorter at a flow rate of 20 microliters/h and evenly spaced by HFE-7500 oil with surfactant flowing at a rate of 180 microliters/h. Their fluorescence was measured as they pass through the detection region onto which a laser was aligned, and their fluorescence was focused onto a photo multiplier tube (Hammamatsu, Bridgewater, NJ). A custom computer Lab View program running on a real-time field-programmable gate array card (National Instruments, Austin, TX) digitized the photomultiplier tube signal. All drops were gated based on detector pulse width to exclude outliers, such as merged or split drops. Since the strongest MDM2 binders were screened, the sorting threshold was set sufficiently high to collect a small number of drops. To prevent evaporation and facilitate liquid handling for downstream processing of the sorted drops, the collection tips were preloaded with 30 microliters of drops containing ddH₂0.

The ability to sort binders by examining the DNA contents of the bright drops was demonstrated using Sanger Sequencing and Next-Generation Sequencing. Since Sanger sequencing required monoclonal DNA, each drop was isolated into a single well. Accordingly, the 30 microliters of drops containing 5 sorted drops were sorted into 30 wells to prevent multicopies. Because Next-Generation Sequencing can characterize single DNA molecules in a DNA mixture, the collection containing 13 sorted drops was processed in a single tube.

RT-PCR amplification and amplicon purification. The drops' contents were separted from the oil by adding 20% of lH, lH,2H,2H-perfluorooctanol (PFO) (Alfa Aesar, Ward Hill, MA) to break the emulsion, followed by vortexing and centrifugation. To prepare the samples for Sanger Sequencing, 5 microliters of ddH20 was added to each well in order to facilitate transfer of the aqueous phase into 25 microliters of the single-step RT-PCR cocktail. This cocktail contains 1 microliter of Qiagen OneStep RT-PCR Enzyme Mix

(Qiagen), lx Qiagen OneStep RT-PCR buffer, 400 micromolar dNTPs, and 0.25 micromolar forward and reverse primers (see, e.g., SEQ ID NOs.: 1-8). For Next-Generation Sequencing, the aqueous phase was directly transferred from the tube into the RT-PCR cocktail.

Thermocycling conditions were 50 °C for 30 min, 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s, followed by 72 °C for 5 min. The PCR products were purified using GenElute™ Gel Extraction Kit (Sigma, St Louis, MO).

Next-Generation Sequencing. The resulting PCR products from the collection tube containing 13 sorted drops were analyzed by Next-Generation Sequencing. Illumina- specific adaptor sequences are attached to the 5'- and 3 '-ends of the PCR products in two consecutive steps of PCR. In the first step, two oligonucleotides constituting each one half of the 5' and 3'- adaptors are attached. Both oligonucleotides contained an overlap of 20 basepairs to the PMI flanking region. After 10 rounds of amplification, two further oligonucleotides which form the second halves of both adaptors were added by 10 PCR cycles. The DNA concentration was measured and adjusted with a High Sensitivity DNA Analysis Kit (Agilent, Santa Clara, CA). Sequencing conditions were set to a read length of 56 basepairs, 20 basepair overlap and 36 basepair PMI. Sequencing was run on the Illumina Genome Analyzer II (GAII) platform at the sequencing-core of New England Biolabs (NEB).

Analysis of sequencing. The Illumina adapter sequence was removed and low quality bases (Q<20) from the 3' end of the raw reads by Cutadapt. Then each read was scanned for constant regions and the random mutated codons were extracted based on sequence syntax by custom perl script. The extracted DNA codons were translated to amino acid sequence using custom perl script. Next the peptide diversity at each mutagenesis position as well as the genotype (DNA sequence) diversity corresponding to each phenotype (peptide) was analyzed in R (R Development Core Team, 2011).

The WebLogo program was applied to both aligned DNA codons and amino acid sequences to check if any particular DNA sequence(s) or amino acid sequence(s) is enriched at certain position.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or

configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

When the word "about" is used herein in reference to a number, it should be understood that still another embodiment of the invention includes that number not modified by the presence of the word "about."

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"

"composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

claimed is:

A method of protein production, comprising:

producing a first fusion protein, wherein the first fusion protein comprises a first portion and a second portion comprising a binding domain;

producing a second fusion protein, wherein the second fusion protein comprises a first portion able to bind to the first portion of the first fusion protein and a second portion comprising an activation domain;

binding the first portion of the first fusion protein and the first portion of the second fusion protein within a compartment having a volume of less than about 1 microliter;

binding an RNA polymerase to the activation domain;

binding the binding domain to a nucleic acid, wherein the RNA polymerase is able to express at least a portion of the nucleic acid to produce a protein; and

expressing the protein.

The method of claim 1, wherein the first portion of the first fusion protein comprises MDM2 and the first portion of the second fusion protein comprises PMI.

The method of any one of claims 1 or 2, wherein the first portion of the second fusion protein comprises MDM2 and the first portion of the first fusion protein comprises PMI.

The method of any one of claims 1-3, wherein the first portion of the first fusion protein and the first portion of the second fusion protein specifically bind to each other.

The method of any one of claims 1-4, wherein the first portion of the first fusion protein and the first portion of the second fusion protein nonspecifically bind to each other.

The method of any one of claims 1-5, wherein the first portion of the first fusion protein and the first portion of the second fusion protein bind with a dissociation constant of less than about 100 nM. The method of any one of claims 1-6, wherein the nucleic acid comprises a promoter that the RNA polymerase binds.

The method of any one of claims 1-7, wherein the nucleic acid comprises an upstream activating sequence that the binding domain binds.

The method of any one of claims 1-8, wherein the nucleic acid encodes a fluorescent protein.

The method of claim 9, comprising determining expression of the protein by determining fluorescence of the fluorescent protein.

The method of any one of claims 1-10, wherein the nucleic acid encodes green fluorescent protein (GFP).

The method of any one of claims 1-11, comprising producing the first fusion protein within the compartment.

The method of any one of claims 1-12, comprising producing the second fusion protein within the compartment.

The method of any one of claims 1-13-, wherein the compartment is a microfluidic droplet.

The method of claim 14, wherein the first fusion protein, the second fusion protein, the RNA polymerase, and the nucleic acid are contained within the microfluidic droplet.

The method of any one of claims 14 or 15, wherein determining expression of the protein comprises determining fluorescence of the microfluidic droplet to determine expression of the protein. The method of any one of claims 14-16, further comprising sorting the microfluidic droplet based on the expression of the protein.

The method of any one of claims 14-17, wherein the microfluidic droplet is one of a plurality of droplets.

The method of claim 18, wherein the droplets have a distribution in diameters such that no more than 5% of the droplets have a diameter greater than about 110% and/or less than about 90% of the overall average cross- sectional dimension of the droplets.

The method of any one of claims 18 or 19, wherein the droplets have a characteristic dimension of no more than about 1 micrometer.

The method of any one of claims 1-20, wherein the acts are performed in the order recited.

A method, comprising:

providing a plurality of droplets, at least some of which droplets comprise a nonconstant DNA sequence and a constant DNA sequence, wherein the nonconstant DNA sequences within the plurality of droplets together form a library of DNA sequences having at least 20 distinguishable members and at least about 50% homology, and wherein the constant DNA sequence is substantially identical in the at least some droplets;

in at least some of the droplets, producing a first protein from the nonconstant DNA sequence and a second protein from the constant DNA sequence;

forming a complex comprising the first protein, the second protein, and an RNA polymerase;

binding the complex to a nucleic acid to express a protein encoded by the nucleic acid; and

expressing the protein.

23. The method of claim 22, wherein the homology is at least about 75%.

24. The method of any one of claims 22 or 23, wherein the homology is at least about 90%.

25. The method of any one of claims 22-24, wherein the nonconstant DNA sequence encodes PMI.

26. The method of any one of claims 22-25, wherein the constant DNA sequence encodes MDM2.

27. The method of any one of claims 22-26, wherein at least a portion of the first protein is able to bind to at least a portion of the second protein. 28. The method of claim 27, wherein the first protein and the second protein are able to

specifically bind to each other.

29. The method of claim 27, wherein the first protein and the second protein are able to

nonspecifically bind to each other.

30. The method of any one of claims 27-29, wherein the first protein and the second protein bind with a dissociation constant of less than about 100 nM.

31. The method of any one of claims 22-30, wherein the nucleic acid comprises a promoter that the RNA polymerase binds.

32. The method of any one of claims 22-31, wherein the nucleic acid comprises an upstream activating sequence that the binding domain binds. 33. The method of any one of claims 22-32, wherein the nucleic acid encodes a fluorescent protein.

34. The method of claim 33, comprising determining expression of the protein by determining fluorescence of the fluorescent protein. 35. The method of any one of claims 22-34, wherein the nucleic acid encodes green

fluorescent protein (GFP).

36. The method of any one of claims 22-35, wherein the library of DNA sequences having at least 100 distinguishable members.

37. The method of any one of claims 22-36, wherein the library of DNA sequences having at least 1,000 distinguishable members.

38. The method of any one of claims 22-37, wherein determining expression of the protein comprises determining fluorescence of the droplet to determine expression of the protein.

39. The method of any one of claims 22-38, further comprising sorting the droplet based on the expression of the protein. 40. The method of any one of claims 22-39, wherein the droplet is one of a plurality of

droplets.

41. The method of any one of claims 22-40, wherein the droplets have a distribution in

diameters such that no more than 5% of the droplets have a diameter greater than about 110% and/or less than about 90% of the overall average cross- sectional dimension of the droplets.

42. The method of any one of claims 22-41, wherein the droplets have a characteristic

dimension of no more than about 1 micrometer.

43. The method of any one of claims 22-42, wherein the acts are performed in the order recited.

A method, comprising:

producing a first fusion protein;

producing a second fusion protein;

forming a complex comprising the first fusion protein, the second fusion protein, and an RNA polymerase within a compartment having a volume of less than about 1 microliter;

expressing the protein.

45. The method of claim 44, wherein the first fusion protein comprises MDM2 and the second fusion protein comprises PMI.

46. The method of any one of claims 44-45, wherein at least a portion of the first protein is able to bind to at least a portion of the second protein. 47. The method of any one of claims 44-46, wherein the first fusion protein and the second fusion protein are able to specifically bind to each other.

48. The method of any one of claims 44-46, wherein the first fusion protein and the second fusion protein are able to nonspecifically bind to each other.

49. The method of any one of claims 44-48, wherein the first protein and the second protein bind with a dissociation constant of less than about 100 nM.

50. The method of any one of claims 44-49, wherein the nucleic acid comprises a promoter that the RNA polymerase binds.

51. The method of any one of claims 44-50, wherein the nucleic acid comprises an upstream activating sequence that the binding domain binds.

52. The method of any one of claims 44-51, wherein the nucleic acid encodes a fluorescent protein.

53. The method of claim 52, comprising determining expression of the protein by

determining fluorescence of the fluorescent protein. 54. The method of any one of claims 44-53, wherein the nucleic acid encodes green

fluorescent protein (GFP).

55. The method of any one of claims 44-54, wherein the compartment is a droplet. 56. The method of claim 55, wherein the first fusion protein, the second fusion protein, the RNA polymerase, and the nucleic acid are contained within the droplet.

57. The method of any one of claims 55 or 56, wherein determining expression of the protein comprises determining fluorescence of the droplet to determine expression of the protein.

58. The method of claim 57, further comprising sorting the droplet based on the expression of the protein.

59. The method of any one of claims 55-58, wherein the droplet is one of a plurality of

droplets.

60. The method of any one of claims 55-59, wherein the droplets have a distribution in

diameters such that no more than 5% of the droplets have a diameter greater than about

110% and/or less than about 90% of the overall average cross- sectional dimension of the droplets.

61. The method of any one of claims 55-60, wherein the droplets have a characteristic dimension of no more than about 1 micrometer.

The method of any one of claims 55-61, wherein the acts are performed in the order recited.

A method, comprising:

producing a first fusion protein, wherein the first fusion protein comprises a first portion comprising MDM2 and a second portion comprising a binding domain;

producing a second fusion protein, wherein the second fusion protein comprises a first portion comprising a peptide inhibitor of MDM2 and a second portion comprising an activation domain;

binding an RNA polymerase to the activation domain; and

binding the binding domain to a nucleic acid, wherein the RNA polymerase is able to express at least a portion of the nucleic acid to produce a protein.

The method of claim 63, wherein the first protein and the second protein bind with a dissociation constant of less than about 100 nM.

The method of any one of claims 63 or 64, wherein the nucleic acid comprises a promoter that the RNA polymerase binds.

The method of any one of claims 63-65, wherein the nucleic acid comprises an upstream activating sequence that the binding domain binds.

67. The method of any one of claims 63-66, wherein the nucleic acid encodes a fluorescent protein.

68. The method of claim 67, comprising determining expression of the protein by determining fluorescence of the fluorescent protein.

69. The method of any one of claims 63-68, wherein the nucleic acid encodes green

fluorescent protein (GFP).

70. The method of any one of claims 63-69, wherein the compartment is a droplet.

71. The method of claim 70, wherein the first fusion protein, the second fusion protein, the RNA polymerase, and the nucleic acid are contained within the droplet.

72. The method of any one of claims 70 or 71, wherein determining expression of the protein comprises determining fluorescence of the droplet to determine expression of the protein. 73. The method of claim 72, further comprising sorting the droplet based on the expression of the protein.

74. The method of any one of claims 70-73, wherein the droplet is one of a plurality of

droplets.

75. The method of any one of claims 70-74, wherein the droplets have a distribution in

76. The method of any one of claims 70-76, wherein the droplets have a characteristic

dimension of no more than about 1 micrometer.

77. The method of any one of claims 70-76, wherein the acts are performed in the order recited.

78. A method of protein production, comprising:

providing a first protein;

providing a second protein;

binding at least a portion of the first protein to at least a portion of the second protein to produce a complex within a compartment having a volume of less than about 1 microliter, the compartment being free of cells;

producing a nucleic acid using the complex; and

expressing the nucleic acid as a third protein.

The method of claim 78, wherein the first protein comprises MDM2 and the second protein comprises PMI.

The method of any one of claims 78 or 79, wherein the first protein and the second protein specifically bind to each other.

The method of any one of claims 78-80, wherein the first protein and the second protein nonspecifically bind to each other.

The method of any one of claims 78-81, wherein the first protein and the second protein bind with a dissociation constant of less than about 100 nM.

83. The method of any one of claims 78-82, wherein the nucleic acid comprises a promoter that the RNA polymerase binds. 84. The method of any one of claims 78-83, wherein the nucleic acid comprises an upstream activating sequence that the binding domain binds.

85. The method of any one of claims 78-84, wherein the nucleic acid encodes a fluorescent protein.

86. The method of claim 85, comprising determining expression of the third protein by determining fluorescence of the fluorescent protein.

87. The method of any one of claims 78-86, wherein the nucleic acid encodes green

fluorescent protein (GFP).

88. The method of any one of claims 78-87, comprising producing the first protein within the compartment. 89. The method of any one of claims 78-88, comprising producing the second protein within the compartment.

90. The method of any one of claims 89, wherein the compartment is a microfluidic droplet. 91. The method of claim 90, wherein the first protein, the second protein, the RNA

polymerase, and the nucleic acid are contained within the microfluidic droplet.

92. The method of any one of claims 90 or 91, wherein determining expression of the protein comprises determining fluorescence of the microfluidic droplet to determine expression of the protein.

93. The method of any one of claims 90-92, further comprising sorting the microfluidic droplet based on the expression of the protein. 94. The method of any one of claims 90-93, wherein the microfluidic droplet is one of a plurality of droplets.

95. The method of claim 94, wherein the droplets have a distribution in diameters such that no more than 5% of the droplets have a diameter greater than about 110% and/or less than about 90% of the overall average cross- sectional dimension of the droplets.

96. The method of any one of claims 94 or 95, wherein the droplets have a characteristic dimension of no more than about 1 micrometer.

97. The method of any one of claims 78-96, wherein the acts are performed in the order recited.

98. A method of protein production, comprising:

producing a first protein and a second protein within a microfluidic droplet, the droplet being free of cells;

binding at least a portion of the first protein to at least a portion of the second protein to produce a complex;

producing a nucleic acid using the complex; and

expressing the nucleic acid as a third protein.

99. The method of claim 98, wherein the first protein is able to bind to the second protein.

100. The method of claim 99, wherein the first protein and the second protein are able to specifically bind to each other.

101. The method of claim 99, wherein the first protein and the second protein are able to nonspecifically bind to each other.

102. The method of any one of claims 99-101, wherein the first protein and the second protein bind with a dissociation constant of less than about 100 nM.

103. The method of any one of claims 98-102, wherein the nucleic acid comprises a promoter that the RNA polymerase binds.

104. The method of any one of claims 98-103, wherein the nucleic acid comprises an upstream activating sequence that the binding domain binds.

105. The method of any one of claims 98-104, wherein the nucleic acid encodes a fluorescent protein.

106. The method of claim 105, comprising determining expression of the protein by

determining fluorescence of the fluorescent protein.

107. The method of any one of claims 98-106, wherein the nucleic acid encodes green

fluorescent protein (GFP). 108. The method of any one of claims 98-107, wherein the library of DNA sequences having at least 100 distinguishable members.

109. The method of any one of claims 98-108, wherein the library of DNA sequences having at least 1,000 distinguishable members.

110. The method of any one of claims 98-109, wherein determining expression of the protein comprises determining fluorescence of the droplet to determine expression of the protein.

111. The method of any one of claims 98- 110, further comprising sorting the droplet based on the expression of the protein.

112. The method of any one of claims 98-111, wherein the droplet is one of a plurality of droplets. 113. The method of any one of claims 98-112, wherein the droplets have a distribution in diameters such that no more than 5% of the droplets have a diameter greater than about 110% and/or less than about 90% of the overall average cross- sectional dimension of the droplets. 114. The method of any one of claims 98-113, wherein the droplets have a characteristic

dimension of no more than about 1 micrometer.

115. The method of any one of claims 98-114, wherein the acts are performed in the order recited. 116. A method of protein production, comprising:

producing a first protein and a second protein within a microfluidic droplet;

assembling a complex comprising the first protein and the second protein;

producing a nucleic acid using the complex; and

expressing the nucleic acid as a third protein.

117. The method of claim 116, wherein the first protein is able to bind to the second protein.

118. The method of claim 117, wherein the first protein and the second protein are able to specifically bind to each other.

119. The method of claim 117, wherein the first protein and the second protein are able to nonspecifically bind to each other.

120. The method of any one of claims 99-119, wherein the first protein and the second protein bind with a dissociation constant of less than about 100 nM.

121. The method of any one of claims 116-120, wherein the nucleic acid comprises a promoter that the RNA polymerase binds. 122. The method of any one of claims 116-121, wherein the nucleic acid comprises an

upstream activating sequence that the binding domain binds.

123. The method of any one of claims 116-122, wherein the nucleic acid encodes a fluorescent protein. 124 The method of claim 123, comprising determining expression of the protein by determining fluorescence of the fluorescent protein.

125 The method of any one of claims 116-124, wherein the nucleic acid encodes green

fluorescent protein (GFP).

126. The method of any one of claims 116-125, wherein the library of DNA sequences having at least 100 distinguishable members. 127. The method of any one of claims 116-126, wherein the library of DNA sequences having at least 1,000 distinguishable members.

128. The method of any one of claims 116-127, wherein determining expression of the protein comprises determining fluorescence of the droplet to determine expression of the protein.

129. The method of any one of claims 116-128, further comprising sorting the droplet based on the expression of the protein.

130. The method of any one of claims 116-129, wherein the droplet is one of a plurality of droplets.

131. The method of any one of claims 116-130, wherein the droplets have a distribution in diameters such that no more than 5% of the droplets have a diameter greater than about 110% and/or less than about 90% of the overall average cross- sectional dimension of the droplets.

132 The method of any one of claims 116-131, wherein the droplets have a characteristic dimension of no more than about 1 micrometer. 133. The method of any one of claims 116-132, wherein the acts are performed in the order recited.