Improved fluorescent protein

Abstract

Provided herein are methods and compositions for detecting protein synthesis with a device using fluorescent proteins. The methods are suitable for monitoring on microfluidic devices.

Description

Field of the Invention

[0001] Provided herein are methods and compositions for detecting protein synthesis using fluorescent proteins in devices. The methods are suitable for monitoring on microfluidic devices. Background of the Invention

[0003] When cell-free protein synthesis is performed on a microfluidic scale in a microfluidic device, such as a digital microfluidic device, it is useful to detect proteins synthesized by the cell-free protein synthesis reaction in real time. However, it is difficult to perform real-time detection of proteins in a cell-free protein synthesis reaction environment. The reaction contains many other proteins and biomolecules in high concentrations, making it difficult to detect proteins by standard protein staining methods (e.g., Coomassie Brilliant Blue G-250, SYPRO TM Nonspecific protein detection using immunoprecipitation (e.g., Ruby, silver staining) is difficult. Immunostaining or affinity-based purification followed by nonspecific protein staining is also unhelpful because extensive washing must be performed on the solid support to prevent background interference. Background interference can become troublesome because washing is known to be difficult in microfluidic and digital microfluidic devices.

[0004] Currently available luminescence complementation methods cannot achieve long-term real-time monitoring in cell-free protein synthesis reactions, which often exceed 16 hours. This limitation is due to multiple reasons, including the 2 O that requires competing luminescent enzymes 2 Depletion and temporary or permanent depletion of luminescent substrate within 3-24 hours of recombinant protein expression detection.

[0005] The protein of interest can also be expressed as a fusion with a fluorescent protein, such as green fluorescent protein (GFP). However, GFP is a 26.9 kDa protein, which is the typical size of most fluorescent proteins. The label of this size increases the total size of the protein of interest, especially if the protein of interest must be labeled with other large fusion proteins, such as 42.5 kDa maltose binding protein (MBP). In view of the fact that the average size of human proteins is about 52 kDa, and the average size of E. coli proteins is about 35 kDa (Kim, YE et al., Annu. Rev. Biochem. 2013. 82: 323-355), adding a fluorescent protein label of comparable size will significantly change the biological function and biophysical properties of the protein.

[0006] Many prior arts disclose the use of subcomponent tags for monitoring expression in cell systems. For example, US Pat. No. 7,666,606 discloses a detection system for protein-protein interactions using microdomains.

[0007] Schinn et al., Biotechnol. Bioeng 11410, October 2017, 2412-2417 ( https: / / onlinelibrary.wiley.com / doi / 10.1002 / bit.2630 5) Publication of rapid in vitro screening of position-dependent effects of unnatural amino acids on protein expression and activity-Schinn-2017-Biotechnology and Bioengineering-Wiley Online Library.

[0008] Split green fluorescent protein (GFP) has been used for a variety of applications. ccGFP is a green fluorescent protein engineered from a tetrameric GFP found in Corynactis califomica, a bright red, colonizing coral polyp that resembles sea anemones and scleractinian stony corals, as described in Nguyen et al.; Nature Scientific Reports volume 11, Article number: 18440 (2021). The inventors herein have optimized the use of ccGFP for cell-free protein synthesis. SUMMARY OF THE INVENTION

[0010] The separate ccGFP variants can be used as a general method for detecting proteins of interest that contain ccGFP at either end of their coding sequence. 11 v1 peptide. A disadvantage of using the previously described split ccGFP system is the low solubility of the protein, necessitating purification of the protein from inclusion bodies and limiting the working concentration that can be used in detection assays.

[0011] The present invention describes herein improved ccGFP variant amino acid sequences and components thereof, nucleic acid sequences for their expression, and uses of improved fluorescent proteins. Mutations in strands adjacent to strand 11 can be modified to improve the properties of the protein. Beta-barrel strands 3 and 10 have H-bond interactions with strand 11. These changes should not disrupt GFP 1-10 :GFP 11 complementation, but may alter the exposed hydrophobic core of the split GFP, which may be responsible for the insoluble expression.

[0012] ccGFP 1-11 The wild-type amino acid sequence is 221 amino acids (chain 3 is underlined):

[0013]

[0014] Disclosed is a ccGFP having a mutation in the strand 3 region GQKTLKLRV (residues 40-48) 1-11 Sequence. Disclosed is a ccGFP having a mutation at position K45 1-11 Sequence, the mutation may be K45E. Disclosed is an amino acid sequence having position 45E having at least 90% homology to the following sequence:

[0015]

[0016]

[0017] This sequence can be used in separate form, for example as ccGFP 11 and ccGFP 1-10 Use. The sequence may be truncated so that amino acids can be removed from the N or C termini without affecting the activity or fluorescence of the folded core. When the sequence is truncated, the modified position may change numbering, and references to K45 refer to the 221 amino acid wild-type sequence. References to chain 3 refer to amino acids 40-48 of the 221 amino acid wild-type sequence.

[0018] Chain 3 amino acid changes produce promising amino acid change positions. An example of an amino acid position is K45. This can be changed to K45E. The chain 3 sequence can include GEQELELRV.

[0019] Describes the specific ccGFP 1-10 Variant K45E. Once combined with ccGFP 11 Mixed with ccGFP 11 Binding complements the missing beta sheet11, generating a detectable and quantifiable fluorescent signal. This enables the detection of ccGFP in vitro and in vivo 11 The tagged protein is similar to the original ccGFP 1-10 Compared to the parental ccGFP 1-10 The K45E protein variant showed improved solubility. The solubility could be further improved by adding a C-terminal MBP fusion protein.

[0020] The changes herein improve the physical properties of the protein (e.g. solubility; complementation rate) without affecting other properties (e.g. fluorescence color; sequence of separated chains, etc.). For example, the parent ccGFP 1-100The solubility is 5-10% when expressed. The mutation described herein increases it to about 55%. Adding MBP as a fusion protein even further improves solubility. The parent protein (27KDa) causes precipitation when the concentration exceeds 1mg / mL. Using the variants described herein, the mutant protein can be concentrated to 65mg / mL in its elution buffer without obvious precipitation. This higher concentration enables direct application of in situ (real-time and endpoint) detection in a small amount of liquid without the need for a large amount of final dilution to add enough protein for endpoint reading.

[0021] Here, we report the use of an improved split peptide system for fluorescence-based monitoring of the expression of a protein of interest in a cell-free protein synthesis reaction. Monitoring can be performed in real time, as well as providing endpoint measurements.

[0022] The present invention discloses a ccGFP variant comprising a K45E mutation. The variant may be ccGFP 1-10 Variant or ccGFP 1-11 Variant. The ccGFP 1-10 Variants can be used with ccGFP 11 Variant compound.

[0023] ccGFP 1-10 The K45E variant sequence may comprise the following sequence:

[0024]

[0025] Underlined E is the altered site of wild-type K

[0026]

[0027] The protein ccGFP K45E variant sequence may comprise a E Sequences with greater than 95% homology to:

[0028]

[0029] or its C-terminal or N-terminal truncations, which are similar to ccGFP 11 Bind to emit fluorescence.

[0030] The protein has a high solubility. Mutants can be used at a concentration greater than 10 mg / mL. Mutants can be used at a concentration greater than 50 mg / mL. Mutants can be used at a concentration greater than 65 mg / mL. The protein can be connected to another sequence, such as maltose binding protein (MBP), to further increase solubility.

[0031] Also disclosed are nucleic acid sequences encoding amino acid sequences having a K45E mutation.

[0032] The nucleic acid sequence may comprise:

[0033]

[0034] Also disclosed is a method for detecting a protein of interest, the method comprising taking a 11 The protein of interest is ccGFP 11 Binding to ccGFP 1-10 variant K45E and by detecting ccGFP from the assembly 1-11 The fluorescent signal of ccGFP is also disclosed. 1-10 Use of variant K45E as a soluble tag to facilitate soluble expression of a protein of interest.

[0035] Disclosed herein is a method for monitoring cell-free protein synthesis in droplets on a digital microfluidics device, comprising:

[0036] a. Cell-free transcription and translation with ccGFP 11 a peptide tag fused to a protein of interest; and

[0037] b. Use of an alternative ccGFP with K45E amino acid change 1-10 Peptide monitoring for the presence of a peptide tag, the addition of ccGFP 1-10 Peptide in the ccGFP 11 The presence of the peptide tag generates a detectable signal.

[0038] The terms "in vitro" and "cell-free" are used interchangeably herein.

[0039] Any in vitro transcription and translation, such as extract-based systems derived from rabbit reticulocyte lysate, human lysate, Chinese hamster ovary lysate, wheat germ lysate, HEK293 lysate, E. coli lysate, insect lysate, yeast lysate can be used.

[0040] Alternatively, in vitro transcription and translation can be assembled from purified components, such as the Purified Recombinant Elements system (PURE).

[0041] In vitro transcription and translation can be coupled or uncoupled.

[0042] The sequence may have greater than 90% homology to any of the sequences mentioned herein. The sequence may have greater than 95% homology to any of the sequences mentioned herein.

[0043] The variant sequence may contain greater than 95% homology to the following sequence:

[0044]

[0045] It has one or more amino acid changes.

[0046] The sequence may contain E Sequences with greater than 95% homology:

[0047]

[0048] Complementation of GFP 11 The amino acid sequence of the peptide may be as follows:

[0049] 1.KRDHMVLLEFVTAAGITGT

[0050] 2.KRDHMVLHEFVTAAGITGT

[0051] 3.KRDHMVLHESVNAAGIT

[0052] 4.RDHMVLHEYVNAAGIT

[0053] 5.GDAVQIQEHAVAKYFTV

[0054] 6.GDTVQLQEHAVAKYFTV

[0055] 7.GETIQLQEHAVAKYFTE

[0056] Or a truncated form thereof. The truncation may include shortening by up to 5 amino acids from the N-terminus, the C-terminus, or a combination thereof.

[0057] GFP 11 GFP 1-10 It can be fused to the protein of interest via an amino acid linker. In one embodiment, the oligopeptide, peptide or polypeptide linker can be 0-50 amino acids.

[0058] Also disclosed is a nucleic acid sequence for expressing a specific tag. The nucleic acid sequence includes

[0059]

[0060] These sequences can be repeated one or more times to generate multiple GFP 11 Domain-containing proteins.

[0061] Proteins can be fused to multiple tags. For example, a protein can be fused to multiple ccGFP 11 Peptide tag fusion and multiple ccGFP 1-10The protein of interest can be synthesized in the presence of one or more sfCherry 11 Peptide tag and one or more GFP 11 Peptide tag fusion and one or more GFP 1-10 Peptide and one or more sfCherry 1-10 Synthesis occurs in the presence of polypeptides.

[0062] Any protein of interest can be synthesized. The protein can be an enzyme, such as terminal deoxynucleotidyl transferase (TdT) or a truncated form thereof, or a homologous amino acid sequence of other species of terminal deoxynucleotidyl transferase (TdT), or a homologous amino acid sequence of any species of Polμ, Polβ, Polλ and Polθ, or a homologous amino acid sequence of any species of X family polymerase.

[0063] The synthesis can be performed in a microfluidic device, such as an electrowetting on dielectric (EWoD) device. Alternatively, the synthesis can be performed in a microtiter plate format. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 One embodiment of the present invention is schematically shown. The cell-free protein synthesis reaction comprises a nucleic acid template containing an expression cassette of a gene of interest fused to a detectable tag, which is then expressed into a protein of interest by coupled or uncoupled in vitro transcription and in vitro translation. Therefore, the protein of interest is fused to a detectable peptide tag at the N-terminus or C-terminus. The property of the detectable peptide tag is that it can complement a complementary polypeptide to produce a fluorescent protein.

[0066] Figure 2 PAGE gel showing the comparison of the ratio of proteins in the pellet (P) and the supernatant (SN).

[0067] Figure 3 From Figure 2 ccGFP in the gel 1-10 v3 - Graph of gel intensity without MBP. Variant sequence Cand 14 is more soluble than wild type.

[0068] Figure 4 From Figure 2 ccGFP in the lower gel 1-10 Gel strength of v3 (containing MBP). MBP fusion further enhanced solubility, with a larger soluble fraction for variant Cand 14.

[0069] Figure 5 Absorption spectra of protein K45E plus MBP are shown for the full-length 1-11 form and the assembled 1-10 plus 11 form. The absorbance traces are approximately the same, with the maximum absorbance for each protein at 500 nm.

[0070] Figure 6 Shown using ccGFP fused to MBP 1-10 Images of fluorescence signals in droplets during protein expression of variant K45E.

[0071] Figure 7 Display for detection of ccGFP 11 Complementation data for MBP recombinant protein. The signal from the detector is proportional to the amount of protein expressed. When the detector is in excess, the signal depends on ccGFP 11 The four bars on the left are the same because ccGFP 11 When ccGFP 11 In excess, the signal is limited by the amount of protein being detected. The four bars on the right are different and correspond to the presence of ccGFP 11 Proportional to the amount.

[0072] Figure 8 : Display ccGFP 1-11 The sequence and predicted domain structure of the protein. Chain 3 covers residues GEQELKLRV. Chain 10 covers residues HKIEHRLVR.

[0073] Fig. 9 : Truncated ccGFP 11 Effect of the sequence of the binding domain on the fluorescence signal (MBP-ccGFP 11 construct).

[0074] Fig.10 : Truncated ccGFP 11 Effect of the sequence of the binding domain on the fluorescence signal (MBP-ccGFP 11 construct).

[0075] Fig.11 : Experimental results of 24 different proteins expressed in droplets on an electrowetting on dielectric (EWoD) device in a reconstructed cell-free protein synthesis system. Each construct contained GFP 11 In the row labeled "Screening," GFP 1-10 The test substance is present from the beginning of expression. The row marked "end point" shows the absence of GFP 1-10 The detection substance was expressed for 10 hours, and then GFP was used 1-10 Fluorescence signal was detected for 5 hours of complementation of the substances. This experiment showed a significant difference between expression / complementation in the Screen BioInk compared to the "end point" assay. DETAILED DESCRIPTION OF THE INVENTION

[0077] Here, we report the use of an improved split peptide system for fluorescence-based monitoring of the expression of a protein of interest in a cell-free protein synthesis reaction. Monitoring can be performed on the device during expression and can therefore be used in real time or as an endpoint measurement.

[0078] Disclosed herein is a ccGFP variant comprising one or more mutations in a beta strand adjacent to strand 11. Disclosed herein is a ccGFP variant comprising one or more mutations in beta strands 3 or 10. The amino acid position may include K45, located in strand 3. Strand 3 is amino acids 40-48, sequence GQKTLKLRV ... 1-11 Sequence, which has a mutation in the chain 3 region GQKTLKLRV (residues 40-48).

[0079] Disclosed herein is a ccGFP variant comprising a K45E mutation. The variant may be a ccGFP 1-10 Variant or ccGFP 1-11 Variant. ccGFP 1-10 Variants can be used with ccGFP 11 Variant compound.

[0080] Disclosed herein is a method for monitoring cell-free protein synthesis in droplets on a digital microfluidics device, comprising:

[0081] a. With ccGFP 11 Cell-free transcription and translation of peptide-tag-fused proteins of interest; and

[0082] b. Use of an alternative ccGFP with K45E amino acid change 1-10 Peptide monitoring for the presence of a peptide tag, the addition of ccGFP 1-10 Peptide in ccGFP 11 The presence of the peptide tag produces a detectable signal.

[0083] Since ccGFP 1-10 The solubility of ccGFP is increased, so 1-10 In the presence of 11 Therefore, you can 1-11 Expression was monitored in real time during assembly. Alternatively, ccGFP 1-10 Can be co-expressed.

[0084] The peptide tag can be ccGFP 11 Tags. The sequence of tags can be selected from

[0085]

[0086] or a truncated form thereof.

[0087] The tag should contain at least the LQEHAVAK region. The tag length should be at least 13 amino acids.

[0088] The nucleic acid sequence used for the expression tag may be a truncated version of 5'GGTGAAACCATCCAGTTACAAGAACACGCCGTGGCCAAATATTTCACCGAA.

[0089] The tag sequence LQEHAVAK can be expressed using the sequence 5'TTACAAGAACACGCCGTGGCCAAA.

[0090] The protein sequences disclosed herein can be connected to additional elements to improve solubility. Variants can be connected to one or more solubility enhancing sequences. The solubility enhancing sequence can be a peptide sequence or a naturally occurring sequence. The solubility enhancing sequence can be selected from, for example, maltose binding protein (MBP), small ubiquitin-like modified protein (SUMO), glutathione S-transferase (GST) or thioredoxin (TRX). The tag can be connected to either the C-terminus or the N-terminus, or to both the C-terminus and the N-terminus. Any example of a solubility enhancer can be used. A list of possible proteins is shown below. Any sequence selected from the following list can be selected:

[0091]

[0092]

[0093]

[0094] Disclosed is the use of soluble ccGFP as a solubility enhancer during the expression process to increase the soluble yield of proteins expressed in a CFPS system. The presence of a specific solubility enhancer during the expression process increases the yield of soluble expression. Proteins can be expressed without synthesizing soluble factors as part of the amino acid sequence, and ccGFP 1-10 The detection agent can be used as a soluble factor after protein expression. Attaching a soluble factor by binding a tag means that the expressed protein is immediately soluble and there is no need to wait for the expressed sequence at the POI to fold correctly to enhance solubility. In addition, CFPS resources are not used to synthesize soluble tagged proteins.

[0095] Applicants have appreciated that the presence of solubility factors that rapidly bind to proteins after expression increases the soluble yield of certain proteins. 1-10 Method for increasing the soluble yield of a protein of interest (POI) expressed in a cell or cell-free expression system by expressing the POI in the presence of a moiety, wherein the highly soluble ccGFP 1-10 Partially binds to the binding sequence attached to the POI. ccGFP11 The binding sequence can be linked to the C-terminus or the N-terminus.

[0096] A method for cell-free expression of peptides or proteins in a digital microfluidics device is disclosed. Droplets with the components required for cell-free protein synthesis (CFPS), also known as in vitro protein synthesis, can be manipulated by electrokinesis to achieve and improve protein expression.

[0097] Electrowetting is the process of changing the wetting properties of a surface (which is generally hydrophobic) with an applied electric field. Microfluidic devices based on electrowetting manipulation of droplets or magnetic beads have been widely described. In the case of droplets in a channel, this can be achieved by allowing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by a barrel wall or microfluidic tube. Embedded in the barrel wall or tube wall are electrodes covered by a dielectric layer, each of which is connected to an A / C bias circuit that can be quickly opened and closed at intervals to change the electrowetting field characteristics of the layer. This produces the ability to guide droplets along a given path.

[0098] As an alternative to microfluidic channel systems, digital microfluidics (DMF) can also be used to generate and manipulate droplets on a flat surface. Compared with channel-based microfluidics, DMF uses alternating current on an electrode array to move fluid on the surface of the array. Therefore, liquids can be moved on an open device by electrowetting. Digital microfluidics enables precise control of the movement of droplets, including droplet fusion and separation.

[0099] 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, i.e., without the use of living cells. The in vitro protein synthesis environment is not limited to the cell wall, nor is it limited by the conditions necessary to maintain cell viability, and can rapidly produce any desired protein from a nucleic acid template, usually plasmid DNA or RNA from in vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis includes systems based on crude lysates (Cold Spring Harb Perspect Biol. 2016 Dec; 8 (12): a023853) and systems based on recombinant, purified molecular reagents, such as the PURE system for producing proteins (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires high concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, etc. (Febs Letters 2013, 2, 58, 261-268).

[0100] To date, digital microfluidics, electrowetting on dielectrics (EWoD) and electrokinesis in general have found only limited use in cell-free based biological applications, primarily due to biofouling, where biological components such as proteins, nucleic acids, crude cell extracts and other biological products adsorb and / or denature to hydrophobic surfaces. It is well known in the art that biofouling limits the ability of EWoD devices to manipulate droplets containing biomacromolecules. Wheeler et al. reported a maximum actuation time of 30 minutes for droplets on an EWoD device containing biological media before biofouling inhibited EWoD-based droplet actuation (Langmuir 2011, 27, 13, 8586-8594).

[0101] Digital microfluidics can be performed in air-filled systems, where droplets are manipulated on surfaces in the air. However, at elevated temperatures or over extended periods of time, volatile aqueous droplets dry onto the surface only by evaporation. This problem is complicated by the high surface area to volume ratio of nanoliter and microliter sized droplets. Therefore, 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 synthesis needs to be extended so that the synthesized protein level can be detected.

[0102] Protein expression generally requires an adequate oxygen supply. The most convenient and high-yield way to power CFPS is via oxidative phosphorylation, in which O 2 however, there are other ways that involve supplementation with energy molecules that do not participate in oxidative phosphorylation. In the confined microfluidics of droplets or digital microfluidics systems, there is not enough oxygen available to achieve efficient protein synthesis.

[0103] Described herein is an improved method for achieving cell-free expression of peptides or proteins in a digital microfluidic device. A method for cell-free expression of peptides or proteins in a microfluidic device is included, wherein the method includes one or more droplets containing a nucleic acid template (i.e., DNA or RNA) and a cell-free system having components for expressing proteins in an oil-filled environment, and using electrokinesis to move the droplets. The components for cell-free protein synthesis droplets can be pre-mixed before introduction into the digital microfluidic device, or mixed on the digital microfluidic device.

[0104] The droplets can be repeatedly moved for a period of at least 30 minutes while expressing the protein. The droplets can be repeatedly moved for a period of at least two hours while expressing the protein. The droplets can be repeatedly moved for a period of at least 12 hours while expressing the protein. The action of moving the droplets causes oxygen to be supplied to the droplets and dispersed throughout the droplets. The action of moving improves protein expression levels compared to droplets that remain stationary.

[0105] Any electrical motion means may be used to move the droplets. The droplets may be moved using electrowetting on dielectrics (EWoD). The electrical signals on an EWoD or optical EWoD device may be delivered through segmented electrodes, active matrix thin film transistors, or digital micromirrors.

[0106] The oil in the device can be any water-immiscible liquid. The oil can be a mineral oil, a silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The oil can be oxygenated before or during the expression process. Alternatively, the device can be an air-filled device in which droplets containing cell-free protein synthesis reagents are quickly moved to a position and fixed to an array under a humidified gas to prevent evaporation. Humidification can be achieved by closing or sealing the digital microfluidic device and providing a reagent reservoir on board. In addition, humidification can be achieved by connecting an aqueous reservoir to a closed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration to provide a specific relative humidity (e.g., a saturated potassium sulfate solution at 30°C).

[0107] The source of supplementary oxygen can be supplied to the droplets. For example, during protein expression, droplets or bubbles containing gaseous or dissolved oxygen can be merged with the droplets. In addition, the source of supplementary oxygen can be obtained by oxygenating the oil used as a filler medium. It is well known in the art that oils, such as hexadecane, HFE-7500, etc. can be oxygenated to support cell growth, particularly the oxygen demand of E. coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air. Alternatively, the source of oxygen can be a molecular source that releases oxygen. Alternatively, the droplets can be moved to the air / liquid boundary to achieve an increase in the diffusion of oxygen from the gaseous environment. Alternatively, the droplets can be present in a humidified air-filled device.

[0108] Droplets can be formed prior to entering a microfluidic device and flow into the device. Alternatively, droplets can be merged on the device. A method is included that includes merging a first droplet containing a nucleic acid template, such as a plasmid, with a second droplet containing a cell-free extract having components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0109] The droplet can be split on the device before or after expression. Included herein is a method further comprising splitting the aqueous droplet into a plurality of droplets. If desired, the split droplets can be screened with other additives. Included is a method wherein one or more split droplets are merged with an additive droplet for screening.

[0110] The cell-free expression of peptide or protein can use the cell lysate with the reagent realizing protein expression.The common components of cell-free reaction include energy source, amino acid supply source, auxiliary factor such as magnesium, and relevant enzyme.Cell extract is obtained by cracking the cell of interest and removing cell wall, DNA genome and other fragments by centrifugation.The rest are cell machinery, including ribosome, aminoacyl-tRNA synthetase, translation initiation factor and elongation factor, nuclease etc.Once adding suitable nucleic acid template, the expression machine of cell origin can be used to express nucleic acid template as peptide or protein.

[0111] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates for CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates that can be produced in cells or produced by synthesis. LETs can be prepared via PCR. Although LETs are easier and faster to prepare, the yield of plasmids is generally higher in CFPS. mRNA can be produced by an in vitro transcription system. The method uses a single nucleic acid template per droplet. The method can use multiple droplets with different nucleic acid templates per droplet.

[0112] The energy source is an important part of the cell-free reaction. Typically, a separate mixture containing the required energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. During expression, the energy source can be supplemented by adding other reagents to the droplets during the process.

[0113] The cell-free extract with components for protein expression contains everything required for protein expression except the nucleic acid template. Therefore, the term includes all related ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, the expression of the protein begins without the need for other reagents.

[0114] Therefore, before adding the template, the cell lysate can be supplemented with additional reagents. Cell-free extracts with components for protein expression will generally be made as bulk reagents or 'reaction mixtures (master mix)', which can be formulated into many identical droplets before different templates are added to different droplets respectively. Common cell extracts currently used are made from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Kluyveromyces yeast (D2P system). All of these extracts are commercially available.

[0115] Cell-free systems can be assembled from the required reagents, rather than from cell extracts. Systems based on reconstructed, purified molecular reagents are commercially available, such as the PURE system for making proteins, which can be used as provided. The PURE system consists of all enzymes involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reactions of the PURE system lack proteases and ribonucleases, which are often present as undesirable molecules in cell extracts.

[0116] The term digital microfluidic device refers to a device with a two-dimensional array of planar microelectrodes. The term excludes any device that only has droplets in an oil stream in a channel. By activating specific electrodes, the droplets are moved on the surface by electrodynamic forces. After electrode activation, the dielectric layer becomes less hydrophobic, allowing the droplets to spread onto the surface. The arrangement of digital microfluidic (DMF) devices is known in the art and depends on the substrate used, the electrodes, the configuration of those electrodes, the use of dielectric materials, the thickness of the dielectric material, the hydrophobic layer, and the applied voltage.

[0117] Once the CFPS reagents have been enclosed in a droplet, additional reagents can be provided by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including, for example, an oxygen or 'power' source, or a test agent that is desired to be exposed to the expressed protein.

[0118] The droplets may be aqueous droplets. The droplets may contain an oil-immiscible organic solvent, such as, for example, DMSO. The droplets may be a mixture of water and a solvent, provided that the droplets do not dissolve into the bulk oil.

[0119] The droplets can be in a bulk oil layer. During expression, the dry gas environment simply dries the bubbles to the surface, leaving a comet-like streak of dry material by evaporation. Therefore, the device is used for liquid filling of the expression process. Alternatively, the aqueous droplets can be in a humidified gas environment. The air-filled device can be sealed and humidified to provide an environment that reduces evaporation of the CFPS droplets.

[0120] Thus, droplets containing a cell-free extract with components for protein expression will generally be in an oil-filled environment before the nucleic acid template is added to the droplets. The template can be added by merging droplets on the microfluidic device. Alternatively, the template can be added to droplets outside the device and then flowed into the device for the expression process. For example, the expression process can be initiated on the device by increasing the temperature. Expression systems generally operate best at temperatures above standard room temperature, such as at or above 29°C.

[0121] The expression process generally takes several hours. Therefore, the process should be maintained for at least 30 minutes or 1 hour, generally at least 2 hours. Expression can be maintained for at least 12 hours. During the expression process, the droplets should be moved within the device. Movement can improve the process by mixing the reagents and ensuring that there is enough oxygen available within the droplets. Movement can be continuous, or it can be repeated with intermediate time periods without movement.

[0122] Thus, while expressing a protein, the aqueous droplets may be repeatedly moved for a period of at least 30 minutes or 1 hour. While expressing a protein, the aqueous droplets may be repeatedly moved for a period of at least two hours. While expressing a protein, the aqueous droplets may be repeatedly moved for a period of at least 12 hours. The action of moving the droplets enables mixing within the droplets and enables the supply of oxygen or other agents to the droplets. The action of moving improves protein expression levels compared to droplets that remain stationary.

[0123] A method is disclosed which comprises transcribing a nucleic acid sequence comprising:

[0124]

[0125] This sequence can be repeated to express multiple ccGFP 11 Amino acid labeling. Transcription can be performed in droplets on a digital microfluidics device. When containing (multiple) ccGFP 11 Tag the expressed protein with ccGFP 1-10 When the proteins are complementary, a fluorescent signal is obtained.

[0126] Digital microfluidics (DMF) refers to a two-dimensional planar platform for a lab-on-a-chip system based on the manipulation of microdroplets. Droplets can be distributed, moved, stored, mixed, reacted or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used with analytical analysis processes such as mass spectrometry, colorimetry, electrochemistry and electrochemiluminescence.

[0127] Droplets can be moved using any means of electrical motion. Aqueous droplets can be moved using electrowetting on dielectrics (EWoD). Electrowetting on dielectrics (EWoD) is a variation of the electrowetting phenomenon based on dielectric materials. In the EWoD process, a droplet of a conductive liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes, the dielectric layer becomes less hydrophobic, causing the droplet to spread over the surface.

[0128] 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 and are used to drive droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0129] The oil in the device can be any water-immiscible or hydrophobic liquid. The oil can be a mineral oil, a silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The air in the device can be any humidified gas.

[0130] Through affinity tags, such as FLAG-tags, HIS-tags, GST-tags, MBP-tags, STREP-tags or other forms of affinity tags, CFPS expressed proteins can be immobilized on solid support affinity resins, and fresh batches of CFPS reagents can be delivered on the resin. Therefore, updated reagents can be used to perform protein synthesis, carefully mimicking the industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS manufacturing methods.

[0131] Droplets can be driven on hydrophobic surfaces on digital microfluidic devices (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), TeflonAF (DuPont Inc), CYTOP (AGCChemicals Inc) or FluoroPel (Cytonix LLC). The hydrophobic surface can be modified in such a way as to reduce biofouling, particularly biofouling caused by exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface can also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Compared with general fluorocarbon-based hydrophobic surfaces, superhydrophobic surfaces prevent biofouling. Therefore, superhydrophobic surfaces extend the ability of digital microfluidic devices to move CFPS 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 a porous PTFE membrane with Krtox-103 oil (DuPont) (Lab Chip, 2019, 19, 2275).

[0132] The droplets may also contain additives to reduce the effects of biofouling on the digital microfluidic surface. Specifically, the droplets containing the CFPS component may also contain additives, such as surfactants or detergents, to reduce the effects of biofouling on the hydrophobic or superhydrophobic surfaces of the 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, the droplets containing the CFPS component may contain 0.1% volume / volume TWEEN20, 0.1% volume / volume Triton X-100, and / or 0.05% weight / volume Pluronic F127.

[0133] For electrowetting on dielectrics (EWoD), the change in contact angle of the reagent after application of an electric potential is an inverse function of the surface tension. Therefore, for low voltage EWoD runs, the reduction in surface tension is achieved by adding a surfactant to the reagents, which for CFPS reactions means adding to the lysate and adding to the DNA. This results in dilution of the lysate, and it has been seen in experiments that dilution or otherwise doping the lysate results in reduced expression levels of the protein of interest. Therefore, performing CFPS on DMF, in which a surfactant is added to the solution being moved, will inevitably result in dilution and doping of the lysate, and therefore reduced protein expression levels. In addition to being a problem in itself, this further complicates the extrapolation of results on DMF to protein yield predictions in the tube. Another disadvantage of having to add a surfactant to the sample is that it increases the time required for sample preparation and also increases the possibility of inconsistent results due to 'user error' because there is more reagent handling. Another disadvantage of having to add a surfactant to the sample is that certain downstream operations are hampered. For example, if the protein of interest is mixed with GFP in a cell-free system, 11 (or similar) peptide tags, whose downstream complementarity with GFP1-10 (or similar) detector polypeptides is hampered in the presence of surfactants. Figure 8 and Fig. 9 Therefore, it is advantageous to remove the surfactant from the aqueous phase.

[0134] Instead of adding a surfactant to the aqueous sample, a surfactant, such as a sorbitan ester, such as Span85 (e.g., Sorbitan trioleate, Sigma Aldrich, SKU8401240025), can be added to the oil. This has the advantage of allowing the CFPS reaction to be carried out in DMF without dilution or doping. In addition, it simplifies the sample preparation process for setting up the reaction, improving ease of use and consistency of results. Using 1% weight / weight Span85 in dodecane enables undiluted CFPS reactions in DMF and undiluted detection of expressed non-fluorescent proteins. Other surfactants besides Span85 and oils besides dodecane can be used. A range of concentrations of Span85 can be used. The surfactant can be nonionic, anionic, cationic, amphoteric, or a mixture thereof. The oil can be a mineral oil or a synthetic oil, including silicone oil, petroleum oil, and perfluorinated oil. Surfactants can have an adverse effect on (1) the CFPS reaction and (2) the efficiency of the detection system (if the detection system involves complementation of the label and the detectable substance). For example, by performing the CFPS reaction on DMF with an oil-surfactant mixture, detection of the expressed protein can also be performed without dilution and without the addition of aqueous surfactant. Surfactants have been shown to reduce the expression of some detection systems, including but not limited to separate ccGFP (e.g., ccGFP 11 / ccGFP 1-10 ) system, it would be beneficial to remove the surfactant from the reagent mixture and instead add it to the oil.

[0135] The peptide tag can be attached to the C-terminus or N-terminus of the protein. The protein can be fused to multiple tags. For example, the protein can be fused to multiple ccGFP 11 peptide tags, and synthesized in multiple ccGFP 1-10 occurs in the presence of polypeptides.

[0136] After expression, the protein can be purified on the device. A method is disclosed, comprising

[0137] a. Take a digital microfluidic device having a planar electrode array;

[0138] b. synthesizing a protein of interest with a bound tag in a droplet on the device;

[0139] c. Capturing the protein via the binding tag, thereby immobilizing the protein;

[0140] d. Using electrodes to move the droplets, thereby removing the synthesized protein from the droplets;

[0141] e. optionally washing the immobilized protein; and

[0142] f. Optionally release the protein into other droplets.

[0143] The binding moiety tag can be a subcomponent of the fluorescent protein. Thus, the fully assembled protein becomes fluorescent. For example, if the fixed material contains ccGFP 1-10 , tagged with ccGFP 11 The peptides complement each other to form an immobilized fluorescent ccGFP, allowing for simultaneous monitoring and purification. The immobilized material can be washed and then eluted by disrupting the complementary ccGFP, for example by using salt or temperature. Alternatively, ccGFP can be removed using a protease, such as TEV or 3C protease. 11 Label.

[0144] The protease may be selected from the following: TEV, C3, enterokinase (EK) light chain, factor Xa (FXA), furin (FN) or thrombin. Enterokinase (EK) cleaves the NNNNL motif. Factor Xa cleaves the I(E / D)GR motif. Furin cleaves the RXXR motif. Thrombin cleaves the LVPRGS motif. TEV protease is a cysteine ​​protease that recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly / Ser) and cleaves between the Gln and G1y / Ser residues. C3 protease is a cysteine ​​protease that recognizes Leu-Glu-Val-Leu-Phe-Gln / Gly-Pro (LEVLFQ / GP) and cleaves between the Gln and Gly-Pro residues.

[0145] A method is disclosed, comprising:

[0146] a. Take a digital microfluidic device having a planar electrode array;

[0147] b. Synthesis of one or more ccGFPs in droplets on the device 11 Label the protein of interest;

[0148] c. Via ccGFP 11 The tag captures the protein, thereby immobilizing it;

[0149] d. Using electrodes to move the droplets, thereby removing the synthesized protein from the droplets;

[0150] e. optionally washing the immobilized protein; and

[0151] f. Optionally release the protein into other droplets.

[0152] Fixed ccGFP can be used 1-10 Once fixed via complementation, the protein becomes fluorescent. Therefore, correctly expressed proteins can be identified by binding to the fixed ccGFP1-10 Complex to monitor and purify.

[0153] Affinity tags can be attached to proteins so that they can be purified from their crude biological source using affinity techniques. The purification tag can be selected from, for example, a FLAG tag, a His tag, a GST tag, a MBP tag, a STREP tag. The His tag, also known as the DYKDDDDK tag, is a popular protein tag commonly used in affinity chromatography and protein research. The His tag is a polyhistidine amino acid chain, usually 6 to 9 histidine amino acids in length.

[0154] The binding moiety used for purification may contain four or more amino acids. The binding sequence may contain 4-30 amino acids. The binding moiety may be selected from:

[0155] α-Tag (SRLEEELRRRITE)

[0156] Avi-Tag (GLNDIFEAQKIEWHE)

[0157] C-Tag (EPEA)

[0158] Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL)

[0159] Dogtag(DIPATYEFTDGKHYITNEPIPPK)

[0160] E-Tag (GAPVPYPDPLEPR)

[0161] FLAG(DYKDDDDK)

[0162] G4T(EELLSKNYHLENEVARLKK)

[0163] HA(YPYDVPDYA)

[0164] His(HHHHHH)

[0165] Isopeptide tag (TDKDMTITFTNKKDAE)

[0166] Lanthanide Binding Tag (LBT) (FIDTNNDGWIEGDELLLEEG)

[0167] Myc(EQKLISEEDL)

[0168] NE-Tag(TKENPRSNQEESYDDNES)

[0169] Polyglutamic acid tag (EEEEEEEE)

[0170] Poly-arginine tag (RRRRRRRR)

[0171] Rho1D4-tag (TETSQVAPA)

[0172] SBP-Tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)

[0173] Sdytag(DPIVMIDNDKPIT)

[0174] SH3(STVPVAPPRRRRG)

[0175] SNAC(GSHHW)

[0176] Snooptag (KLGDIEFIKVNK)

[0177] Softag 1(SLAELLNAGLGGS)

[0178] Softag 3(TQDPSRVG)

[0179] Spot-Tag (PDRVRAVSHWSS)

[0180] Spytag(AHIVMVDAYKPTK)

[0181] S-Tag (KETAAAKFERQHMDS)

[0182] Streptavidin-tag (AWAHPQPGG) (AWRHPQFGG)

[0183] Streptavidin-tag II (WSHPQFEK)

[0184] T7 tag (MASMTGGQQMG)

[0185] TC-Tag (EVHTNQDPLD)

[0186] Ty-Tag (CCPGCC)

[0187] VSV-Tag (YTDIEMNRLGK)

[0188] Xpress-Tag (DLYDDDDK)

[0189] Following expression, the protein can be purified, for example, by binding to particles such as magnetic beads.

[0190] The species of ccGFP 1-10Can be linked to a purification tag to allow purification of the expressed protein. 1-10 A soluble tag can be attached to further enhance the solubility of the expressed protein. 1-10 , which is connected to an affinity purification tag and a soluble tag. The soluble tag can be selected from:

[0191]

[0192]

[0193]

[0194] The purification tag can be selected from:

[0195] α-Tag (SRLEEELRRRITE)

[0196] Avi-Tag (GLNDIFEAQKIEWHE)

[0197] C-Tag (EPEA)

[0198] Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL) Dogtag (DIPATYEFTDGKHYITNEPIPPK)

[0199] E-Tag (GAPVPYPDPLEPR)

[0200] FLAG(DYKDDDDK)

[0201] G4T(EELLSKNYHLENEVARLKK)

[0202] HA(YPYDVPDYA)

[0203] His(HHHHHH)

[0204] Isopeptide tag (TDKDMTITFTNKKDAE)

[0205] Lanthanide Binding Tag (LBT) (FIDTNNDGWIEGDELLLEEG) Myc (EQKLISEEDL)

[0206] NE-Tag(TKENPRSNQEESYDDNES)

[0207] Polyglutamic acid tag (EEEEEEEE)

[0208] Poly-arginine tag (RRRRRRRR)

[0209] Rho1D4-tag (TETSQVAPA)

[0210] SBP-Tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)

[0211] Sdytag(DPIVMIDNDKPIT)

[0212] SH3(STVPVAPPRRRRG)

[0213] SNAC(GSHHW)

[0214] Snooptag (KLGDIEFIKVNK)

[0215] Softag 1(SLAELLNAGLGGS)

[0216] Softag 3(TQDPSRVG)

[0217] Spot-Tag (PDRVRAVSHWSS)

[0218] Spytag(AHIVMVDAYKPTK)

[0219] S-Tag (KETAAAKFERQHMDS)

[0220] Streptavidin-tag (AWAHPQPGG) (AWRHPQFGG)

[0221] Streptavidin-Tag II (WSHPQFEK)

[0222] T7 tag (MASMTGGQQMG)

[0223] TC-Tag (EVHTNQDPLD)

[0224] Ty-Tag (CCPGCC)

[0225] VSV-Tag (YTDIEMNRLGK)

[0226] Xpress-Tag (DIYDDDDK)

[0227] Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to an affinity polyhistidine purification tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a P17 soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3 linked to a NEXT soluble tag1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a Fh8 soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a Trx soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a Mocr soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3 linked to a SNUT soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a GST soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3 linked to a CusF soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to an MBP soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a ZZ soluble tag 1-10 Disclosed herein is a ccGFP having one or more mutations in strand 3, linked to a polyHis purification tag and a solubility tag 1-10 In each case, the species of ccGFP may have a modification at K45, such as K45E.

[0228] This article discloses ccGFP 11 A plurality of detection agents are combined, which comprises the following sequence:

[0229] P17

[0230]

[0231] NEXT

[0232]

[0233] Fh8

[0234]

[0235] Trx

[0236]

[0237]

[0238] Mocr

[0239]

[0240] SNUT

[0241]

[0242] GST

[0243]

[0244]

[0245] MBP

[0246]

[0247] FWf

[0248]

[0249] ZZ

[0250]

[0251] The present invention discloses a nucleic acid sequence for expressing the type detection agent, which includes the following nucleic acid sequence:

[0252] MBP

[0253]

[0254]

[0255] P17

[0256]

[0257] NEXT

[0258]

[0259]

[0260] Fh8

[0261]

[0262]

[0263] MBP

[0264]

[0265]

[0266] TRX

[0267]

[0268] MOCR

[0269]

[0270] SNUT

[0271]

[0272]

[0273] GST

[0274]

[0275]

[0276] FWf

[0277]

[0278]

[0279] ZZ

[0280]

[0281] Device

[0282] Manipulation of droplets by applying an electric potential can be achieved on electrodes covered with an insulator or dielectric or a series of insulators or dielectrics. Manipulation of droplets caused by an applied electric potential is called electrowetting. The electromotion occurs due to a non-uniform electric field that affects the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on a solid surface (electrowetting on dielectrics or EWoD). DEP can also be used to generate forces on polarizable particles to induce their movement. The electrical signal can be transmitted to discrete electrodes, transistors, transistor arrays, or semiconductor membranes, whose electrical properties can be modulated by optical signals.

[0283] The EWoD phenomenon occurs when a droplet is driven between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in surface tension, which causes the droplet to move due to a change in the droplet contact angle. The electrowetting effect can be quantitatively treated using the Young-Lippmann equation:

[0284] cosθ-cosθ 0 =(1 / 2γLG)cV 2

[0285] Among them, θ 0 is the contact angle when the electric field across the interfacial layer is zero, γLG is the liquid-gas tension, and c is the specific capacitance (as ε r .ε0 / t is given by, where ε r is the dielectric constant of the insulator / dielectric, ε 0 is the permittivity of a vacuum, t is the thickness), and V is the applied voltage or potential. Therefore, the change in contact angle (induced droplet movement) is a function of surface tension, potential, dielectric thickness, and dielectric constant.

[0286] When a droplet is driven by an EWoD, two sets of opposing forces act on it: the electrowetting force induced by the electric field and the drag force, which consists of the drag force caused by the interaction of the droplet with the filler dielectric and the contact line friction (reference). The minimum voltage (threshold voltage) applied to balance the electrowetting force with the sum of all the drag forces is determined by the thickness of the insulator / dielectric and the dielectric contact ratio (t / ε). r ) 1 / 2 Therefore, in order to reduce the driving voltage, it is necessary to reduce (t / ε r ) 1 / 2 (i.e. increasing the dielectric constant or reducing the thickness of the insulator / dielectric). 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 droplets. Therefore, most academic studies report the use of much higher voltages of >100V on easily manufactured thick dielectric films (>3 microns) to achieve electrowetting.

[0287] However, high voltage EWoD-based devices with thick dielectric films have limited industrial applicability, primarily due to their limited droplet multiplexing capabilities. The use of low voltage devices including thin film transistors (TFTs) and optically activated amorphous silicon layers (a-Si) has paved the way for industrial applications of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplexed manner. The driving voltage of TFTs or optically activated a-Si is low (typically <15V). A bottleneck in the manufacture and therefore adoption of low voltage devices has been the technical challenge of depositing high quality thin film insulators / dielectrics. Therefore, there has been a particular need to improve the manufacture and composition of thin film insulator / dielectric devices.

[0288] Typically, electrodes (or array elements) for EWoDs are covered with either (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings include fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of such materials as hydrophobic coatings on dielectrics is generally <100 nm and may have defects in the form of pinholes or porous structures; therefore, it is particularly important that the insulator / dielectric is pinhole-free to avoid electrical shorts. Teflon has also been used as an insulator / dielectric, but has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole-free. Other hydrophobic insulator / dielectric materials may include polymer-based dielectrics such as those based on silicones, epoxies (such as SU-8), or polyparaxylene (e.g., polyparaxylene N, polyparaxylene C, polyparaxylene D, or polyparaxylene HT). Teflon is still used as a hydrophobic outer coating for these insulator / dielectric polymers due to minimal contact angle hysteresis and large contact angles with aqueous solutions. However, there are difficulties in reliably producing pinhole-free coatings of parylene or SU-8 <1 micron; therefore, the thickness of these materials is generally kept at 2-5 microns, at the expense of increased electrowetting voltage requirements. It has also been reported that conventional EWoD devices with parylene C are prone to breakage and instability for repeated droplet manipulations using cell culture media. Multilayer insulator devices deposited with metal oxides and parylene C films have been used to make more robust insulators / dielectrics and can operate at lower applied voltages. Inorganic materials, such as metal oxides and semiconductor oxides, which are commonly used as "gate dielectrics" in the CMOS industry, have been used as insulators / dielectrics for EWoD devices. They offer the advantage of thin film deposition (<100 nm) using standard clean room methods. These materials are inherently hydrophilic, require additional hydrophobic coatings, and are prone to pinhole formation due to the thin film layer deposition process. Together with the need for lower voltage operation of EWoD, recent development efforts have focused on (1) using materials with improved dielectric properties (e.g., using insulators / dielectrics with high dielectric constants), and (2) optimizing manufacturing methods to make the insulators / dielectrics pinhole-free to avoid dielectric breakdown.

[0289] The operation of EWoD devices is affected by contact angle saturation and hysteresis, which is believed to be caused by one or a combination of the following phenomena: (1) charge entrapment in the hydrophobic membrane or insulator / dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instability, (4) dielectric breakdown of the dielectric layer, (5) electrode-electrode-insulator interface capacitance (caused by double layer effects), and (6) fouling of the surface (such as fouling of the surface by biomacromolecules). One of the adverse effects of such hysteresis is the reduction of the operational lifetime of EWoD-based devices.

[0290] Contact angle hysteresis is believed to be the result of charge accumulation at the interface or within the hydrophobic insulator after a few runs. Due to this charging phenomenon, the required driving voltage increases, leading to an eventual catastrophic dielectric breakdown. The most likely explanation is that pinholes at the insulator / dielectric may allow the liquid to come into contact with the electrode, leading to electrolysis. Hydrophobic insulators that are prone to pinholes or porosity further promote electrolysis.

[0291] Most studies used to understand contact angle hysteresis on EWoD are based on short time scales and conducted with low conductivity solutions. Long duration actuation (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCl) may produce several effects other than electrolysis. Ions in solution can permeate through the hydrophobic coating (under an applied electric field) and interact with the underlying insulator / dielectric. Ion penetration can lead to (1) changes in the dielectric constant due to charge trapping (which is different from interfacial charging) and (2) changes in the surface potential of the pH-sensitive metal oxide. Both can lead to a reduction in the electrowetting force that manipulates aqueous droplets, resulting in contact angle hysteresis. The inventors have previously discovered that damage from high conductivity solutions reduces electrowetting on the electrode by inhibiting modulation of the contact angle when an electric field is applied, or makes electrowetting impossible on the electrode.

[0292] An electrokinetic device includes a first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating, the 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 including a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and a voltage source operably coupled to the matrix electrodes.

[0293] The dielectric layer may include 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 thickness of the dielectric layer may be 10nm to 100μm. A combination of more than one material may be used, and the dielectric layer may include more than one sublayer, which may be made of different materials.

[0294] The conformal layer may comprise parylene, siloxane or epoxy. It may be a thin protective parylene coating between an insulating dielectric and a hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In the present invention, the reason for depositing parylene is not to improve insulating / dielectric properties, such as reducing pinholes, but as a conformal layer between a dielectric layer and a hydrophobic layer. The inventors have found that, in contrast to other similar insulating coatings of the same thickness, such as PDMS (polydimethylsiloxane), parylene prevents contact angle hysteresis caused by high conductivity solutions or solutions that deviate from neutral pH for a long time. The thickness of the conformal layer may be from 10 nm to 100 μm.

[0295] The hydrophobic layer may comprise a fluoropolymer coating, a fluorinated silane coating, a manganese oxide polystyrene nanocomposite, a zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, a carbon nanotube structure, a silica nanocoating, or a slippery liquid-infused porous coating.

[0296] The element may include one or more of a plurality of array elements, each element containing element circuitry; discrete electrodes; a thin film semiconductor whose electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.

[0297] The functional coating may comprise 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.

[0298] The electro-motion device may include a controller for regulating the voltage supplied to the individual matrix electrodes. The electro-motion device may include a plurality of scan lines and a plurality of gate lines, wherein each thin film transistor is coupled to a scan line and a gate line, and the plurality of gate lines are operably connected to the controller. This enables all individual elements to be individually controlled.

[0299] The second substrate may further include a second hydrophobic layer disposed on the second electrode. The first substrate and the second substrate may be disposed such that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining an electromotion working space between the hydrophobic layers.

[0300] The method is particularly suitable for use with aqueous droplets having a volume of 1 μl or less.

[0301] 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 surface coating. These devices are based on devices described in E Ink's patent application entitled "Digital microfluidic device including dual substrates with thin film transistors and capacitive sensing", which is filed as U.S. Patent Application No. 2019 / 0111433 and is incorporated herein by reference.

[0302] An electrokinetic device is described herein, comprising:

[0303] A first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating comprising:

[0304] A dielectric layer in contact with the matrix electrodes,

[0305] a conformal layer in contact with the dielectric layer, and

[0306] a hydrophobic layer in contact with the conformal layer;

[0307] a second substrate including a top electrode;

[0308] A spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0309] A voltage source is operably coupled to the matrix electrodes.

[0310] An electrokinetic device is described herein, comprising:

[0311] A first substrate having an electrode matrix, wherein each matrix electrode is coupled to a thin film transistor, and wherein the matrix electrodes are coated with a functional coating comprising:

[0312] one or more dielectric layers comprising silicon nitride, hafnium oxide, or aluminum oxide in contact with the matrix electrode, a conformal layer comprising parylene in contact with the dielectric layer, and

[0313] a hydrophobic layer in contact with the conformal layer;

[0314] a second substrate including a top electrode;

[0315] A spacer disposed between the first substrate and the second substrate and defining an electrokinetic working space; and

[0316] A voltage source is operably coupled to the matrix electrodes.

[0317] The electrokinetic device may be used with other components such as, for example, means for heating and cooling the device or kits for introducing reagents as needed. Example

[0318] Preparation and analysis of amino acid variants

[0319] Generation of ccGFP 1-10 v3 K45E and ccGFP 1-10v3 (WT) plus and minus the starter culture of MBP. Colonies were selected and incubated overnight at 37°C. The material was lysed and centrifuged at 21000rcf for 10 minutes at 20°C. The supernatant (SN) containing soluble protein was removed. The pellet (P) was resuspended and transferred to a new tube ((insoluble protein)) using a needle and syringe. A protein gel assay was performed to measure the soluble (SN) and insoluble fractions (P):

[0320] Mix 5 μL of each soluble and insoluble sample with 5 μL of protein loading buffer + DTT

[0321] Heat the sample to 95°C for 5 minutes

[0322] Transfer 4 μL of each sample to PAGE

[0323] Perform PAGE at 250 V for 20 min

[0324] • Stain the gel in Coomassie Brilliant Blue for 20-30 minutes.

[0325] The sample bands of PAGE under four conventional conditions were quantified using GelAnalyzer.

[0326] The supernatant and pellet of each sample were added to 100% and the relative ratios were calculated. Figure 2 Shown in.

[0327] ccGFP 1-10 v3(no MBP)( Figure 3 )

[0328] Sediment Soluble part Sediment standard deviation Solubility standard deviation Cand.14 69.1％ 30.9％ 0.26％ 1.64％ WT 78.8％ 21.2％ 22.85％ 2.80％

[0329] Increased solubility Cand.14 45.6％

[0330] ccGFP 1-10 v3-MBP( Figure 4 )

[0331]

[0332]

[0333] Increased solubility Cand.14 41.8％

[0334] ccGFP 1-10 The v3 K45E variant is more soluble than v3 "WT". C-terminal MBP fusion proteins with a rigid α-helical linker are also generally more soluble than the free protein alone. ccGFP 1-10The soluble fraction of v3K45E was 30-40%, while the MBP fusion protein of the variant was 60-75%, which has the advantage of detergent fraction. Changing K45 to E45 increased the solubility by about 40%. MBP fusion ccGFP has been prepared 1-10 v3 K45E (NDET06) was as high as 130 mg / mL without any signs of precipitation.

[0335] CFPS was performed using a variant on an electrowetting device.

[0336] method

[0337] To analyze the variant MBP-K45E ccGFP 1-10 To investigate the detection properties of (NDET06), an electrowetting device (eDrop) experiment was set up to express ccGFP 11 Labeled proteins.

[0338] To test expression and detection levels, a mixture of different nucleic acid templates was expressed in four different protein expression systems. NDET06 was present in the expression buffer. Unmodified ccGFP was used. 1-10 It was not possible to perform comparable experiments because the test protein was not sufficiently soluble even as an MBP conjugate.

[0339] The results can be seen in Figure 6 Each droplet contained the same amount of NDET06. The difference in fluorescence was measured with ccGFP 11 Therefore, the NDET06 assay can measure protein expression in real time as well as as an endpoint measurement.

[0340] To analyze the optimal NDET06 concentration in the expression system, additional experiments were set up using four known concentrations of recombinant MBP-ccGFP. 11 . The amount of detection agent is different. Figure 7 The results for the fluorescence intensity from the droplets are shown in Figure 2. The signal from the detector is proportional to the amount of protein expressed. When the detector is in excess, the signal depends on the ccGFP 11 The four bars on the left are the same because ccGFP 11 When ccGFP 11 In excess, the signal is limited by the amount of protein detected. The four bars on the right are different and correspond to the presence of ccGFP 11 Proportional to the amount.

[0341] Summary of findings

[0342] NDET06 can detect ccGFP 11The labeled protein was in 1.5-fold molar excess of the soluble protein.

[0343] NDET is highly soluble and can be used directly in expression systems at concentrations up to at least 200 μM. When NDET06 is added to lysates, the optimal concentration detected is 50 μM.

[0344] • Considering the determined optimal NDET06 concentration, the assay is able to quantify soluble protein at least up to 30 μM.

[0345] in conclusion

[0346] Adding variant K45E with MBP fusion (NDET06) to the expression system enables:

[0347] Shorten time to results, as NDET06 detects soluble protein from the beginning of expression;

[0348] Achieve higher sensitivity because the CPFS reaction and the fluorescent signal based on it are not diluted by the addition of NDET06;

[0349] Loading the device is easy because NDET06 and the expression system are loaded as one reagent;

[0350] More space is obtained on the device because NDET06 does not take up space on the device as a separate reagent.

[0351] ccGFP 11 Truncation of labels.

[0352] Full-length and 18 shortened MBP-ccGFP 11 Purified DNA of (v1) variants was compared with negative (-DNA) control and positive (ccGFP 11 (v1) purified protein) controls were used together with the reconstituted in vitro transcription / translation reactions. Aliquots of the samples were analyzed and then expressed in ccGFP 1-10 Before complementation, MBP-ccGFP was quantified by PAGE. 11 Relative expression of (v1). Fig. 9 and 10 Display shortened ccGFP 11 Effect of the GETIQLQEHAVAKYFTE tag. The GETIQ region can be removed, leaving LQEHAVAKYFTE. The YFTE region can be removed, leaving GETIQLQEHAVAK. Up to three amino acids can be removed from each end, leaving IQLQEHAVAKY.

[0353] Expression and endpoint detection on the EWoD device

[0354] Fig.11Results of experiments showing 24 different proteins expressed in droplets on an electrowetting on dielectric (EWoD) device in a reconstituted cell-free protein synthesis system. Each construct contained GFP 11 In the row labeled "Screening," GFP 1-10 The test substance is present from the beginning of expression. The row marked "end point" shows the absence of GFP 1-10 The detection substance was expressed for 10 hours, and then GFP was used 1-10 Fluorescence signal after 5 hours of complementation of the detection material. This experiment shows a significant difference between expression / complementation in the "Screening Bio-Ink" compared to the "End Point" detection. The detection of protein clusters formed after expression using only the endpoint detection means that the protein aggregates after expression, thereby reducing the soluble yield. It can be clearly seen from the image that some constructs have spots, indicating that protein aggregates are likely present. The level of aggregation can identify conditions that are worth further testing and identify conditions that have high levels of aggregation of proteins, from which further purification is unlikely to obtain material. The high solubility of the modified ccGFP protein makes such an experiment possible, meaning that concentrated droplets of detection agent can be merged with droplets of expressed protein and the levels of expressed protein measured.

Claims

1. A ccGFP variant comprising one or more mutations in beta strand 3.

2. The ccGFP variant according to claim 1, comprising a K45 mutation. The ccGFP variant according to claim 1 , comprising a K45E mutation.

4. The ccGFP variant according to any one of claims 1 to 3, which is ccGFP 1-10 Variant.

5. The ccGFP variant according to any one of claims 1 to 3, wherein the ccGFP 1-10 ccGFP 11 complex.

6. The variant according to claim 4, wherein the ccGFP K45E variant sequence comprises a fixed E Sequences with greater than 95% homology to: or its C-terminal or N-terminal truncations, which are similar to ccGFP 11 Bind to emit fluorescence.

7. The variant according to any one of claims 1 to 6, wherein the ccGFP K45E variant sequence comprises the sequence:

8. The variant of any one of claims 1 to 7, further comprising a solubility enhancing sequence.

9. The variant of claim 8, wherein the solubility enhancing sequence is selected from glutathione S-transferase (GST), small ubiquitin-like modifier protein (SUMO), maltose binding protein (MBP), Fasciola hepatica 8kDa antigen (FH8), thioredoxin (TRX), solubility enhancing ubiquitous tag (SNUT), seventeen kilodalton protein (SKP), monomeric bacteriophage T7orc protein (MOCR), Escherichia coli secreted protein A (ESPA), T7 phage tail (P17), metal binding protein (CUSF) or a 53 amino acid long N-terminal extension sequence (NEXT).

10. The variant according to claim 9, comprising a sequence selected from the group consisting of:

11. The variant of any one of claims 1 to 10, wherein the concentration of the protein is greater than 10 mg / mL.

12. The variant of any one of claims 1 to 11, wherein the concentration of the protein is greater than 50 mg / mL.

13. A nucleic acid sequence encoding the amino acid sequence according to any one of claims 1 to 10.

14. A nucleic acid sequence according to claim 13, comprising:

15. A method for detecting a protein of interest, comprising: taking a 11 The protein of interest is ccGFP 11 According to claim 4 or any one of claims 6 to 10, ccGFP1- 10 Variant binding and detection of ccGFP from the assembly 1-11 The fluorescent signal is used to monitor the presence of the protein of interest.

16. A method for using ccGFP according to claim 4 or any one of claims 6 to 10 1-10 Method for expressing a protein of interest (POI) in the presence of a ccGFP variant to increase the soluble yield of the expressed POI 1-10 Variants with ccGFP linked to POI 11 Binding sequence binding.

17. The method of claim 15 or claim 16, wherein the attachment to ccGFP is performed in a droplet on a digital microfluidics device. 11 Expression of the protein of interest.

18. The method according to claim 17, comprising the ccGFP according to claim 4 or any one of claims 6 to 10. 1-10 In the presence of 11 proteins and by detecting ccGFP from the assembly 1-11 The fluorescent signal is used to monitor the presence of the protein of interest.

19. A method according to claim 17 or claim 18, wherein the digital microfluidics device is an active matrix thin film transistor (AM-TFT) based device.

20. The method according to any one of claims 17 to 19, wherein the digital microfluidics device comprises two parallel plates separated by a spacer to define a fluid volume filled with a hydrophobic liquid or a non-ionic liquid.

21. The method of claim 20, wherein the protein is in aqueous droplets in an oil layer and the hydrophobic liquid or non-ionic liquid contains a surfactant.

22. The method of claim 21, wherein the surfactant is a sorbitan ester.

23. The method according to any one of claims 20 to 23, wherein the hydrophobic liquid or non-ionic liquid is decane, dodecane or dodecamethylpentasiloxane.

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