WO2023227913A1

WO2023227913A1 - Creating nucleic acids for in-vitro protein synthesis

Info

Publication number: WO2023227913A1
Application number: PCT/GB2023/051411
Authority: WO
Inventors: Michael Chun Hao CHEN; Gordon Ross MCINROY; Tobias William Barr Ost
Original assignee: Nuclera Ltd
Priority date: 2022-05-27
Filing date: 2023-05-30
Publication date: 2023-11-30
Also published as: GB202207816D0

Abstract

Provided herein are methods, and compositions for the synthesis of genes and proteins. The methods are applicable to synthesis of nucleic acids and proteins on a microfluidic device.

Description

CREATING NUCLEIC ACIDS FOR IN-VITRO PROTEIN SYNTHESIS

FIELD OF THE INVENTION

Provided herein are methods for the amplification of nucleic acids and compositions for the on- device expression and detection of protein synthesis. The methods are applicable to monitoring on a microfluidic device.

BACKGROUND TO THE INVENTION

Nucleic acid synthesis is vital to modern biotechnology. The rapid pace of development in the biotechnology arena has been made possible by the scientific community's ability to artificially synthesise DNA, RNA and proteins.

Artificial DNA synthesis allows biotechnology and pharmaceutical companies to develop a range of peptide therapeutics, such as insulin for the treatment of diabetes. It allows researchers to characterise cellular proteins to develop new small molecule therapies for the treatment of diseases our aging population faces today, such as heart disease and cancer. It even paves the way forward to creating life, as the Venter Institute demonstrated in 2010 when they placed an artificially synthesised genome into a bacterial cell.

However, current DNA synthesis technology does not meet the demands of the biotechnology industry. Despite being a mature technology, it is highly challenging to synthesise a DNA strand greater than 200 nucleotides in length in viable yield, and most DNA synthesis companies only offer up to 120 nucleotides routinely. In comparison, an average protein-coding gene is of the order of 2000-3000 contiguous nucleotides, a chromosome is at least a million contiguous nucleotides in length and an average eukaryotic genome numbers in the billions of nucleotides. In order to prepare nucleic acid strands thousands of base pairs in length, all major gene synthesis companies today rely on variations of a 'synthesise and stitch' technique, where overlapping 40-60-mer fragments are synthesised and stitched together by enzymatic copying and extension. Current methods generally allow up to 3 kb in length for routine production.

Described herein are improved methods for preparing nucleic acid samples, which can optionally then be used for protein synthesis.

Cell-free protein synthesis (CFPS) regimes are attractive alternatives to cell-based expression systems as they can be treated as reagents rather than organisms, making them amenable to in- vitro experimentation techniques. Additionally, cell-free systems are less sensitive to toxic protein synthesis; are open systems that can be modulated via addition of elements due to the lack of a cell membrane; are adaptable to high-throughput experiments; and can be used to good effect in small volumes. However, many of the cellular expression regulatory control paradigms still apply (e.g. incorrect ribosome binding motifs can lead to poor binding and poor transcription; incorrect codon usage can lead to inefficient translation etc).

Efficient protein synthesis relies on having the correct nucleic acid expression construct in the correct conditions. Protein synthesis and purification can be improved by attaching additional amino acids to the protein of interest, for example sequences improving solubility or tags for purification. In order to efficiently screen the optimal cell-free conditions for expression of a particular protein sequences it is desirable to provide a population of nucleic acid expression constructs. Furthermore, in order to identify the best DNA construct to generate a protein of interest it is desirable to provide a population of nucleic acid expression constructs. The invention herein describes methods for the preparation of nucleic acid constructs suitable for cell-free protein expression, and the use thereof.

When performing cell-free protein synthesis at microfluidic scale in a microfluidic device, such as a digital microfluidic device, it is useful to detect in real-time the proteins that are synthesized from said cell-free protein synthesis reaction. 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 at high concentration, making non-specific protein detection via standard protein staining methods difficult (e.g., Coomassie Brilliant Blue G-250, SYPRO™ Ruby, Silver staining). Immunostaining or affinity-based purification followed by nonspecific proteins staining are equally unhelpful as significant washing on a solid support must be performed to prevent background interference. As washing is known to be difficult in microfluidic and digital microfluidic devices, background interference may become debilitating.

Currently existing luminescent complementation approaches cannot achieve prolonged realtime detection in cell-free protein synthesis reactions, which often extend beyond 6 hours. This limit is due to a combination of reasons including O₂ consumption by the luminescencegenerating enzyme that competes with cell-free protein synthesis O₂ requirements and temporary or permanent exhaustion of luminescent substrate over 3 - 24 hours of recombinant protein expression detection. Proteins of interest may also be expressed as a fusion to a fluorescent protein, such as green fluorescent protein (GFP). However, GFP is a 25.9 kDa protein, which is the typical size for most fluorescent proteins. Tags of this size increase the total size of the protein of interest, especially if the protein of interest must be tagged with other large fusion proteins such as maltose-binding protein (MBP), which is 42.5 kDa. Given the average size of a human protein is ~52 kDa and the average size of an E. coli protein is ~35 kDa (Kim, Y. E. et al. Annu. Rev. Biochem. 2013. 82:323- 355), the addition of a comparably sized fluorescent protein tag can significantly change the biological function and biophysical characteristics of a protein.

Many pieces of prior art disclose the use of sub-component tags for monitoring expression in a cellular system. For example US 7,666,606 discloses protein-protein interaction detection systems using microdomains.

Schinn et al. BiotechnoL Bioeng 114 10 October 2017 2412-2417.

(https://onlinelibrarv.wilev.com/doi/10.1002/bit.263Q5) discloses Rapid in-vitro screening for the location-dependent effects of unnatural amino acids on protein expression and activity - Schinn - 2017 - Biotechnology and Bioengineering - Wiley Online Library.

Method for obtaining expression constructs include for example htps://www.biotechrabbit.com/media/wvsiwyg/files/btrproductinsert/RTS Manuais/PIN- 14008-002 RTS Ecoli LTGS Histag Manual.pdf. Disclosed herein are improved methods for making populations of linear expression constructs and obtaining proteins using these linear expression constructs.

Amplification techniques such as PCR have been implemented on microfluidic systems. For example Hua et al, Anal. Chem. 2010, 82, 2310-2316 describes a multiplexed real-time polymerase chain reaction on a digital microfluidic platform. Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting into temperature zones where thermocycled amplification can occur.

SUMMARY

Here we report the preparation of nucleic acid amplicons in a droplet on a digital microfluidic device. The amplification uses a mixed biological sample such as a cDNA library as the source of nucleic acids, and adds primers allowing for selective amplification in particular droplets. The amplicon sequence is chosen by the primers, which allow selective amplification of the desired sequence. The amplification provides adapted templates suitable for cell-free protein synthesis, either in a single amplification or via multiple simplification steps.

Described is a method for the amplification of nucleic acids in a droplet on a digital microfluidic device having an array of electrodes comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using the one or more primers to amplify a target nucleic acid in the merged droplet.

The amplification can be thermally cycled or isothermal. Two primers can be used to control the sequence of interest being amplified. The primers can be merged from two droplets into the droplet having a mixture of nucleic acids.

The primers can be synthesised off the device. Alternatively the primers can be synthesised on the device in separate droplets. The amplification can use multiple droplets to amplify different sequences. Multiple droplets having a mixture of nucleic acids can be mixed with multiple droplets having different primer sequences. Different genes may be amplified in different droplets. Different regions of the same gene may be amplified in different droplets.

The mixture of nucleic acids can be any biological source, for example a cDNA library or genomic sample, which may be human. The amplified material may be at least lkB in length. The amplified material may be at least 3kb in length. The amplified material may be used to express a protein. The expression may be in droplets on the device.

Disclosed is a method for the amplification of a nucleic acid sequence and expression of a protein in droplets on a digital microfluidic device having an array of electrodes comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; c. using the one or more primers to selectively amplify a nucleic acid in the merged droplet; and d. expressing a protein using the amplified nucleic acid sequence.

Described is a method for the monitoring of cell-free protein synthesis in a droplet on a digital microfluidic device comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; c. using the one or more primers to amplify a target nucleic acid in the merged droplet; d. cell-free transcription and translation of the target nucleic acid to produce a protein of interest fused to a peptide tag; and e. monitoring the presence of the peptide tag using a further polypeptide which in the presence of the peptide tag produces a detectable signal.

The detectable signal may be fluorescence or luminescence.

The transcription and translation system may be in human lysate system, a rabbit reticulocyte lysate (RRL) system, a Chinese Hamster Ovary lysate system, a wheat germ cell-free system, a E. coli whole cell lysate system or in a system of purified recombinant elements (PURE) or a mix thereof.

The peptide 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, GFP, eGFP, ccGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. 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 sfCherryii and the further polypeptide sfCherryno. The peptide tag may be CFASTH or CFASTio and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.

The droplets may be in an oil layer, which may contain surfactant. The surfactant in the oil layer may be a non-ionic surfactant. The surfactant in the oil layer may be a sorbitan ester. The surfactant in the oil layer may be Span85.

A split peptide system can be engineered to perform in situ, fluorescence-based monitoring of the expression of a protein of interest in cell-free protein synthesis reactions. The monitoring can be performed on device during the course of the expression, so can be used in real-time or as an end-point measurement.

The use of the terms "in-vitro" and "cell-free" may be used interchangeably herein. The detectable signal may be for example fluorescence or luminescence. The detectable signal may also be caused by the binding of a ligand to the complemented oligopeptide, peptide, or polypeptide tag fused to the protein of interest.

The detectable signal may also be caused by the binding of the polypeptide to the protein of interest fused to a His-tag.

The peptide tag may also be one component of a protein that forms a detectable substrate, such as a luminescent or colorigenic substrate. The protein could include beta-galactosidase, betalactamase, or luciferase.

The protein may be fused to multiple tags. For example the protein may be fused to multiple GFPn 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-io polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPu peptide tags and the synthesis occurs in the presence of one or more GFPi-io polypeptides and one or more sfCherryi-io polypeptides.

Any protein of interest may be synthesised. The protein may be an enzyme, for example a terminal deoxynucleotidyl transferase (TdT) enzyme or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of Polp, Poip, PolA, and Pol0 of any species or the homologous amino acid sequence of X family polymerases of any species.

The synthesis may be performed in a digital microfluidic device, for example an electrowetting- on-dielectric (EWoD) device. Alternatively the synthesis may be performed in a microtitre plate format.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: A schematic outlining the process of preparing an expression cassette using a two stage amplification process. The first stage introduces universal sequences (AO and BO). In the example shown the sequences code for a protease cleavage sites such as TEV and 3C. The amplification gives a double stranded amplicon having ends AO and BO (which happen to code for a protease cleavage site in the example shown). This amplicon can be further amplified using the megaprimers, the megaprimers having sequences which hybridise to AO and BO. The amplification using the primers TEV (AO) and 3C (BO) can be performed on a digital microfluidic device. Each primer can be prepared on or off the device. The primers can be supplied as premixed droplets, or can be merged on the device from two distinct droplets each having a single sequence.

Figure 2 shows a further embodiment of the invention. Specifically where, in situ on device:

1) eDNA synthesis is performed in parallel on two different initiators immobilized to a solid matrix (shown as UF and UR). The initiator can be the same sequence, or can contain variable regions.

2) Writing two different loci-specific sequences onto the end of the initiators using eDNA synthesis.

3) Contacting the loci-specific oligos to a template sample (the oligos could be immobilised or cleaved into solution).

4) Using the loci-specific oligos as FWD and REV PCR primers to make a gene-specific amplicon tailed with UF (top strand) and UR (bottom strand).

5) Using the UF/UR tailed amplicon as the input to a second assembly reaction with long megaprimers to make a full length competent expression construct.

The amplification steps could involve thermocycling or be an isothermal process.

Step 2 above describes how a single pair of F/R loci-specific primers can be written in parallel. In principle this paradigm could be extended for F/R primer pairs for a plurality of regions of interest, in parallel on the same device, e.g. to target multiple genes from the same template in parallel.

Figure 3 shows a standardized "mastermix reagent". The mastermix makes the manufacture of universal expression constructs very simple. In order to make robust, the megaprimers are supplemented with single stranded terminal primers at a much higher concentration to enrich for the full-length amplicons. This way, the megaprimers provide the specificity (i.e. enable a functional construct to be generated) but the inclusion of the terminal primers allows the number of moles of amplicon to be dramatically increased (compared to if they are not present in the mix). Figure 4 shows an exemplary 12 construct library. Each protein of interest is flanked by a variety of optional solubility tags, purification tags, detections tags, buffer sequences, promoter sequences and binding sites, either on the C or N terminus of the expressed protein. The library mix can be screened in parallel to determine the optimal conditions for protein expression and isolation.

DETAILED DESCRIPTION OF THE INVENTION

Here we report the preparation of nucleic acid amplicons in a droplet on a digital microfluidic device. The amplification uses a mixed biological sample as the source of nucleic acids, and adds primers allowing for selective amplification in particular droplets. The amplicon sequence is chosen by the primers, which allow selective amplification of the desired target sequence. Different amplicons may be amplified in different droplets as different primers can be added to different droplets.

Definitions:

• Target nucleic acid sequence = the sequence coding for a protein that already has priming sequences.

• Priming sequence = the sequence (for example AO/BO) which the I eft/right flank primers will bind to. Sequence AO and BO are universal and hybridise to the ends of the flank adapters.

• Left/right flank primer = primers that will install the left and right flanks (long sequences) of the construct to enable protein expression (by hydridising to Al and Bl or AO and BO).

• Starting nucleic acid sequence = a biological source sequence from which a target nucleic acid sequence can be generated by appending priming sequences (e.g. installing AO/BO)

• Adapter priming sequence = the variable loci sequence (Al/Bl) in the starting nucleic acid sequence which the forward/reverse adapter primers will bind to. Al and Bl are variable and hybridise to the source sequence

The terms 'left' and right' are used herein to symbolizing opposing ends of a template, and could equally be marked as 'end 1' and 'end 2' or 'start codon flank' and 'stop codon flank'. The term left and right have no positional meaning and are used to aid interpretation of the claims in relation to diagrams. The left flank and right flank elements could be transposed without affecting the meaning of the terms (for example the right flank could have a start codon and the left flank a stop codon).

The terms AO, Al etc are used to signify regions of nucleic acid sequence, and apply equally to the complementary sequences Al' and AO' which hybridise thereto. Al and Al' are loci specific sequences. AO and BO are universal sequences.

Thus the flow can be envisaged as:

Starting sequence (biological sample) -> Target sequence (short adapters attached having known priming sequences) -> Construct suitable for CFPS (long flanks attached). The primer sequences AO and BO can be 'attached' (via amplification) to starting sequences to make target sequences. The target sequences are then amplified using universal flank adapters specific to AO and BO. Alternatively the source sequences can be amplified using variable left and right flanks containing Al and Bl.

Priming sequences AO/BO enable universal left/r ight flank primers to bind and install left/right flanks. The priming sequences can include a sequence coding for a protease cleavage site.

Adapter priming sequences Al/Bl enable forward/reverse adapter primers to bind and install priming sequences AO/BO in the amplified target. AO and BO are 'loci specific' and vary depending on the starting nucleic acid.

The amplification can be done in a single step having multiple primers. Thus primers A0/A1 and BO/B1 can be used in a composition with the left and right flank primers and the amplification primers to obtain the constructs ready for CFPS.

Disclosed is a method for the expression of proteins in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet having a mixture of nucleic acids; b. adding three pairs of nucleic acid primer sequences to the droplet, the pairs comprising; i. primers to amplify a target nucleic acid in the merged droplet, wherein a forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and a reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; ii. a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; and iii. amplification primers complementary to the left and right flanks to selectively amplify the full-length constructs and reduce the proportion of residual left flank primers, wherein the amplification uses at least 100 fold concentration of amplification primers in proportion to the flanking primers c. using the primers to selectively amplify a nucleic acid in the merged droplet to produce a nucleic acid expression construct in the droplet; and d. using the expression construct to produce a protein.

The primers can be synthesised off the device. The primers can be synthesised using any known synthetic method, for example phosphoramidite synthesis. The primers can be supplied to the device, for example by dispending from reservoirs into droplets. The primers can be mixed on or off the device.

Primers can be a desired length sufficient to selectively amplify from the nucleic acid mixture. For example the primers may be 10-30 nucleotides in length. The primers may be 15-25 nucleotides in length. The primers may be approximately 20 bases in length.

The primers can be synthesised to have distinct loci (Al and Bl) attached to a universal sequence (A0 and B0).

The amplification can use multiple droplets to amplify different sequences on the same device. Multiple droplets having a mixture of nucleic acids can be mixed with multiple droplets having different primer sequences. Different genes may be amplified in different droplets. Different regions of the same gene may be amplified in different droplets. The amplification can be obtained using thermocycling. Different droplets may be thermocycled separately via localised heating, or the whole device may be heated and cooled to amplify all droplets simultaneously. Alternatively isothermal methods of amplification may be used.

The mixture of nucleic acids can be any biological source, for example a genomic sample, which may be human. The mixture of nucleic acids may be a cDNA library.

The amplified material may be at least IkB in length. The amplified material may be at least 3kB in length. The amplified material may be used to express a protein. The expression may be in droplets on the device.

The primers may be synthesised on the device. Disclosed is a method of nucleic acid synthesis, which comprises the steps of:

(a) providing an initiator oligonucleotide;

(b) adding a 3'-blocked nucleotide to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT);

(c) removal of all reagents from the initiator oligonucleotide;

(d) cleaving the blocking group in the presence of a cleaving agent; and

(e) removal of the cleaving agent.

The method can add greater than 1 nucleotide by repeating steps (b) to (e).

The method can assemble multiple sequences within separate droplets on the device, which can then be merged with droplets containing the nucleic acid sample to be selectively amplified. Described is a method for the amplification of nucleic acids in a droplet on a digital microfluidic device having an array of electrodes comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using the one or more primers to amplify a target nucleic acid in the merged droplet, wherein the forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and the reverse adapter primer comprises at its 3' end a matching sequence BO which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence Bl; to produce a double-stranded target nucleic acid having ends AO and BO.

Described is a method for the amplification of nucleic acids in a droplet on a digital microfluidic device having an array of electrodes comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using the one or more primers to amplify a target nucleic acid in the merged droplet, wherein the forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and the reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; to produce a double-stranded target nucleic acid having ends AO and BO; and d. adding further primers to the droplet and amplifying the double-stranded target nucleic acid with a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression.

Disclosed herein is a method for the expression of proteins in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using the one or more primers to amplify a target nucleic acid in the merged droplet, wherein the forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5’ end a sequence AO; and the reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; to produce a double-stranded target nucleic acid having ends AO and BO; and d. adding further primers to the droplet and amplifying the double-stranded target nucleic acid with a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression; and e. using the expression construct to produce a protein.

Disclosed herein is a method for the expression of proteins in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using the one or more primers to amplify a target nucleic acid in the merged droplet, wherein the forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and the reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; to produce a double-stranded target nucleic acid having ends AO and BO; and d. adding further primers to the droplet and amplifying the double-stranded target nucleic acid with a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression; and e. cell-free transcription and translation of the expression construct to produce a protein of interest fused to a peptide tag; and f. monitoring the presence of the peptide tag using a further polypeptide which in the presence of the peptide tag produces a detectable signal.

The reaction can be performed in a single amplification, which can introduce ends AO and BO in a single amplification also using the left and right flank primers and the terminal amplification primers to produce the nucleic acid expression constructs.

Alternatively the left and right flank primers can be different for each amplification and include template specific ends Al and Bl.

Constructs produced require at least a promoter region such as a T7 promoter, one or more ribosome binding sites, a protein of interest and a stop codon. The constructs may also contain one or more solubility tags, purification tags or detection tags. The constructs may contain coding and non coding sequences acting as linkers, buffers etc.

The left flank primers are typically longer than 200 nucleotides in length. The left flank primers can be longer than 500 nucleotides in length. The left flank primers can be longer than 1000 nucleotides in length.

References herein to 'nucleoside triphosphates' refer to a molecule containing a nucleoside (i.e. a base attached to a deoxyribose or ribose sugar molecule) bound to three phosphate groups. Examples of nucleoside triphosphates that contain deoxyribose are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP). Examples of nucleoside triphosphates that contain ribose are: adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridine triphosphate (UTP). Other types of nucleosides may be bound to three phosphates to form nucleoside triphosphates, such as naturally occurring modified nucleosides and artificial nucleosides. Therefore, references herein to '3'-blocked nucleotide' include nucleoside 5' -triphosphates (e.g., dATP, dGTP, dCTP or dTTP) which have an additional group on the 3'-OH which prevents further addition of nucleotides, i.e., by replacing the 3'-OH group with an O-protecting group.

It will be understood that references herein to '3'-block', '3'-blocking group' or '3'-protecting group' refer to the group attached to the 3' end of the nucleotide or nucleoside triphosphate which prevents further nucleotide addition. The present method uses reversible 3'-blocking groups which can be removed by cleavage to allow the addition of further nucleotides. By contrast, irreversible 3'-blocking groups refer to dNTPs where the 3'-OH group can neither be exposed nor uncovered by cleavage.

The 3' -blocked nucleoside can be blocked by any chemical group that can be unmasked to reveal a 3'-OH. The 3' -blocked nucleoside can be blocked by a 3'-O-azidomethyl, 3'-aminooxy, 3'-O-(N- oxime) (3'-O-N=CRIR2, where Ri and R2 are each a C1-C3 alkyl group, for example CH3, such that the oxime can be O-N=C(CH3h (N-acetoneoxime)), 3'-O-al ly I group, 3'-O-cyanoethyl, 3'-O-acetyl, 3'-O-nitrate, 3'-phosphate, 3'-O-acetyl levulinic ester, 3'-O-tert butyl dimethyl silane, 3'-O- trimethyl(silyl)ethoxymethyl, 3'-O-ortho-nitrobenzyl, and 3'-O-para-nitrobenzyl.

The 3' -blocked nucleoside can also be blocked by any chemical group that can be directly utilized in chemical ligations, such as copper-catalyzed or copper-free azide-alkyne click reactions and tetrazine-alkene click reactions. The 3'-blocked nucleotide or nucleoside triphosphate can include chemical moieties containing an azide, alkyne, alkene, and tetrazine.

References herein to 'cleaving agent' refer to a substance which is able to cleave the 3'-blocking group from the 3'-blocked nucleotide. In one embodiment, the cleaving agent is a chemical cleaving agent. In an alternative embodiment, the cleaving agent is an enzymatic cleaving agent. The cleaving can be done in a single step, or can be a multi-step process, for example to transform an oxime (such as for example 3'-O-(N-oxime), 3'-O-N=C(CH3)2, into aminooxy (O- NH2), followed by cleaving the aminooxy to OH.

It will be understood by the person skilled in the art that the selection of cleaving agent is dependent on the type of 3'-nucleotide blocking group used. For instance, tris(2- carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THPP) can be used to cleave a 3'-O-azidomethyl group, palladium complexes can be used to cleave a 3'-O-allyl group, or sodium nitrite can be used to cleave a 3'-aminooxy group. Therefore, in one embodiment, the cleaving agent is selected from: tris(2- carboxyethyl)phosphine (TCEP), a palladium complex or sodium nitrite.

In one embodiment, the cleaving agent is added in the presence of a cleavage solution comprising a denaturant, such as urea, guanidinium chloride, formamide or betaine. The addition of a denaturant has the advantage of being able to disrupt any undesirable secondary structures in the DNA. In a further embodiment, the cleavage solution comprises one or more buffers. It will be understood by the person skilled in the art that the choice of buffer is dependent on the exact cleavage chemistry and cleaving agent required.

References herein to an 'initiator oligonucleotide' or 'initiator sequence' refer to a short oligonucleotide with a free 3'-end which the 3'-blocked nucleotide can be attached to. In one embodiment, the initiator sequence is a DNA initiator sequence. In an alternative embodiment, the initiator sequence is an RNA initiator sequence.

References herein to a 'DNA initiator sequence' refer to a small sequence of DNA which the 3'- blocked nucleotide can be attached to, i.e., DNA will be synthesised from the end of the DNA initiator sequence.

In one embodiment, the initiator sequence is between 5 and 50 nucleotides long, such as between 5 and 30 nucleotides long (i.e. between 10 and 30), in particular between 5 and 20 nucleotides long (i.e., approximately 20 nucleotides long), more particularly 5 to 15 nucleotides long, for example 10 to 15 nucleotides long, especially 12 nucleotides long.

In one embodiment, the initiator sequence is single-stranded. In an alternative embodiment, the initiator sequence is double-stranded. It will be understood by persons skilled in the art that a 3'-overhang (i.e., a free 3'-end) allows for efficient addition.

In one embodiment, the initiator sequence is immobilised on a solid support. This allows TdT and the cleaving agent to be removed (in steps (c) and (e), respectively) without washing away the synthesised nucleic acid. The initiator sequence may be attached to a solid support stable under aqueous conditions so that the method can be easily performed via a flow setup.

In one embodiment, the initiator sequence is immobilised on a solid support via a reversible interacting moiety, such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin or streptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K.

In one embodiment, the initiator sequence contains a base or base sequence recognisable by an enzyme. A base recognised by an enzyme, such as a glycosylase, may be removed to generate an abasic site which may be cleaved by chemical or enzymatic means. A base sequence may be recognised and cleaved by a restriction enzyme.

In a further embodiment, the initiator sequence is immobilised on a solid support via a chemically-cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker. Therefore, in one embodiment, the method additionally comprises extracting the resultant nucleic acid by cleaving the chemical linker through the addition of tris(2- carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for a disulfide linker; palladium complexes or an allyl linker; or TCEP for an azide-masked hemiaminal ether linker.

Disclosed herein is a method for the monitoring of in-vitro protein synthesis comprising a. in-vitro transcription and translation of a protein of interest fused to a peptide tag; and b. monitoring the presence of the peptide tag using a further polypeptide which in the presence of the peptide tag produces a detectable signal.

Disclosed is a method for the cell-free expression of peptides or proteins in a digital microfluidic device. The droplets having the components required for cell-free protein synthesis (CFPS), otherwise known as in-vitro protein synthesis, can be manipulated by electrokinesis in order to effect and improve protein expression.

Disclosed is a method of a method of providing a nucleic acid expression construct suitable for cell-free protein expression on a digital microfluidic device, wherein the method comprises: i. taking a double-stranded target nucleic acid having ends AO and BO; ii. amplifying the double-stranded target nucleic acid with a left flank primer and a right flank primer in droplets on the device wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression.

Disclosed is a method of providing a nucleic acid expression construct suitable for cell-free protein expression on a digital microfluidic device, wherein the method comprises: i. amplifying a starting nucleic acid sequence with a forward adapter primer and a reverse adapter primer in droplets on the device wherein: the forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and the reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; to produce a first double-stranded target nucleic acid sequence having ends AO and BO; ii. amplifying the double-stranded target nucleic acid with a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression.

Disclosed is a method of providing a variety of nucleic acid expression constructs suitable for cell-free protein expression, wherein the method comprises: i. taking one or more double stranded target nucleic acids having ends AO and BO; ii. amplifying the target nucleic acid with multiple left flank primers and one or more right flank primers to produce a population of constructs having different solubility tags or ribosome binding sites, wherein: each left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site for a particular species, an optional solubility tag and, at its 3’ end, a sequence complementary to AO; and the right flank primer comprises a detection tag, an optional solubility tag, a terminator sequence, a sequence encoding for a stop codon and, at its 3’ end, a sequence complementary to BO; iii. amplifying the products produced having the left and right flanks using amplification primers complementary to the left and right flanks to selectively amplify the full-length constructs and reduce the proportion of residual left flank primers, wherein the amplification uses at least 100 fold concentration of amplification primers in proportion to the flanking primers; to produce a population of linear double-stranded expression constructs having a variety of solubility tags or ribosome binding sites suitable for cell-free protein expression of proteins which can be detected.

The matching sequences Al and Bl and AO and BO can independently between x and y nucleotides, more preferably 10 and 50 nucleotides. These matching sequences may or may not be fully complementary. Depending on whether the input amplicon is double or single stranded, the primers may be complementary to the sense or antisense strands. Where the template used is ssDNA, the one primer would only be complementary once the first copy of the template strand was made. Thus one primer is complementary to one strand and one primer to the complementary strand.

Sequences AO and BO can encode for protease cleavage sites in an expressed amino acid sequence. The protease can be a cysteine, serine, or threonine protease, an aspartic protease, glutamic protease or metallo protease.

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

The primer sequences can include sequences:

5'-GAGAACCTGTACTTCCAGAGC-3' (TEV cleavage sequence ENLYFQS) 5'-TCCTTGGAACAGAACCTCGAG-3' (3' -5' LEVLFQ.G 3C cleavage sequence) 5'-CTCGAGGTTCTGTTCCAAGGACCT-3' (LEVLFQGP 3C cleavage sequence)) The left flank primer may further comprise a sequence or plurality of sequences encoding for ribosome interactions sites selected from alternative ribosome binding sites (RBS) or internal ribosome entry sites. The left flank primer may code for a selection of solubility tags. The left flank primer may end with the AO complementary sequence 5'- CTCGAGGTTCTGTTCCAAGGACCT-3'. This sequence will express the amino acid sequence LEVLFQGP, a 3C protease cleavage sequence.

The left flank primer and/or the right flank primer may further comprise a DNA sequence or plurality of DNA sequences encoding for additional peptide structures selected from detection tags, purification tags, solubility tags, linkers and/or spacers.

The detection tags may be selected from a component part of a fluorescent protein.

Affinity tags may be appended to proteins so that they can be purified from their crude biological source using an affinity technique The purification tags may be selected from for example FLAG- tag, His-tag, GST-tag, MBP-tag, STREP-tag. The Flag® tag, also known as the DYKDDDDK-tag, is a popular protein tag that is commonly used in affinity chromatography and protein research. His tags are polyhistidine strings of amino acids, typically between 6 and 9 histidine amino acids in length.

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:

Alfa-tag (SRLEEELRRRLTE)

Avi-tag (GLNDIFEAQ.KIEWHE)

C-tag (EPEA)

Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)

Dogtag (DIPATYEFTDGKHYITNEPIPPK)

E-tag (GAPVPYPDPLEPR)

FLAG (DYKDDDDK)

G4T (EELLSKNYHLENEVARLKK)

HA (YPYDVPDYA)

His (HHHHHH)

Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG)

Myc (EQ.KLISEEDL)

NE-Tag (TKENPRSNQEESYDDNES)

Poly Glutamate-tag (EEEEEEE)

Poly Arginine-tag (RRRRRRR)

RholD4-tag (TETSQ.VAPA)

SBP-tag (MDEKTTGWRGGHVVEGLAGELEQ.LRARLEHHPQGQREP)

Sdytag (DPIVMIDNDKPIT)

SH3 (STVPVAPPRRRRG)

SNAC (GSHHW)

Snooptag (KLGDIEFIKVNK)

Softag 1 (SLAELLNAGLGGS)

Softag 3 (TQ.DPSRVG)

Spot-tag (PDRVRAVSHWSS)

Spytag (AHIVMVDAYKPTK)

S-tag (KETAAAKFERQ.HM DS)

Strep-tag (AWAHPQ.PGG) (AWRHPQFGG)

Strep-tag II (WSHPQ.FEK)

T7tag (MASMTGGQQMG)

TC-tag (EVHTNQ.DPLD)

Ty-tag (CCPGCC)

VSV-tag (YTDIEMNRLGK)

Xpress-tag (DLYDDDDK)

The expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:

The left flank primer and/or the right flank primer may further comprise protective elements that inhibit digestion of the left flank and/or right primers and the resulting expression construct by nucleases. The protective elements may be buffer sequences that absorb nuclease digestion without affecting the operationally important regions of the construct such as the start and stop codons.

The protective elements may be selected from the following: internal phosphorothioate bonds, terminal capping groups (e.g. 5' -alkylamino, 3'-phosphate, 3'-inverted T etc.) or modified nucleotides (e.g. methylated bases, 2-aminoadenosine, base-modified bases etc.), hairpin motifs or g-quadruplexes. The protective elements may enable circularisation of the expression construct to thereby protect the expression construct from terminal nucleases.

The left flank primer and/or the right primer may further comprise isolation elements for pulldown enrichment of the left flank and/or right primer and the resulting expression construct.

The left flank primer can be between 500 and 3000 nucleotides in length. More preferably, the left flank primer is at least 1000 nucleotides in length. Most preferably, the left flank primer is between 1000 and 3000 nucleotides in length.

The right flank primer can be between 100 and 3000 nucleotides in length.

The right primer may end with the B0 complementary sequence 5'- GAGAACCTGTACTTCCAGAGC-3'. Such sequences express the TEV protease cleavage site ENLYFQS.

The left flank primer may comprises a sequence or plurality of sequences encoding for ribosome interaction sites selected from alternative ribosome binding sites or internal ribosome entry sites. The method may have multiple amplification steps which are in separate droplets. Further primers or reagents may be added following a first amplification in order to enable a second amplification. The second amplification may be performed using a plurality of left flank primers and a single right flank primer to produce a population of expression constructs having different ribosome binding sites or 5'-UTR's.

The method may further contain a third amplification step to selectively amplify the full length construct.

The amplification process may be performed with additional primers which selectively amplify full length constructs. Thus the amplification primers can be used in excess compared to the flanking primers. For example at least 100 fold excess in concentration or at least 1000 fold excess of the amplification primers can be used in order to convert the flanking primers into full length amplicons and lower the presence of truncated transcripts.

The amplification using multiple primer sets can be performed in a single step. Thus primers A0/A1 and B0/B1 can be used with the left and right flank primers and the amplification primers to selectively produce full length constructs.

Disclosed is an expression construct or population of expression constructs in droplets prepared according to the methods described herein.

Disclosed is a method of expressing a protein using a construct or population of constructs using a cell-free system on a DMF device containing an array of electrodes.

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. 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.

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).

To date, digital microfluidics, electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-free biological-based applications, mostly due to biofouling, where biological components such as proteins, nucleic acids, crude 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 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).

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 proteins levels to be detectable.

Protein expression typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis.

Described herein are improved methods allowing for the cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for the cell-free expression of peptides or proteins in a microfluidic device wherein the method comprises one or more droplets containing a nucleic acid template (i.e., DNA or RNA) and a cell-free system having components for protein expression in an oil-filled environment, and moving said droplets using electrokinesis. The components for the cell-free protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device.

The droplet 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 to be supplied to the droplet and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

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.

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 (DMPS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85.

The oil in the device can be any water immiscible liquid. The oil can be mineral oil, silicone oil, an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The oil can be oxygenated prior to or during the expression process. Alternatively, the device can be an airfilled device where droplets containing cell-free protein synthesis 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).

A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the droplets during the protein expression. 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 (RSCAdv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air.

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 cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis.

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.

The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.

Any particular nucleic acid template can be generated and expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in-vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet.

An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed 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. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process.

The cell-free extract having the components for protein expression includes everything required for protein expression apart from the nucleic acid template. Thus the term includes all the relevant ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, protein expression is initiated without further reagents being required.

Thus the cell-lysate can be supplemented with additional reagents prior to the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or 'master mix' which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available.

Rather than originating from a cell extract, the cell-free system can be assembled from the required reagents. Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied. The PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.

The protein synthesis reaction reagent can be a mixture of cell lysate and purified components, for example a system of purified recombinant elements i.e. protein synthesis using recombinant elements (PURE). Particularly the enzymes used for protein expression can be a mixture of cell lysates and purified enzymes.

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.

Once the CFPS reagents have been enclosed in the droplets, 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.

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.

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 CFPS droplets. The droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets. 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 expression process. For example the expression process can be initiated on the device by increasing the temperature. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.

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 is available within the droplet. The moving can be continuous, or can be repeated with intervening periods of nonmovement.

Thus the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed. 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.

"Droplet" refers to a volume of liquid that electrowets a hydrophobic surface and is at least partially bounded by carrier fluid and/or, in some instances, a gas or gaseous mixture such as ambient air. For example, a droplet may be completely surrounded by carrier fluid or may be bounded by carrier fluid and one or more surfaces of an EWoD device. Droplets may take a wide variety of shapes; non-limiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more working surface of an EWoD device. Droplets may include typical polar fluids such as water, as is the case for aqueous or non-aqueous compositions, or may be mixtures or emulsions including aqueous and non-aqueous components. Droplets may also include dispersions and suspensions, for example magnetic beads in an aqueous solvent. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes.

"Droplet operation" refers to any manipulation of one or more droplets on a microfluidic device. A droplet operation may, for example, include: loading a droplet into the DMF device; dispensing one or more droplets from a source reservoir; splitting, separating or dividing a droplet into two or more droplets; moving a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; holding a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a microfluidic device; other droplet operations described herein; and/or any combination of the foregoing. The terms "merge," "merging," "combine," "combining" and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations that are sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, "merging droplet A with droplet B," can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms "splitting," "separating" and "dividing" are not intended to imply any particular outcome with respect to volume of the resulting droplets (i.e., the volume of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term "mixing" refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of "loading" droplet operations includes but is not limited to microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles.

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.

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.

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.

A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen 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 from a gaseous environment. Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device.

The droplet 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 cell-free system having the components for protein expression to form the droplet.

The droplets can be split on the device either before, during or after expression. Included herein is a method further comprising splitting the droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one of more of the split droplets are merged with additive droplets for screening. Through an affinity tag, such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag, CFPS-expressed proteins can be immobilized to a solid-support affinity resin and fresh batches of CFPS reagent can be delivered over the said resin. Thus, renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS production methods.

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 CFPS reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or UltraEver 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 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 Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275).

Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing CFPS components 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 CFPS components may contain TWEEN 20 at 0.1% v/v, Triton X-100 at 0.1% v/v, and/or Pluronic F127 at 0.08% w/v.

For electrowetting on dielectrics (EWoD), the change in contact angle of reagent upon the application of electric potential is an inverse function of surface tension. Thus, for low voltage EWoD operations, reduction in surface tension is achieved by addition of surfactants to reagents, which for CFPS reactions means to the lysate and to the DNA. This results in a dilution of the lysate, and it has been seen, in experiments, that diluting or otherwise adulterating the lysate results in a decrease in expression level of the protein of interest. Thus performing CFPS on DMF where the surfactants are added to the solutions being moved will necessarily result in a dilution and adulteration of the lysate and thus a decrease in the level of protein expression. In addition to being a problem in its own right, this further complicates extrapolation of on-DMF results to in-tube predictions of protein yield. An additional detriment of having to add surfactants to the samples is that this increases the time required for sample preparation, as well as increasing the potential for inconsistent results due to 'user error,' as there is more handling of reagents. An additional detriment of having to add surfactants to the samples is that certain downstream operations are hindered. For example, if a protein of interest is expressed in a cell-free system with a GFP 11 (or similar) peptide tag, it's downstream complementation with a GFP1-10 (or similar) detector polypeptide is hindered in the presence of surfactant. This is shown in Figures 8 and 9. Removal of the surfactant from the aqueous phase is therefore advantageous.

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 CFPS 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 allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane 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 CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS 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. GFP11/GFP1-10) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial.

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 GFPi-io. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi-io. The protein may be fused to multiple tags. For example the protein may be fused to multiple GFPn peptide tags and the synthesis occurs in the presence of multiple GFPi-io polypeptides. For example the protein may be fused to multiple sfCherryu peptide tags and the synthesis occurs in the presence of multiple sfCherryi-io polypeptides. The protein of interest may be fused to one or more sfCherryu peptide tags and one or more GFPu peptide tags and the synthesis occurs in the presence of one or more GFPi-io polypeptides and one or more sfCherryi-io polypeptides.

The complementary ccGFP/GFPu peptide amino acid sequence could be the following:

1. KRDHMVLLEFVTAAGITGT

2. KRDHMVLHEFVTAAGITGT

3. KRDHMVLHESVNAAGIT

4. RDHMVLHEYVNAAGIT

5. GDAVQIQEHAVAKYFTV

6. GDTVQLQ.EHAVAKYFTV

7. GETIQLQ.EHAVAKYFTE or a truncated version thereof. Truncations may involve a shortening of up to 5 amino acids from the N terminus, the C terminus or a combination thereof.

GFPn or GFPi-io 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.

Also disclosed are nucleic acid sequences for expressing particular tags. Nucleic acid sequences include

5'GGTGATACCGTTCAGCTGCAAGAACATGCAGTTGCAAAATACTTTACCGTG

5'GGTGAAACCATCCAGTTACAAGAACACGCCGTGGCCAAATATTTCACCGAA

These sequences may be repeated one or more times to produce a protein having multiple GFPn domains.

Devices 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.

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 - cos0_o= (l/2yLG) c.V² where 0_O 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 s_r. s₀/t, where s_r is 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.

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.

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.

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. SU-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 SU-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.

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.

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.

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.

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.

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.

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 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.

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. 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.

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.

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.

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

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.

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;

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.

Claims

CLAIMS:

1. A method for the amplification of a nucleic acid sequence and expression of a protein in droplets on a digital microfluidic device having an array of electrodes comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; c. using the one or more primers to selectively amplify a nucleic acid in the merged droplet; and d. expressing a protein using the amplified nucleic acid sequence.

2. The method according to claim 1 wherein at least two primer sequences are used for amplification.

3. The method according to claim 2 wherein each primer is in a separate droplet and the primer droplets are merged with the droplet having a mixture of nucleic acids.

4. The method according to any one preceding claim, wherein the primers are synthesised in droplets on the device.

5. The method according to any one preceding claim, wherein the mixture of nucleic acids is a genomic sample.

6. The method according to any one preceding claim, wherein the mixture of nucleic acids is a cDNA library.

7. The method according to claim 5 or claim 6, wherein the genomic sample or cDNA library is human.

8. The method according to any one preceding claim, wherein the amplification is isothermal.

9. The method according to any one preceding claim, wherein the primer amplified material is further amplified into an expression construct which is suitable for expressing a protein in a single amplification step with multiple primers.

10. The method according to any one preceding claim, wherein multiple droplets having a mixture of nucleic acids are mixed with multiple droplets having different primer sequences.

11. The method according to claim 10 wherein different genes are amplified in different droplets.

12. The method according to claim 10 wherein different regions of the same gene are amplified in different droplets.

13. A method according to any one preceding claim for the monitoring of cell-free protein synthesis in a droplet on a digital microfluidic device comprising a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; c. using the one or more primers to amplify a nucleic acid in the merged droplet; d. cell-free transcription and translation of a protein of interest fused to a peptide tag; and e. monitoring the presence of the peptide tag using a further polypeptide which in the presence of the peptide tag produces a detectable signal.

14. A method as claimed in claim 13 wherein the detectable signal is fluorescence or luminescence.

15. A method as claimed in claim 13 or claim 14 wherein the wherein the transcription and translation occurs in human lysate system, a rabbit reticulocyte lysate (RRL) system, a Chinese Hamster Ovary lysate system, a wheat germ cell-free system, a E. co// whole cell lysate system or in a system of purified recombinant elements (PURE) or a mix thereof.

16. A method as claimed in any one of claims 13 to 15 wherein the peptide tag is one component of a fluorescent protein (GFP).

17. A method as claimed in claim 14 wherein the peptide tag is ccGFPu and the further polypeptide is ccGFPi-io.

18. A method as claimed in claim 14 wherein the peptide tag is one component of sfCherry and the further polypeptide is sfCherryi-i₀.

19. The method of any one of claims 1 to 18, wherein the droplets are in an oil layer and the oil layer contains surfactant.

20. The method of claim 19 wherein the surfactant in the oil layer is a non-ionic surfactant.

21. The method according to claim 20 wherein the surfactant is a sorbitan ester.

22. The method according to claim 21 wherein the surfactant is Span85.

23. A method according to any one preceding claim for the expression of proteins in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using primers to amplify a target nucleic acid in the merged droplet, wherein a forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and a reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; to produce a double-stranded target nucleic acid sequence having ends AO and BO; d. using further primers to amplify the double-stranded target nucleic acid sequence with a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression; and e. using the expression construct to produce a protein. A method according to claim 23 for the expression of proteins in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet having a mixture of nucleic acids; b. adding one or nucleic acid primer sequences to the droplet; and c. using primers to amplify a target nucleic acid in the merged droplet, wherein a forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and a reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; to produce a double-stranded target nucleic acid sequence having ends AO and BO; d. adding further primers to the droplet and amplifying the double-stranded target nucleic acid sequence with a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; to produce a double-stranded expression construct suitable for cell-free protein expression; and e. using the expression construct to produce a protein. The method according to claim 23 wherein AO and/or BO contains a sequence which codes for a protease cleavage site. The method according to claim 23 or 24 wherein the left flank primer is between 500 and 3000 nucleotides in length. The method according to any one of claims 23 to Tl further comprising using amplification primers complementary to the left and right flanks to selectively amplify the full-length constructs and reduce the proportion of residual left flank primers, wherein the amplification uses at least 100 fold concentration of amplification primers in proportion to the flanking primers. A method according to any one preceding claim for the expression of proteins in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet having a mixture of nucleic acids; b. adding three pairs of nucleic acid primer sequences to the droplet, the pairs comprising; i. primers to amplify a target nucleic acid in the merged droplet, wherein a forward adapter primer comprises at its 3' end a matching sequence Al which can bind to a first region of the nucleic acid sequence, and at its 5' end a sequence AO; and a reverse adapter primer comprises at its 3' end a matching sequence Bl which can bind to a second region of the nucleic acid sequence, and at its 5' end a sequence BO; ii. a left flank primer and a right flank primer wherein: the left flank primer comprises at least a promoter sequence, a sequence encoding for a ribosome binding site and, at its 3' end, a sequence complementary to AO; and the right flank primer comprises a terminator sequence, a sequence encoding for a stop codon and, at its 3' end, a sequence complementary to BO; and iii. amplification primers complementary to the left and right flanks to selectively amplify the full-length constructs and reduce the proportion of residual left flank primers, wherein the amplification uses at least 100 fold concentration of amplification primers in proportion to the flanking primers c. using the primers to selectively amplify a nucleic acid in the merged droplet to produce a nucleic acid expression construct in the droplet; and d. using the expression construct to produce a protein.