Protein expression systems

Abstract

Provided herein are methods of protein synthesis, protein synthesis reagents for the expression of eukaryotic and mammalian proteins and methods for optimizing protein expression yields for mammalian proteins. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by merging droplets on the device in order to screen a selection of expression compositions in parallel and then identify the optimum conditions for producing the stable folded protein in a cell-based system.

Description

[0001] PROTEIN EXPRESSION SYSTEMS

[0002] FIELD OF THE INVENTION

[0003] Provided herein are methods of protein synthesis, protein synthesis reagents for the expression of eukaryotic and mammalian proteins and methods for optimising protein expression yields for mammalian proteins. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by merging droplets on the device in order to screen a selection of expression compositions in parallel and then identify the optimum conditions for producing the stable folded protein in a cell-based system.

[0004] BACKGROUND TO THE INVENTION

[0005] Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The in-vitro protein synthesis environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in- vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261-268).

[0006] However such cell-free systems are not suitable for expression of many mammalian proteins in a stable form, for example transmembrane proteins, and are incapable of performing post translational modifications (PTM’s). Therefore in many instances cell-free expression of desired proteins is not possible and expression in cells is required. Furthermore, the reagents for cell-free protein synthesis are expensive to use when large amount of proteins are required. A scale up reaction using for example 1 mL of reconstituted cell-free expression reagent will likely give at most a few mg of the desired protein. For a significant amount of protein, expression in living cells is required.

[0007] To date, digital microfluidics, electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-based applications, mostly due to denaturing and biofouling, where biological components such as cells, proteins, nucleic acids, cell extracts and other bioproducts adsorb and / or denature to hydrophobic surfaces. Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing live cells and biomacromolecules. Wheeler and colleagues report that the maximum actuation time for droplets on EWoD devices containing biological media is 30 min before biofouling inhibits EWoD-based droplet actuation (Langmuir 2011 , 27, 13, 8586-8594).

[0008] On an EWoD device, cell expression leads to variable outcomes. The level of protein expression in different droplets on the same device is not consistent. The inventors herein have improved the reliability of expression levels of proteins in cell-based systems. Additionally, purified component mixtures are typically not capable of performing downstream maturation of the produced protein into the desired form. For example, purified component mixtures are not capable of effecting post-translational modification of proteins, such as glycosylation.

[0009] Described herein are methods for handling cells on digital microfluidic devices and the use of said cells to produce proteins, particularly mammalian proteins.

[0010] SUMMARY OF THE INVENTION

[0011] Disclosed herein are method of measuring protein expression in cells on a digital microfluidic device. Disclosed is a method of producing proteins comprising the steps of: a. taking a population of cells in droplets on a digital microfluidic device, wherein the cells contain transfected circular nucleic sequences expressing proteins having a desired amino acid sequence and a detection tag; b. allowing the cells to express the proteins; and c. measuring the level of the expressed protein using a detector species which binds to the detection tag.

[0012] The cells may be transfected on the device. Disclosed herein is a method of producing proteins comprising the steps of: a. taking circular nucleic sequences expressing proteins having a desired amino acid sequence and a detection tag; b. taking a population of cells in droplets on a digital microfluidic device; c. transfecting the circular nucleic sequences into the cells; d. allowing the cells to express the proteins; and e. measuring the level of the expressed protein using a detector species which binds to the detection tag. The detection of the protein may occur in the cells. Alternatively the protein may be secreted from the cells and detected outside the cells. Alternatively the cells may undergo lysis in order to release the contents for detection. The cells may be lysed on the digital microfluidic device. The detector species may be present in the cell culture media in order to detect expression, for example of secreted proteins or cell surface proteins. The purification beads may be present in the cell culture media in order to bind to expressed proteins, for example secreted proteins or cell surface proteins, which many be optionally then quantified. The detector species may be added to the droplets after lysis, or may be present in the lysis solution. The detector tag may be a sub-component of a fluorescent protein. The detection may be via assembly of a split fluorescent protein.

[0013] Typical methods of lysis include chemical treatments such as the use of detergents or alkaline solutions, the use of hypotonic (low ionic strength) solutions to cause bursting of the cells, the use of enzymes to degrade the cell walls of the use of physical methods such as shearing or vibration. After secretion or lysis the protein may be purified, for example using an affinity tag. The detection of expression levels may take place before and / or after purification.

[0014] Rather than lysing the cells, the cells may co-express the detector species, thus enabling detection inside the living cells. The expression may use the same or different plasmids. The method may use the same plasmid as the desired amino acid sequence.

[0015] Rather than lysing the cells, the cells may secrete the protein after expression. The protein may be a cell-surface protein. Detection may take place in the presence of the competent cells.

[0016] In order to expose the cells to multiple reagents, the cells may be immobilised. Immobilisations may be via trapping inside a hydrogel matrix, for example an alginate, agarose or polyacrylamide matrix. Alternatively the cells may be immobilised via attachment to a solid support, for example magnetic or paramagnetic beads. Alternatively the cells could be adhered to the top-plate or TFT surface of the digital microfluidic device.

[0017] Droplets containing cells may be separated and concentrated using a filter or molecular sieve. For example a polymer matrix such as a hydrogel can be used to retain cells and separate the bulk of the liquid not having cells, thereby removing spent media and allowing fresh media and nutrients to be exchanged. Droplets may be drawn through an immobilised hydrogel in order to remove the bulk of the liquid volume and separate the cells from the media. Any cell type may be used, for example the cells used for expression may be selected from mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa. The cells may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

[0018] Any type of protein may be produced. Proteins may include eukaryotic, mammalian or human proteins. The protein may be an antibody or nanobody. The method may use a series of droplets having varying reagent compositions, thereby identifying a composition suitable for expression of the protein of interest. The optimum conditions help for example identify the optimum expression parameters for growth of cells in order to obtain the desired amino acid sequence in a correctly folded state.

[0019] The device may be an electrowetting-on-dielectric (EWoD) device. The device may comprise an active-matrix of electrodes.

[0020] The method may identify conditions for protein synthesis, folding and / or isolation which are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence. In such instances the presence of the protein additives may be required for the cellbased expression. Where additional protein additives are beneficial, these may optionally be co-expressed in the cell. The co-expression may use the same plasmid as the desired amino acid sequence. Alternatively the cells used for expression may contain genes for the expression of the additional proteins which aid expression, folding and / or isolation of the desired amino acid sequence. The additional proteins may be for example one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

[0021] Disclosed herein is a method of producing proteins comprising the steps of: a. taking circular nucleic sequences expressing proteins having a desired amino acid sequence and a detection tag which is a sub-component of a fluorescent protein; b. taking a population of cells in droplets on a digital microfluidic device, wherein the cells are immobilised in a hydrogel matrix; c. transfecting the circular nucleic sequences into the cells; d. allowing the cells to express the proteins; e. lysing the cells to release the proteins; f. adding a detector species which is a sub-component of a fluorescent protein and which binds to the detection tag to the droplets containing the lysate; and e. measuring the level of the expressed protein using the assembled fluorescent protein.

[0022] The screening may use a variety of circular nucleic sequence templates or plasmids. The variety of plasmid templates may include the same nucleic acid sequence coding for the desired amino acid sequence flanked by a variety of different for example solubility tags or purification tags. Alternatively the codon sequences may be chosen such that varying codons express the desired amino acid sequence in the chosen cell-type.

[0023] The screening method may identify the optimum conditions for the growth of cells, for example the best temperature or growth media in order to obtain the desired amino acid sequence in a correctly folded state. The growth media may include for example metal ions or co-factors for expression.

[0024] Also disclosed is a kit comprising: i. a circular nucleic acid template coding sequence for a protein of interest and a detection tag; ii. a population of cells suitable for expression proteins upon transfection with the nucleic acid template; and iii. a digital microfluidic device.

[0025] The kit may comprise a variety of nucleic acid template sequences coding for the same amino acid sequence, for example having varying flanking sequences and / or varying coding sequences; and a variety of cell-lines for expressing the nucleic acid template coding sequence for a protein of interest.

[0026] The kit may further comprising a reagent for transfection. The kit may comprise a cationic, lipid-based transfection reagent.

[0027] Where the synthesis is performed on a microfluidic device, the mixture can be optimised by combining reagents on the device. For example concentrations of reagents that give optimal expression can be identified by blending components at different ratios in a large number of droplets and the expression monitored in parallel to identify optimal compositions. For example a variety of cell types and growth conditions can be used to express each of the one or more sequences. The expressed protein may be fused to a peptide tag. The peptide tag may contain sequences for detection and / or purification. The peptide tag may contain a sequence enabling detection of the expressed protein. The detection tag may be one component of a fluorescent protein and the further polypeptide a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmllRFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFPi. 10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi- . The peptide tag may be CFASTn or CFASTw and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.

[0028] The tags can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids.

[0029] FIGURES

[0030] Figure 1 This experiment shows Alginate hydrogel patches in 192 droplet reaction zone format on digital microfluidic devices along with negative control, some of which are shown with arrows. The negative control did not receive the CaCh for crosslinking alginate to form hydrogel, but instead a wash buffer. The hydrogels are seen as translucent patches within a reaction zone droplet in the image. The image on the right shows the reaction zones after the hydrogel has been dissolved on demand as a result of fusion and mixing with a metal chelating chemical droplet (EDTA). This demonstrates that hydrogel patches can be formed on digital microfluidic devices and dissolved on demand, and can be used for trapping E.Coli cells in the gel patches so that they can have buffer exchange or other operations as needed for application of growing cells.

[0031] Figure 2 shows 25 .m particles functionalized with 488 Atto-dye trapped in alginate hydrogel patches in 192 droplet reaction zone format. The negative controls (without hydrogel) as shown in the box. The presence of hydrogel traps particles; while absence of hydrogel keeps them suspended within the aqueous phase.

[0032] Figure 3 shows a schematic method for demonstrating cell viability on a digital microfluidic device. E. Coli transfected with plasmid for fluorescent protein ccGFP cells prepared on plate, cultured overnight, induced with IPTG to start the production of fluorescence and introduced into droplets on an electrowetting device. An increase in fluorescence in the droplets overtime indicates the cells on the device are viable and producing proteins. Figure 4 shows panels of (L) 192 reaction zone droplets containing hydrogel trapped and nontrapped E.Coli cells at OD -2 with alternating double rows of induced and non-induced cells at t=0; (middle) at t=10 hrs and (R)heatmap of reaction zone droplets showing percentage change in fluorescence after 10 hrs. There was an average increase of -180% in fluorescence over 10 hours incubation showing viability of cells on the devices. The non-induced cells had % change of 9%. Cells expressing in hydrogel and without a hydrogel had similar fluorescence change (155 vs 220%); and this is comparable to on-bench overnight incubation where the fluorescence increased by 215%.

[0033] Figure 5 shows an image of transfected CHO cells expressing fluorescent protein on an electrowetting device.

[0034] Figure 6 shows the median fluorescence intensities grouped by plasmid. The droplets in the left column of Fig. 5 were separated into a separate group termed Plasmid + ExFec but excluded. Note an offset was included for Time for ease of reading. The readings were taken at approximately 12 hour intervals.

[0035] Figure 7 shows the mean fluorescence values for off device transfection. The error bars represent the standard deviation. Note an offset was included for Time for ease of reading. The readings were taken at the same time. The Plasmid + ExFec was the Plasmid and ExpiFectamine transfected in the standard protocol. The Plasmid + ExFec at 37 was the Plasmid and ExpiFectamine that was incubated at 37°C in separate tubes for 30 minutes prior to transfection.

[0036] Figure 8 shows the proportional fluorescence of on and off device transfection. The data is the same data from Fig. 6 and Fig. 7 plotted on the same axes.

[0037] Figure 9 shows a schematic for nutrient exchange for cells in droplets on an electrowetting device.

[0038] Figure 10 shows that expression on cartridge is well correlated to expression in flasks.

[0039] Figure 11 shows that cells are more metabolically active (as seen by increase in fluorescence signal) in CO2 incubated basefluid. DETAILED DESCRIPTION OF THE INVENTION

[0040] Disclosed herein is an improved method for screening expression of proteins using cells on a digital microfluidic device. The proteins may be eukaryotic proteins. The eukaryotic proteins may be mammalian or human.

[0041] One potentially problematic aspect of culturing mammalian cells in a digital microfluidic device cartridge is the ability to exchange liquid culture medium the cells are suspended in. Typically this is required for macro cell culture to keep mammalian cells healthy and able to express transfected nucleic acid sequences. Cells can be held in a defined place on the cartridge to enable diffusion of media to / from the cells. The cells are immobilised in order to exchange the cell culture medium, for example to introduce fresh reagents and remove metabolites. The cells can be immobilised by trapping in a hydrogel or by using solid supports as an example. For immobilisation on solid supports, the supports may be functionalised with an affinity matrix that binds to cell-surface proteins.

[0042] The solid support to which the cells may be immobilised may include either the top or bottom surfaces of the digital microfluidic device, or may be a further solid support such as magnetic or paramagnetic particles. The surfaces of the device may be modified prior to assembly of the device, for example using printing techniques. Cells may be immobilised via electrostatic charge, physical entrapment or via affinity binding. Where the cells are held with an electrostatic charge, then may be released using the opposite charge.

[0043] 3D cell culture (cells embedded in hydrogel) in flasks is known and is capable of maintaining mammalian cell culture for many days. As cells are trapped within a porous hydrogel matrix, media can freely diffuse to / from cells, and as the matrix is a solid, removal of spent media and refresh with new media is simple, as it can be poured out / in to the flask, and obviates the need for centrifugation. Such a system is amenable to cartridge-based mammalian cell expression.

[0044] Hydrogels are three-dimensional networks of hydrophilic polymers that can retain a large amount of water while maintaining their structure. They are formed through various methods, depending on the desired properties and applications. Methods of forming hydrogels include ionic cross-linking, hydrogen bonding, hydrophobic interactions, free-radical polymerisation, click chemistry, photo-polymerisation, thermo-sensitive gelation or pH sensitive gelation.

[0045] Any suitable hydrogel can be used, for example alginate, agarose or polyacrylamide. Hydrogels can be made in situ using many methods, for example blending two or more components to initiate gelling. After formatting of the gels, which remain stationary inside the cartridge, the aqueous droplets can be removed from the cells and replenished with fresh droplets having new reagents.

[0046] The gels can have functionalisation to capture protein upon release either from secretion or from lysis. Some gels can optionally be de-gelled, for example using further chemical treatment, or changing temperature, for example after expression the cells can be released for lysis and detection.

[0047] In one embodiment, cells can be embedded in calcium-alginate hydrogel patches by adding calcium to a solution of alginate. For example a nucleic acid template may be supplied in a solution of calcium which causes gelling of a solution containing cells in alginate. If desired the gel can be broken by adding citrate or EDTA to chelate the calcium.

[0048] The gels may be immobilised and held in place on the electrowetting device. Alternatively the gels may be held in place using electrowetting actuation. Where the device is used for long periods, continuous driving of a droplet (saline) with electric field may cause the dielectric to break and cause the droplets to have electrolysis / pinning and crashing. The gel may be immobilised using magnet particles within the gel. Magnetic beads or particles may be dispensed with the gel so the gel is unable to move. Any magnet can be used, for example a magnetic plate or bar to pin all the droplets / reaction zones simultaneously. The gel, and any cells therein may be pinned using a magnet without using any electrowetting actuation. The field generated by the magnet would pull the beads towards itself but not cause the beads to clump into a pellet or break free of the gel. The gel is thus pinned without electrical power being supplied to the array.

[0049] The immobilised cells can be transfected with the circular nucleic acid template containing the sequence for the protein of interest and detector tag. Transfection is the process of introducing foreign DNA into eukaryotic cells. Various methods can be used, each with its own advantages and applications. The methods can be broadly categorized into physical, chemical, and biological techniques, and may include for example electroporation, chemical methods such as calcium phosphate, lipofection, for example using cationic, lipid-based transfection reagent and transfection enhancers, polymer based transfection using polymers such as polyethylenimine (PEI) or physical methods such as using magnets where DNA is attached to magnetic nanoparticles and guided into cells using a magnetic field or optoperforation, which uses a laser to create temporary pores in the cell membrane, allowing DNA to enter. The transfection may use commercial reagents, for example ExpiFectamine™ kits. The ExpiFectamine™ 293 Transfection Kit is a core component of the Expi293™ Expression System. It is designed for transient transfection of high density cultures of HEK (human embryonic kidney) 293 cells. The high-efficiency, cationic, lipid-based transfection reagent and transfection enhancers are designed to power the highest possible level of protein expression from Expi293F™ cells cultured in Expi293™ Expression Medium.

[0050] T ransfection can add one or more plasmids. A single plasmid may be used to express multiple proteins. The plasmid may express both the protein of interest attached to a detector tag and a further protein which binds to the detector tag.

[0051] After transfection the cell is maintained in a viable state in order to express proteins. Where the cell has expressed both components of a detector species and tag, the level of expression can be monitored directly.

[0052] The cell may be lysed in order to release the contents. Lysis may involve de-gelling of the hydrogel. The contents of the lysate may be mixed with the detector species in order to measure the level of expression.

[0053] Lysis conditions may involve physical or chemical treatments or combinations thereof. Chemical treatments may include:

[0054] Detergents: Detergents like Triton X-100, SDS, and NP-40 solubilize cell membranes, making them permeable. These are often used for eukaryotic cells and can be mild or harsh depending on the detergent.

[0055] Osmotic Lysis: Cells are placed in a hypotonic (low ionic strength) solution, causing them to swell and burst due to osmotic pressure.

[0056] Chaotropic Agents: Chemicals like urea and guanidine hydrochloride disrupt hydrogen bonds, denaturing proteins and lysing cells.

[0057] Combinations of chemicals may be used as needed depending on the cells. Commercial buffers for cell lysis are available, for example RIPA Lysis and Extraction Buffer and M-PER™ Mammalian Protein Extraction Reagent, both from ThermoFisher. The device allows screening of different lysis conditions, for example to measure the level of expressed proteins after lysis under varying conditions.

[0058] Physical conditions may include heat or forcing the cells through gaps smaller than the size of the cells or related method of shearing. Physical method may include sonication. Physical method may include the high speed motion or heating of particles to which the cells are attached.

[0059] The expressed protein may also have an affinity tag to enable purification, for example a tag for attaching to a solid support. Droplets having lysate or secreted proteins may be split, and one droplet taken for detection and one for purification.

[0060] Disclosed is a method of synthesising a protein in a digital microfluidic device using immobilised cells. Protein expression is dependent on the conditions and reagents used for expression. The best expression system for a given protein of interest is not predictable, and may require screening of a large number of similar conditions in order to identify the optimal expression system. The use of EWoD devices can screen large numbers of closely related conditions in parallel in droplets on the device. The droplets can be blended on the device in order to prepare the reagents. For example a cell growing media can be supplemented with a variety of additional components at a selection of concentrations. For example a salt screen, buffer screen or pH screen can be performed across a range of conditions and at variable concentrations. Described herein is the preparation of a variety of different conditions on a single device for the purposes of simultaneously screening a variety of cell growing conditions.

[0061] Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) with an applied electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting have been extensively described. In the case of droplets in channels this can be achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by the walls of a cartridge or microfluidic tubing. Embedded in the walls of the cartridge or tubing are electrodes covered with a dielectric layer each of which are connected to an A / C biasing circuit capable of being switched on and off rapidly at intervals to modify the electrowetting field characteristics of the layer. This gives rise to the ability to steer the droplet along a given path.

[0062] As an alternative to microfluidic channel systems, droplets can also be generated and manipulated on planar surfaces using digital microfluidics (DMF). In contrast to channel based microfluidics, DMF utilizes alternating currents on an electrode array for moving fluid on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics allows precise control over the droplet movements including droplet fusion and separation. Digital microfluidics can be carried out in an air-filled system where the liquid drops are manipulated on the surface in air. However, at elevated temperatures or over prolonged periods, the volatile aqueous droplets simply dry onto the surface by evaporation. This issue is compounded by the high surface area to volume ratio of nanoliter and microliter sized drops. Hence air-filled systems are generally not suitable for protein expression where the temperature of the system needs to be maintained at a temperature suitable for enzyme activity and the duration of the synthesis needs to be prolonged for synthesized protein levels to be detectable.

[0063] Cell growth typically requires a supply of oxygen, carbon dioxide and other nutrients. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen, CO2 and nutrients are available to enable efficient growth.

[0064] Described herein are improved methods allowing for the growth of cells in a digital microfluidic device. Included is a method for the growth of cells in a microfluidic device wherein the method comprises transfecting an immobilised cell and moving droplets containing growth media and other reagents around the immobilised cells using electrokinesis. The components for the protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device.

[0065] The droplets can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows oxygen and nutrients to be supplied to the cells and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0066] The droplet can be moved using any means of electrokinesis. The droplet can be moved using electrowetting-on-dielectric (EWoD). The electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.

[0067] The filler liquid may be a hydrophobic or non-ionic liquid. For example the filler liquid may be decane or dodecane. The filler fluid may be a silicone oil such as dodecamethylpentasiloxane (DM PS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85. The oil can be oxygenated prior to or during the expression process. The oil can be exposed to carbon dioxide prior to the expression process. Alternatively, the device can be an air-filled device where droplets containing reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs. Additionally, humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30 °C).

[0068] The droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a population of cells to form a combined droplet capable of protein synthesis.

[0069] The droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with additive droplets for screening. Lysis can be performed before or after splitting.

[0070] In order to optimise expression, the cell; growing media can be supplemented with additional components, including purified enzymes. The additional components may include salts, cofactors, buffers, surfactants, chaperones or additional protein components. The additional protein components may be selected from for example chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases. Such components may be added to the growing media for subsequent cell-based growth and expression. Alternatively the additional protein components may be co-expressed inside the cells.

[0071] The additional protein components may be involved in any post-translational modification process, for example methylation, ubiquitinylation, sumoylation, isoprenylation or glycosylation.

[0072] Common post-translation modifications include phosphorylation, methylation, sulfation, acetylation, ubiquitylation, prenylation, myristoylation, SUMOylation, palmitoylation, different types of glycosylation (N-glycosylation, O-glycosylation, C-glycosylation and S-glycosylation), phosphoglycosylation and glycosylphosphatidylinositol (GPI anchored). Protein phosphorylation is an important reversible regulatory mechanism that plays a key role in the activities of many enzymes, membrane channels and many other proteins in prokaryotic and eukaryotic organisms. Phosphorylation target sites are Ser, Thr, Tyr, His, Pro, Arg, Asp and Cys amino acid residues, but mainly happens on Ser, Thr, Tyr and His residues. Phosphorylation involves transferring a phosphate group from adenosine triphosphate to the receptor residues by kinase enzymes. Conversely, dephosphorylating or removal of a phosphate group is an enzymatic reaction catalyzed by phosphatases. Phosphorylation can change the function of proteins via one of the two principal ways: by allostery or by binding to interaction domains.

[0073] Acetylation is typically catalyzed via lysine acetyltransferase (KAT) and histone acetyltransferase (HAT) enzymes. Acetyltransferases use acetyl CoA as a cofactor for adding an acetyl group (COCH3) to the e-amino group of lysine side chains, whereas deacetylases (HDACs) remove an acetyl group on lysine side chains. Forms of acetylation include Na- acetylation, Ne-acetylation and O-acetylation, and may occur on Lys, Ala, Arg, Asp, Cys, Gly, Glu, Met, Pro, Ser, Thr and Vai residues with different frequencies, although the acetylation is more reported on Lysine residue. Ne-acetylation is more biologically significant compared to the other types of acetylation. Acetylation has an essential role in biological processes such as chromatin stability, protein-protein interaction, cell cycle control, cell metabolism, nuclear transport and actin nucleation.

[0074] Ubiquitylation is an important reversible PTM and can occur on all 20 amino acids, however, it occurs on lysine more frequently. This PTM has a major role in the degradation of intracellular proteins via the ubiquitin (Ub)-proteasome pathway. Ubiquitylation is catalyzed by an enzyme complex that contains ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes. Ubiquitinated proteins may be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues. Ubiquitylation modification in substrate proteins can be removed by several specialized families of proteases called deubiquitinases. Ubiquitination plays important roles in stem cell preservation and differentiation by regulation of the pluripotency, and plays a role in many various cell activities such as proliferation, regulation of transcription, DNA repair, replication, intracellular trafficking and virus budding, the control of signal transduction, degradation of the protein, innate immune signaling, autophagy and apoptosis.

[0075] Methylation is a reversible PTM, which often occurs in the cell nucleus and on the nuclear proteins such as histone proteins. Methylation occurs on the Lys, Arg, Ala, Asn, Asp, Cys, Gly, Glu, Gin, His, Leu, Met, Phe and Pro residues in target proteins, with lysine and arginine the two main target residues, at least in eukaryotic cells. One important role of methylation is in histone modification. Histone proteins, after synthesis of their polypeptide chains, are methylated at Lys, Arg, His, Ala or Asn residues. N ysine methylation is one of the most abundant histone modifications in eukaryotic chromatin, involves transferring the methyl groups from S-adenosylmethionine to histone proteins via methyltransferase enzyme. In eukaryotes, methylated arginine has been observed in histone and non-histone proteins. Recent studies have shown that methylation is associated with fine tuning of various biological processes ranging from transcriptional regulation to epigenetic silencing via heterochromatin assembly.

[0076] Glycosylation occurs in multiple subcellular locations, such as endoplasmic reticulum, the Golgi apparatus, cytosol and the sarcolemma membrane. Glycosylation occurs in eukaryotic and prokaryotic membranes and secreted proteins, and nearly 50% of the plasma proteins are glycosylated. In this modification, oligosaccharide chains are linked to specific residues by a covalent bond. This enzymatic process, which is catalyzed by a glycosyltransferase enzyme, usually occurs in the side chain of residues such as Trp, Ala, Arg, Asn, Asp, lie, Lys, Ser, Thr, Vai, Glu, Pro, Tyr, Cys and Gly; however, it occurs more frequently on Ser, Thr, Asn and Trp residues in proteins and lipoproteins. According to the target residues, glycosylation can be classified into six groups: N-glycosylation, O-glycosylation, C-glycosylation, S-glycosylation, phosphoglycosylation and glypiation (GPI-anchored). N-glycosylation and O-glycosylation are two major types of glycosylation and have important roles in the maintenance of protein conformation and activity. Glycosylation has a great role in many important biological processes such as cell adhesion, cell-cell and cell-matrix interactions, molecular trafficking, receptor activation, protein solubility effects, protein folding and signal transduction, protein degradation, and protein intracellular trafficking and secretion.

[0077] Small Ubiquitin-Related Modifier (SUMO) ylation has been discovered in a wide range of eukaryotic organisms. SUMOylation can occur in both cytoplasm and nucleus on lysine residues. SUMOylation occurs as a modifier in e-amino group of lysine residues in target protein through a multi-enzymatic cascade. In this reaction, SUMO is connected to a lysine residue in substrate protein by covalent linkage via three enzymes, namely activating (E1), conjugating (E2) and ligase (E3). Often, SUMOylation modifications occur at a consensus motif WKxE (where W represents Lys, lie, Vai or Phe and X any amino acid). SUMOylation plays a major role in many basic cellular processes like transcription control, chromatin organization, accumulation of macromolecules in cells, regulation of gene expression and signal transduction and is necessary for the conservation of genome integrity. Lipidation involves the covalent attachment of lipids to proteins. These PTMs may use a variety of lipids, including octanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, cholesterol, etc. Myristoylation, palmitoylation and prenylation can be considered as the three main types of these lipid modifications. Palmitoylation is the covalent attachment of fatty acids, like palmitic acid on the Cys, Gly, Ser, Thr and Lys. S-palmitoylation contains a reversible covalent addition of a 16-carbon fatty acid chains, palmitate, to a cysteine via a thioester linkage. Palmitoyl-CoA (as the lipid substrate) is attached to the target protein. Mostly, S-palmitoylation occurs in eukaryotic cells and plays critical roles in many different biological processes including protein function regulation, protein-protein interaction, membrane-protein associations, neuronal development, signal transduction, apoptosis and mitosis.

[0078] Myristoylation (N-myristoylation) is an irreversible PTM that occurs mainly on cytoplasmic eukaryotic proteins. Myristoylation happens approximately in 0.5-1.5% of eukaryotic proteins. In myristoylation, after removal of the initiating Met, a 14-carbon saturated fatty acid, called myristic acid, is attached to the N-terminal glycine residue via a covalent bond. This attachment is often observed in Met-Gly-X-X-X- Ser / Thr motif and is catalyzed by an N- myristoyl transferase (NMT) (there are at least two types of NMT enzymes, NMT 1 and NMT2, in humans). Proteins that undergo this PTM play critical roles in regulating the cellular structure and many biological processes such as stabilizing the protein structure maturation, signalling, extracellular communication, metabolism and regulation of the catalytic activity of the enzymes.

[0079] Prenylation is another important lipid-based PTM, which occurs after translation as an irreversible covalent linkage mainly in the cytosol. This reaction occurs on cysteine and near the carboxyl-terminal end of the substrate protein. Prenylation has two main forms: farnesylation and geranylation. These two forms contain the addition of two different types of isoprenoids to cysteine residues: farnesyl pyrophosphate (15-carbon) and geranylgeranyl pyrophosphates (20-carbon), respectively. In prenylated proteins, one can find a consensus motif at the C-terminal; the motif is CAAX where C is cysteine, A is an aliphatic amino acid and X is any amino acid. This process is catalyzed by three prenyltransferase enzymes: farnesyltransferase (FT) and two geranyl transferases (GT1 and GT2). Prenylation is known as a crucial physiological process for facilitating many cellular processes such as proteinprotein interactions, endocytosis regulation, cell growth, differentiation, proliferation and protein trafficking. N-sulfation or O-sulfation includes the addition of a negatively charged sulfate group by nitrogen or oxygen to an exposed residue on the target protein. Currently, PTS is observed mainly in secreted and transmembrane proteins in multicellular eukaryotes. This reaction is catalyzed by two transmembrane enzymes, tyrosyl protein sulfotransferases 1 and 2 (TPST1 and TPST2). TPSTs govern the transfer of an activated sulfate from 3-phospho adenosine 5- phosphosulfate to residues within acidic motifs of polypeptides.

[0080] The expressed protein may contain an AVI tag. The Avi-tag peptide (GLNDIFEAQKIEWHE) is recognized by BirA ligase which enzymatically attaches a biotin molecule to a single lysine residue within the Avi-tag sequence. The enzyme can be present during expression to attach the biotin to the expressed tag after expression of the POI.

[0081] The expressed protein may contain a variety of flanking tags to enhance solubility or purification. The expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:

[0082]

[0083] The expressed protein may contain a sequence acting as binding moiety for purification. The binding moiety for purification may contain four or more amino acids. The binding sequences may contain 4-30 amino acids. The binding moiety may be selected from:

[0084] Alfa-tag (SRLEEELRRRLTE)

[0085] Avi-tag (GLNDIFEAQKIEWHE)

[0086] C-tag (EPEA) Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0087] Dogtag (DIPATYEFTDGKHYITNEPIPPK)

[0088] E-tag (GAPVPYPDPLEPR)

[0089] FLAG (DYKDDDDK)

[0090] G4T (EELLSKNYHLENEVARLKK)

[0091] HA (YPYDVPDYA)

[0092] His (HHHHHH)

[0093] Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0094] Myc (EQKLISEEDL)

[0095] NE-Tag (TKENPRSNQEESYDDNES)

[0096] Poly Glutamate-tag (EEEEEEE)

[0097] Poly Arginine-tag (RRRRRRR)

[0098] Rho1 D4-tag (TETSQVAPA)

[0099] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)

[0100] Sdytag (DPIVMIDNDKPIT)

[0101] SH3 (STVPVAPPRRRRG)

[0102] SNAC (GSHHW)

[0103] Snooptag (KLGDIEFIKVNK)

[0104] Softag 1 (SLAELLNAGLGGS)

[0105] Softag 3 (TQDPSRVG)

[0106] Spot-tag (PDRVRAVSHWSS)

[0107] Spytag (AHIVMVDAYKPTK)

[0108] S-tag (KETAAAKFERQHMDS)

[0109] Strep-tag (AWAHPQPGG) (AWRHPQFGG)

[0110] Strep-tag II (WSHPQFEK)

[0111] T7tag (MASMTGGQQMG)

[0112] TC-tag (EVHTNQDPLD)

[0113] Ty-tag (CCPGCC)

[0114] VSV-tag (YTDIEMNRLGK)

[0115] Xpress-tag (DLYDDDDK)

[0116] The expression composition may be assembled on the device from mixing a variety of droplets in order to screen a variety of compositions in parallel.

[0117] By way of example, the screening reagents may include,

[0118] ■ Chaperone mix (e.g., GrpE mix) ■ Kinase 1 (e.g., NEB CK2)

[0119] ■ Kinase 2 (e.g., NEB PKA)

[0120] ■ Protease 1 (e.g., NEB TEV)

[0121] ■ Protease 2 (e.g., Merck HRV 3C)

[0122] ■ N-acetyl transferase

[0123] ■ Common metal ions cocktail

[0124] ■ Common co-factors cocktail

[0125] The compositions can be blended by the user and the level of expression of the protein of interest monitored in each of the blended conditions.

[0126] Metal ions may include one or more of the following: MgCh, CuCh, ZnCh, CaCh, MnCh, NiCh, CoCI2.

[0127] Co-factors may include one or more of Nicotinamide adenine dinucleotide (NAD), Flavin adenine dinucleotide (FAD), S-adenosyl methionine (SAM), pyridoxal phosphate (PLP) Coenzyme A (CoA), thiamine pyrophosphate (TPP) or haem.

[0128] Chaperones may include one or more of DnaK, DnaJ, GroE, GrpE, heat shock proteins, protein disulfide isomerase (PDI), human protein disulfide isomerase (hPDI), disulfide bond C (DsbC), a thioredoxin, such as TRXB1 , Caseinolytic peptidase B protein homolog (CLPB) or FK506 binding proteins (FKBPs).

[0129] The additive may be for example one or more reducing agents. The additive may be selected from DTT, glutathione (GSH) or glutathione disulfide (GSSG).

[0130] The added protease may cleave the protein of interest from flanking regions. The flanking regions may include tags used for detection or solubility or other buffer regions. The flanking regions may be cleaved by any protease. The protease may be a TEV or 3C protease.

[0131] The TEV protease may act upon the amino acid sequence ENLYFQS. The template for expression of the POI may include the nucleic acid sequence GAGAACCTGTACTTCCAGAGC.

[0132] The 3C protease may act upon the amino acid sequence LEVLFQGP. The template for expression of the POI may include the nucleic acid sequence CTCGAGGTTCTGTTCCAAGGACCT. The method may be used to perform a surfactant screen to identify the best surfactant for expression of a particular protein. The surfactant may be ionic, nonionic, or zwitterionic.

[0133] Surfactant molecules are composed of a hydrophobic region and a hydrophilic region. This amphiphilic structure enables surfactants to obtain a discoidal conformation in the solution, known as micelles. Micelles solubilize membrane proteins by encompassing the transmembrane domains of integral membrane proteins, with the loops and hydrophilic regions exposed to solvent. The minimum concentration of a surfactant necessary to form micelles and extract membrane proteins is called critical micelle concentration or CMC.

[0134] Depending on the charge of hydrophilic group, surfactants are classified into three groups: ionic, nonionic, and zwitterionic surfactants. Ionic surfactants carry a charged group, either negative (anionic) or positive (cationic), and historically have been the most efficient group of detergents in extracting membrane proteins from lipid bilayers. However, ionic detergents can have deleterious effects on protein-protein interactions and often lead to protein denaturation. Sodium dodecyl sulfate (SDS) and sodium cholate are two common examples of ionic detergents.

[0135] Nonionic surfactants are currently the most popular and successful group of surfactants in solubilizing membrane proteins for both functional and structure determination purposes. This is due to their nondisruptive nature, which enables them to preserve the native structure of the target protein by breaking protein-lipid interactions instead of protein-protein interactions. Alkyl glycoside surfactants such as n-dodecyl-B-D-maltoside (DDM), n-decyl-B-D-maltoside (DM), n-Octyl-B-D-Glucopyranoside (OG), and n-Nonyl-B-D-Glucopyranoside (NG) by contributing to the purification and crystallization of about 70% of membrane proteins are the most common nonionic surfactants for protein studies. Another advantage of nonionic surfactants is that they do not interfere with optical measurements, which enables fluorescence-based experiments on expressed proteins.

[0136] Zwitterionic surfactants typically have an intermediate level of harshness between ionic and nonionic detergents. They carry both positive and negative charged groups in their polar regions with an overall net charge of zero. An example of a Zwitterionic surfactant is lauryldimethylamine-N-oxide or LDAO.

[0137] Membrane mimetic systems, such as nanodiscs and styrene malic acid lipid particles (SMALPs), provide an alternative platform for stabilization of membrane proteins and hence eliminate the deleterious effects of detergents on these macromolecules. Nanodiscs are composed of phospholipid patches surrounded by two copies of membrane scaffold protein (MSP), a genetically engineered version of human serum apolipoprotein A-l.

[0138] Plasmids are circular templates, which can be produced either in cells or synthetically.

[0139] The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any devices simply having droplets in a flow of oil in a channel. The droplets are moved over the surface by electrokinetic forces by activation of particular electrodes. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface. A digital microfluidic (DMF) device set-up is known in the art, and depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage.

[0140] Once the cells have been enclosed in the droplets and immobilised, additional reagents can be supplied by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including for example oxygen or ‘power’ sources, or test reagents to which it is desired to expose to the expressed protein.

[0141] The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by merging droplets on the device in order to screen a selection of expression compositions in parallel. The compositions may include varying transfected template nucleic acid sequences which express a desired amino acid sequence.

[0142] The droplets can be aqueous droplets. The droplets can contain an oil immiscible organic solvent such as for example DMSO. The droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk oil.

[0143] The droplets can be in a bulk oil layer. A dry gaseous environment simply dries the bubbles onto the surface during the expression process, leaving comet type smears of dried material by evaporation. Thus the device is filled with liquid for the expression process. Alternatively, the aqueous droplets can be in a humidified gaseous environment. A device filled with air can be sealed and humidified in order to provide an environment that reduces evaporation of droplets.

[0144] The droplets containing the cells will therefore typically be in the oil filled environment before the circular nucleic acid templates are added to the droplets and transfected into the cells. The templates can be added by merging droplets on the microfluidic device. Alternatively, the templates can be added to the droplets outside the device and then flowed into the device for the transfection process. For example the transfection process can be initiated on the device by adding calcium phosphate. The cell growth typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C or at or above 37 °C.

[0145] The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the droplets should be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen, carbon dioxide and nutrients are available within the droplet such that the cells are viable. The moving can be continuous, or can be repeated with intervening periods of non-movement.

[0146] Thus the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed in the cells. The aqueous droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows mixing within the droplet, and allows oxygen or other reagents to be supplied to the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0147] Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a- chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.

[0148] The droplet can be moved using any means of electrokinesis. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWoD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.

[0149] The electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors. Optically-activated s-Si EWoD devices are well known in the art for actuating droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771). The oil in the device can be any water immiscible or hydrophobic liquid. The oil can be mineral oil, silicone oil, an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The air in the device can be any humidified gas.

[0150] A source of supplemental oxygen and / or carbon dioxide can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen or CO2 can be merged with the aqueous droplets during the protein expression. Alternatively the source of oxygen can be a molecular source which releases oxygen. Alternatively the droplets can be moved to an air / liquid boundary to enable increased diffusion of oxygen or carbon dioxide from a gaseous environment. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E.Coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air.

[0151] Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device.

[0152] A source of carbon dioxide to the cells can be delivered via exposing the basefluid / oil to an atmosphere of CO2 prior to use. Disclosed herein is a basefluid composition comprising a silicone oil containing dissolved carbon dioxide via exposure to an atmosphere having greater than 5% CO2. Disclosed herein is a basefluid composition comprising a silicone oil containing dissolved carbon dioxide via exposure to an atmosphere having 8% CCh.The silicone oil may be dodecamethylpentasiloxane (DMPS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85.

[0153] The droplets can be actuated on a hydrophobic surface on the digital microfluidic device (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPei (Cytonix LLC). The hydrophobic surface may be modified in such a way to reduce biofouling, especially biofouling resulting from exposure to cellular reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Superhydrophobic surfaces prevent biofouling compared with typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus prolong the capability of digital microfluidic devices to move droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633-49648). The hydrophobic surface can also be a slippery liquid infused porous surface (SLIPS), which can be formed by infusing Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275).

[0154] Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing cells can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011 , 27, 13, 8586-8594). Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and / or Pluronic F127. Specifically, droplets containing celular components may contain TWEEN 20 at 0.1 % v / v, Triton X-100 at 0.1 % v / v, and / or Pluronic F127 at 0.05% w / v.

[0155] Rather than adding surfactants to the aqueous sample, it is instead possible to add surfactant, such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the oil. This has the advantages of enabling reactions to proceed on-DMF without dilution or adulteration. Additionally, it simplifies the sample preparation procedure for setting up the reactions, increasing the ease of use and the consistency of results. Using 1 % w / w Span85 in dodecane or DM PS allows for dilution-free reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane or DMPS could be used. A range of concentrations of Span85 could be used. Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof. Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils. Surfactants can have a detrimental effect on (1) the cell viabilty and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the cellular reaction on- DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the Split GFP (e.g. ccGFPn / ccGFPi-io) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial.

[0156] The peptide tag can be attached to the C or N terminus of the protein. The peptide tag may be one component of a green fluorescent protein (GFP). For example the peptide tag may be GFPn and the further polypeptide GFP1-10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi-10. For example the peptide tag may be ccGFPn and the further polypeptide ccGFPi- . The protein may be fused to multiple tags. For example the protein may be fused to multiple GFP11 peptide tags and the synthesis occurs in the presence of multiple GFP1-10 polypeptides. For example the protein may be fused to multiple sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi-10 polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more GFP1-10 polypeptides and one or more sfCherryi-10 polypeptides.

[0157] Devices

[0158] The manipulation of droplets by the application of electrical potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. Droplet manipulation as a result of an applied electrical potential is known as electrowetting. Electrokinesis occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to create forces on polarizable particles to induce their movement. The electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semiconductor film whose electrical properties can be modulated by an optical signal.

[0159] EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young- Lippmann equation: cos0 - cos0o= (1 / 2 / LG) c.V2where 0o is the contact angle when the electric field across the interfacial layer is zero, yLG is the liquid-gas tension, c is the specific capacitance (given as sr. so / t, where sris dielectric constant of the insulator / dielectric, so is permittivity of vacuum, t is thickness) and V is the applied voltage or electrical potential. The change in contact angle (inducing droplet movement) is thus a function of surface tension, electrical potential, dielectric thickness, and dielectric constant.

[0160] When a droplet is actuated by EWoD, there are two opposing sets of forces that act upon it: an electrowetting force induced by electric field and resistant forces that include the drag forces resulting from the interaction of the droplet with filler medium and the contact line friction (ref). The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is variably determined by the thickness-to-dielectric contact ratio of the insulator / dielectric, (t / £r)1 / 2. Thus, to reduce actuation voltage, it is required to reduce (t / £r)1 / 2(i.e. , increase dielectric constant or decrease insulator / dielectric thickness). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, the deposition of high quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved. Most academic studies thus report the use of much higher voltages >100V on easily fabricated, thick dielectric films (>3 pm) to effect electrowetting.

[0161] High voltage EWoD-based devices with thick dielectric films, however, have limited industrial applicability largely due to their limited droplet multiplexing capability. The use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a-Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion. The driving voltage for TFTs or optically-activated a-Si are low (typically <15 V). The bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators / dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator / dielectric devices.

[0162] Typically, the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator / dielectric is pinhole free to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. Sll-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator / dielectric polymers. However, there are difficulties in reliably producing <1 micron pinhole-free coatings of parylene or Sll-8; thus, the thickness of these materials is typically kept at a 2-5 microns at the cost of increased voltage requirements for electrowetting. It has also been reported that traditional EWoD devices with parylene C are easily broken and unstable for repeated droplet manipulation with cell culture medium. Multi-layer insulator devices deposited with metal-oxide and parylene C films have been used to produce a more robust insulator / dielectric and enable operations with lower applied voltages. Inorganic materials, such metal oxides and semiconductor oxides, commonly used in the CMOS industry as “gate dielectrics”, have been used as insulator / dielectric for EWoD devices. They offer the advantage of utilizing standard cleanroom processes for thin film depositions (<100 nm). These materials are inherently hydrophilic, requiring an additional hydrophobic coating, and can be prone to pinhole formation as a result of thin film layer deposition process. Together with the need for lower voltage operations of EWoD, recent developmental work has focused on (1) using materials with improved dielectric properties (e.g., using high-dielectric constant insulators / dielectrics), (2) optimizing the fabrication process to make the insulator / dielectric pinhole free to avoid dielectric breakdown.

[0163] Operation of EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator / dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules). One of the adverse effects of this hysteresis is reduced operational lifetime of the EWoD-based device.

[0164] Contact angle hysteresis is believed to be a result of charge accumulation at the interface or within the hydrophobic insulator after several operations. The required actuation voltage increases due to this charging phenomenon resulting in eventual catastrophic dielectric breakdown. The most probable explanation is that pinholes at the insulator / dielectric may allow the liquid to come into contact with the electrode causing electrolysis. Electrolysis is further facilitated by pinhole-prone or porous hydrophobic insulators.

[0165] Most of the studies to understand contact angle hysteresis on EWoD have been conducted on short time scales and with low conductivity solutions. Long duration actuations (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCI) could produce several effects other than electrolysis. The ions in solution can permeate through the hydrophobic coat (under the applied electric field) and interact with the underlying insulator / dielectric. Ion permeation can result in (1) change in dielectric constant due to charge entrapment (which is different from interfacial charging) and (2) change in surface potential of a pH sensitive metal oxide. Both can result in reduction of electrowetting forces to manipulate aqueous droplets, leading to contact angle hysteresis. The inventors have previously found that the damage from high conductivity solutions reduces or disables electrowetting on electrodes by inhibiting the modulation of contact angle when an electric field is applied.

[0166] An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes.

[0167] The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.

[0168] The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick. The conformal layer may be between 100 nm and 200 nm thick.

[0169] The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating.

[0170] The elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.

[0171] The functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer. This has been found to be a particularly advantageous combination.

[0172] The electrokinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled.

[0173] The second substrate may also comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers.

[0174] The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.

[0175] The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019 / 0111433, incorporated herein by reference.

[0176] Described herein are electrokinetic devices, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes; Described herein is an electrokinetic device, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes, a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0177] The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed.

[0178] Cell-Based Expression

[0179] Following the screening assay, the best conditions for production of soluble proteins are identified in the cells. The selected conditions can then be used to produce the protein in a host cell to which the nucleic acid sequence to be expressed is added, for example as an expression vector or plasmid. The screening assay may identify conditions for protein synthesis, folding and / or isolation which are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence. In such instances the presence of the protein additives may be required for the cell-based expression. Where additional protein additives are beneficial, these may optionally be co-expressed in the cell. The co-expression may use the same plasmid as the desired amino acid sequence. Alternatively the cells used for expression may contain genes for the expression of the additional proteins which aid expression, folding and / or isolation of the desired amino acid sequence.

[0180] The additional protein additives may be for example one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

[0181] The screening method may identify the optimum conditions for the growth of cells, for example the best temperature or growth media in order to obtain the desired amino acid sequence in a correctly folded state.

[0182] Disclosed is a kit comprising: i. a circular nucleic acid template coding sequence for a protein of interest and a detection tag; ii. a population of cells suitable for expression proteins upon transfection with the nucleic acid template; and iii. a digital microfluidic device.

[0183] The kit may comprise a variety of nucleic acid template sequences coding for the same amino acid sequence and a variety of cell-lines for expressing the nucleic acid template coding sequence for a protein of interest.

[0184] Examples

[0185] Figure 1 This experiment shows Alginate hydrogel patches in 192 droplet reaction zone format on digital microfluidic devices along with negative control, some of which are shown with arrows. The negative control did not receive the CaCh for crosslinking alginate to form hydrogel, but instead a wash buffer. The hydrogels are seen as translucent patches within a reaction zone droplet in the image. The image on the right shows the reaction zones after the hydrogel has been dissolved on demand as a result of fusion and mixing with a metal chelating chemical droplet (EDTA). This demonstrates that hydrogel patches can be formed on digital microfluidic devices and dissolved on demand, and can be used for trapping E.Coli cells in the gel patches so that they can have buffer exchange or other operations as needed for application of growing cells.

[0186] Figure 2 shows 25 .m particles functionalized with 488 Atto-dye trapped in alginate hydrogel patches in 192 droplet reaction zone format. The negative controls (without hydrogel) as shown in the box. The presence of hydrogel traps particles; while absence of hydrogel keeps them suspended within the aqueous phase.

[0187] Figure 4 shows panels of (L) 192 reaction zone droplets containing hydrogel trapped and nontrapped E.Coli cells at OD -2 with alternating double rows of induced and non-induced cells at t=0; (middle) at t=10 hrs and (R)heatmap of reaction zone droplets showing percentage change in fluorescence after 10 hrs. There was an average increase of -180% in fluorescence over 10 hours incubation showing viability of cells on the devices. The non-induced cells had % change of 9%. Cells expressing in hydrogel and without a hydrogel had similar fluorescence change (155 vs 220%); and this is comparable to on-bench overnight incubation where the fluorescence increased by 215%. The reagent stocks used were prepared as follows; all reagents on device had 0.05% F-127. The device was primed with DM PS with 0.1% Span85 (w / v) prior to loading aqueous reagents.

[0188] 0.5% alginate stock preparation:

[0189] 10X wash buffer = IBA, Buffer W (10X), 2-1003-100, 1003-0215

[0190] 10% F127 = Invitrogen, P6866, 2418436

[0191] All water is MQ water

[0192] Prepare IX wash buffer with F127:

[0193] 0.05% F127 = 10% F127 (0.2 mL) + MQ (40 mL)

[0194] Prepare IX wash buffer in F127:

[0195] 10X Wash buffer (5 mL) + 0.05% F127 (45 mL)

[0196] Alginate = Sodium Alginate, NOVAMATRIX, PRONOVA UP LVG, 42000001-5G, BP-2102-09

[0197] (100 mg) Alginate into (10 mL)1X wash buffer 0.05% F127

[0198] The volume of the buffer added is corrected for the actual volume weighed out.

[0199] Vortexed and sonicated to solution

[0200] CaCh 0.3 M preparation:

[0201] CaCI2.2H2O (Mr = 147) (Mol. biology grade) = Merck, 208291 , 3847886

[0202] (441 mg) CaCI2.2H2O into (10 mL) IX wash buffer 0.05% F127

[0203] The volume of the buffer added is corrected for the actual volume weighed out.

[0204] Vortexed to solution.

[0205] CaCI20.1 M preparation:

[0206] CaCI2.2H2O (Mr = 147) (Mol. biology grade) = Merck, 208291 , 3847886

[0207] (147 mg) CaCI2.2H2O into (10 mL) IX wash buffer 0.05% F127

[0208] The volume of the buffer added is corrected for the actual volume weighed out.

[0209] Vortexed to solution. Experiment 1

[0210] 0.25% Aliqinate preparation:

[0211] 100 pL 0.5% + 100 pL Wash buffer (0.1 M Tris-CI, 0.15 M NaCI, 0.05% F127, pH 8.0)

[0212] 0.2% Aliqinate preparation:

[0213] 100 pL of 0.2% Alginate was prepared by adding 40 pL of Alginate to 60 pL wash buffer

[0214] 0.1 % Alginate preparation:

[0215] 50 pL (0.5% alginate solution) + 200 pL (0.1 M Tris-CI, 0.15 M NaCI, 0.05% F127, pH 8.0) Vortexed before use.

[0216] E. Coli preparation:

[0217] BL21 cells (NEB, C2527) cells were transformed with 10 ng of either pJL-sfGFP or pET41a- ccGFP plasmid. Transformation was performed as follows:

[0218] • BL21 cells (50 pl) were taken from -80 °C and thawed on ice for 5 minutes.

[0219] • 10 ng of plasmid DNA was added to the cells. Cells and DNA were mixed by gently tapping on the tubes.

[0220] • Cells were incubated on ice for 30 minutes.

[0221] • Heat-shock was given to the cells at 42 °C for 30 seconds and cells were quickly transferred to ice for another five minutes.

[0222] • 950 pl of SOC media was added to the cells. Cells were incubated on 37 °C for one hour on a heat block with mixing at 300 rpm.

[0223] • 200 pl of the transformed cells were inoculated to LB media with kanamycin. The culture was incubated at 37 °C and 220 rpm in an incubator.

[0224] • NEXT day, the OD was recorded on a spectrophotometer.

[0225] • Before use, 5 pL of 10% F-127 was added to 995 pL of cells to give final concentration of 0.05% F-127.

[0226] IPTG (4 pl / mL, 100 mM stock)

[0227] 0.8 pl IPTG added to the 200 pl cells (100 pl cells + 100 pl Wash buffer (0.1 M Tris-CI, 0.15 M NaCI, 0.05% F127, pH 8.0)_or Alginate as above.

[0228] Figure 1 :

[0229] Ports assignment:

[0230] A12- F12- 0.25% Alginate G12, H12- 0.1% Alginate

[0231] A1-A4: Buffer

[0232] A5-A8: CaCI20.1 M

[0233] B1-B8: CaCI20.1 M

[0234] C1-C8: CaCI20.1 M

[0235] A10, B10, G10 and H10: 0.2 M EDTA, pH 8

[0236] Figure 2:

[0237] A12- F12: 0.25% Alginate + 5% w / v Strep beads (tagged with Atto 488)

[0238] G12, H12: 0.1% Alginate + + 5% w / v Strep beads (tagged with Atto 488)

[0239] A1-A8: CaCI20.1 M

[0240] B1-B8: CaCI20.1 M

[0241] C1-C8: CaCI20.1 M

[0242] H5 and H6: 0.1 M Tris-CI, 0.15 M NaCI, 0.05% F127, pH 8.0)

[0243] Figure 4:

[0244] A12- 10 pL of 0.2% Alginate + 10 pL of 3.6 OD (IPTG induced to express ccGFP) B12- 10 pL of 0.2% Alginate + 10 pL of 3.6 OD (no induction)

[0245] C12- 10 pL of 0.2% Alginate + 10 pL of 3.6 OD (IPTG induced to express ccGFP)

[0246] D12- 10 pL of 0.2% Alginate + 10 pL of 3.6 OD (no induction)

[0247] E12- 10 pL of wash buffer + 10 pL of 3.6 OD (IPTG induced to express ccGFP)

[0248] F12- 10 pL of wash buffer + 10 pL of 3.6 OD (no induction)

[0249] G12- 10 pL of wash buffer + 10 pL of 3.6 OD (IPTG induced to express ccGFP)

[0250] H12- 10 pL of wash buffer + 10 pL of 3.6 OD (no induction)

[0251] A1, A3, A5, A7: CaCI20.3 M

[0252] A2, A4, A6 and A8: Wash buffer

[0253] B1-B8: CaCI20.3 M

[0254] C1-C8: CaCI20.3 M

[0255] Control H4- Wash buffer, Control H5- OD4 control E.Coli expressing sfGFP

[0256] A10, B10, G10 and H10- Wash buffer.

[0257] Transfection on device with DNA and Transfectamine incubation

[0258] Summary

[0259] • ExpiCHO cells were successfully transfected on device. • Using the proportion of the total amount of fluorescence as a metric the off and on device transfection exhibited a similar trend in protein production.

[0260] The mixing of ExpiFectamine and plasmid DNA was used in an attempt to increase transfection efficiency. Additionally, an off device transfection was also completed. Here, the ExpiFectamine and Plasmid DNA were incubated at 37°C for 30 minutes in separate tubes. This was to determine if on device loading, which can take around 30-40 minutes prior to mixing, affected transfection efficiency.

[0261] Results

[0262] The cultured CHO cells and other reagents were prepared in order to perform an eProtein discovery run according to commercial instructions. Cells were made to a concentration of 6x106cells / ml in ExpiCHO Expression Media with 0.05% F-127 added. The measured concentration of cells was 6.87x106cells / ml. 874 pl of cells were collected and suspended in 1000 pl of ExpiCHO Expression Media with 0.05% F-127.

[0263] Plasmid DNA expressing a green fluorescent protein (GFP) was loaded into ports B1 , and B2. B3 was loaded with OptiPRO buffer (the buffer the plasmid was diluted in). B4 was loaded with ExpiFectamine. B5 and B6 were loaded with OptiPRO buffer (the buffer the ExpiFectamine was diluted in). All reagents contained 0.05% F-127. The cells were maintained on device in order to express the transfected fluorescent protein. There was evidence of transfection on device (Fig. 5). The mean fluorescence for each of the droplets in the groups was determined over 12 hour intervals. The analysis indicated that only two droplets had clear increases in fluorescence. When the analysis was restricted to these droplets there was a clear increase in fluorescence. The cause of this was suspected to be due to cell heterogeneity during dispense (Fig. 6).

[0264] An off device transfection was started just prior to the loading of the on device transfection. Off device transfection were prepared for a volume of 900 pl in a 12 well plate. Tubes of plasmid DNA diluted in OptiPRO SFM and ExpiFectamine diluted in OptiPRO SFM were incubated at 37°C for 30 minutes. The purpose of this was to mimic the on device loading, which can take around 30-40 minutes with the device heated to 37°C at this time. The objective was to determine then comparative transfection efficiency. The amount of GFP produced was used as a measure of transfection efficiency. Cells producing GFP were pipetted into a 384 well plate and the GFP fluorescence was measured (Fig. 7). The Plasmid and ExpiFectamine transfected as standard and the Plasmid and ExpiFectamine transfected after a 37°C incubation in separate tubes had similar fluorescence indicating that the incubation of the plasmid DNA and ExpiFectamine in separate tube at 37°C prior to mixing and transfection as standard had little effect on the protein production.

[0265] The off device transfection and on device transfection were compared. Since the fluorescence values were different and there was no GFP control on the device transfection for comparison the fluorescence values were plotted as a proportion. First the fluorescence values were blanked by subtracting the minimum value observed for off and on device transfections. This was effectively a blank subtraction. Then a proportional value was calculated by dividing the maximum fluorescence observed in on or off device conditions after minimum subtraction and dividing each fluorescence value by the calculated maximum. This made all of the values a proportional value of the maximum value observed. These values were plotted (Fig. 8). The error bars of on device transfection were larger than the off device transfection. However, the overall trend of on and off device transfection was proportional.

[0266] Conclusions

[0267] From the data presented here it was concluded that cells were successfully transfected on device. Improving the cell density heterogeneity would be expected to improve the transfection efficiency of the other droplets that received both Plasmid and ExpiFectamine.

[0268] Expression of fluorescent proteins in cells on a cartridge shows cells are viable (Figure 10).

[0269] • T ransfected ExpiCHO-S cells with ccGFP plasmids having a secretory protein tag were grown in a CO2 incubator for 24 hours.

[0270] • The cells were loaded and kept on two devices: one with continuous base fluid exchange, and the other without base fluid exchange.

[0271] • ccGFP expression was monitored over several days.

[0272] Results ccGFP expression continued to increase over six days on the device. Transfected cells were grown in the CO2 incubator for a day. Next day feed and enhancer solutions were added to the cells and then cells were loaded on the Nuclera eProtein Discovery™ device. The base fluid used was equilibrated with CO2 overnight. Remaining transfected cells after loading the device were kept in CO2 incubator and ccGFP expression was quantified on plate reader on day 2, 3, and 6. ccGFP fluorescence intensity on the device was quantified and normalized with the value from standard ccGFP fluorescence (100ug / mL (3.6 .M)) to get ccGFP expression in nM.

[0273] Correlation of ccGFP expression between the eDrop device and on bench

[0274] The mean ccGFP expression (in nM) is plotted for expression from day 2, day 3 and day6. The correlation between bench and on device expression is high with R2 value of 0.97 and P- value of the correlation is close to 0 (Figure 10).

[0275] Conclusions

[0276] • ccGFP expression continued to increase for six days without any media or base fluid exchange.

[0277] • The protein expression from the device correlates with the expression on the bench.

[0278] • The expression between the droplets for the same protein varies significantly (2-4 fold).

[0279] • From day 3 onwards, the protein expression was detectable on the device.

[0280] Detection of growth on device after 24 hour incubation shows the benefits of adding carbon dioxide to the basefluid (Figure 11).

[0281] Summary

[0282] • The infusion of CO2 into the base fluid resulted in increased cell viability.

[0283] • Performance is improved with base fluid that has been incubated in an 8% CO2 environment.

[0284] Results

[0285] ExpiCHO Cells and Heat Killed cells (HKC) were loaded onto a Nuclera eProtein Discovery™ cartridges at a concentration of 1x106cells per ml. The array was split in two. The first half, ports loaded from A12, B12, C12, and D12, were used for the first time point at 0 hours post array formation and the second half, ports loaded from E12, F12, H12, and G12, were used for the second time point at 22 hours post array formation. Cell viability stain PrestoBlueHS was loaded as standard during loading and then loaded for a second time after the device had been incubated for 22 hours (Fig. 11). There was a difference in droplet fluorescence between droplets incubated in the CO2 infused base fluid and the standard base fluid (Fig. 11).

[0286] Conclusions

[0287] The data here suggest that infusing the base fluid with CO2 by incubating the base fluid in an atmosphere of 8% CO2 enables cell growth.

Claims

Claims1. A method of producing proteins comprising the steps of: a. taking circular nucleic sequences expressing proteins having a desired amino acid sequence and a detection tag; b. taking a population of cells in droplets on a digital microfluidic device; c. transfecting the circular nucleic sequences into the cells; d. allowing the cells to express the proteins; and e. measuring the level of the expressed protein using a detector species which binds to the detection tag.

2. The method according to claim 1 , wherein the cells are lysed to release the expressed proteins.

3. The method according to claim 2, wherein the expressed proteins are detected by adding the detector species after cell lysis.

4. The method according to claim 2 or claim 3, wherein the lysis is performed by chemical, enzymatic or hypotonic methods.

5. The method according to claim 2 or claim 3, wherein the lysis is performed using one or more detergents.

6. The method according to claim 1 , wherein the detector species is co-expressed in the cells or is present in the media in which the cells are expressing the protein.

7. The method according to any one of claims 1 to 6, wherein the cells are immobilised.

8. The method according to claim 7, wherein the cells are immobilised in a hydrogel matrix.

9. The method according to claim 8, wherein the hydrogel is an alginate gel.

10. The method according to claim 7, wherein the cells are immobilised on a solid support.

11. The method according to claim 10, wherein the solid support is a magnetic or paramagnetic bead.

12. The method according to any one of claims 1 to 11 , wherein the cells used for expression are selected from mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa.

13. The method according to claim 12, wherein the cells are derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

14. The method according to any one of claims 1 to 13 for the expression of eukaryotic or human proteins.

15. The method or use according to any one of claims 1 to 14, wherein the protein expression is performed on an electrowetting-on-dielectric (EWoD) device.

16. The method according to any one of claims 1 to 15 comprising a series of droplets having varying reagent compositions, thereby identifying a composition suitable for expression of the protein of interest.

17. The method or use according to any one of claims 1 to 16, wherein the identified conditions for protein synthesis, folding and / or isolation are aided by the presence of one or more protein sequences in addition to the desired amino acid sequence.

18. The method according to claim 17, wherein the additional proteins required are coexpressed in the cells.

19. The method according to claim 18, wherein the additional proteins are expressed using the same plasmid as the desired amino acid sequence.

20. The method according to any one of claims 17 to 19, wherein the additional proteins are one or more of chaperones, glycosylating enzymes, proteases, redox active enzymes, phosphorylases and kinases.

21. The method according to any one of claims 1 to 20 which identify the optimum expression parameters for growth of cells in order to obtain the desired amino acid sequence in a correctly folded state.

22. The method according to any one of claims 1 to 21 , wherein the expressed proteins are purified before and / or after the step of measuring the level of the expressed protein.

23. A kit comprising: i. a circular nucleic acid template coding sequence for a protein of interest and a detection tag; ii. a population of cells suitable for expression proteins upon transfection with the nucleic acid template; and iii. a digital microfluidic device.

24. The kit according to claim 23 further comprising a reagent for transfection.