WO2023063892A2

WO2023063892A2 - High-throughput combinatorial dna assembly via microfluidics

Info

Publication number: WO2023063892A2
Application number: PCT/SG2022/050736
Authority: WO
Inventors: Chwee Teck Lim; Ying Zhou; Ming Wang; Viet Linh DAO; Chueh Loo Poh
Original assignee: National University of Singapore
Current assignee: National University of Singapore
Priority date: 2021-10-14
Filing date: 2022-10-14
Publication date: 2023-04-20
Anticipated expiration: 2024-04-14
Also published as: WO2023063892A3

Abstract

The invention relates to a microfluidic device for creating recombinant plasmid constructs, comprising a first layer comprising a plurality of sample channels and an air channel arranged to converge with an outlet; a second layer comprising a first plurality of controllable valve channels arranged to control each sample channel, wherein the first layer is adjacent to the second layer, each sample channel is in fluidic connection with the outlet and a sample reservoir; the air channel is in fluidic connection with the outlet and an air inlet. The invention also relates to a method of creating recombinant plasmid constructs using a microfluidic device.

Description

HIGH-THROUGHPUT COMBINATORIAL DNA ASSEMBLY VIA MICROFLUIDICS

Field of the Invention

The invention relates to a microfluidic device for automated high-throughput DNA assembly and library construction, and a method of creating recombinant plasmid constructs using a microfluidic device.

Background of the Invention

Besides the traditional benchtop manual pipetting, the most commonly used method for combinatorial DNA assembly is the liquid handling system, which uses a robotic arm to perform pipetting by moving, ascending, and descending the robotic arm with a pipette tip attached. This automated system can dispense and mix reagents in a large scale, at the same time reducing error occurrence and laborious effort significantly. However, robotic technology is typically expensive; apart from the cost of the hardware, it also requires the use of customised consumables such as pipette tips embedded with marker to allow precise locating of robotic arm. For example, the cost for materials and hands- on time for DNA assembly using a liquid-handling system is estimated to be ~$ 141 000 to synthesize 13 824 constructs.

Therefore, there is a great demand for a highly automated system for high-throughput DNA assembly and large-scale DNA library construction with low cost.

Summary of the Invention

In a first aspect, the present invention provides for a microfluidic device for creating recombinant plasmid constructs, comprising: a first layer comprising a plurality of sample channels and an air channel arranged to converge with an outlet; a second layer comprising a first plurality of controllable valve channels arranged to control each sample channel, wherein the first layer is adjacent to the second layer, each sample channel is in fluidic connection with the outlet and a sample reservoir; the air channel is in fluidic connection with the outlet and an air inlet. The valve channels may control each sample channel independently or any combination of channels simultaneously.

The proposed microfluidic device can be loaded with user-defined DNA fragments to perform mixing without time-consuming liquid handling. Through controlling the microvalves to allow the reagents to flow through the microchannel, the desired fragments can be mixed and collected in a high-throughput fashion with a wide volume dispensing range (0.5-20 pl). The inventors have created a 16 microchannels system to build 65 different plasmid constructs in less than an hour with a high success rate verified by sequencing. This platform can be easily automated and scaled to accommodate up to 1000 3-fragment constructs with 30 channels. This device offers high throughput while ensuring high accuracy by eliminating cross-contamination of samples. The proposed technique offers potential for automation of synthetic biology procedures with significantly reduced cost and time. Air channels allows air purging to avoid cross-contamination of fluid within the channels, to ensure high accuracy while maintaining high throughput.

In an embodiment, the first layer further comprises a plurality of wash channels; and the second layer further comprises a second plurality of controllable valve channels arranged to control each wash channel, wherein each wash channel is in fluidic connection with a sample channel or air channel, the outlet and a wash fluid.

The separate wash channels minimize cross-contamination between wash fluid and samples. This is particularly important when handling small volumes in the microliter range. Each washing step may be followed by purging with air to further reduce the likelihood of cross-contamination. The valve channels may control each wash channel independently or any combination of channels simultaneously.

In an embodiment, the wash fluid comprises air, oil or water.

The type of wash fluid may be chosen to avoid mixing with any remnants of sample in the channels to further reduce the likelihood of cross-contamination.

In an embodiment, the sample reservoir is arranged to comprise a DNA fragment selected from the group consisting of: a replication origin, an antibiotic resistance gene, and a target gene.

Each sample reservoir may comprise a different DNA fragment for creating a recombinant plasmid construct, so that different plasmid constructs can be created easily by controlling the input from different sample reservoirs, and each sample reservoir can be used repeatedly until it is depleted. This eliminates the need to make multiple mixes of each DNA fragment, thus saving time and costs. In a second aspect, the invention provides for a method of creating recombinant plasmid constructs, comprising the steps of: a) providing a microfluidic device comprising a first layer comprising a plurality of sample channels and an air channel arranged to converge with an outlet; a second layer comprising a first plurality of controllable valve channels arranged to control each sample channel, wherein the first layer is adjacent to the second layer, each sample channel is in fluidic connection with the outlet and a sample reservoir; the air channel is in fluidic connection with the outlet and an air inlet; b) releasing at least two samples through the sample channels into the outlet; c) stopping sample flow in the sample channels; d) purging the sample channels and the outlet; and e) collecting a sample mixture from the outlet. The valve channels can control each sample channel independently or any combination of channels simultaneously.

Advantageously, this method allows high throughput plasmid construction while maintaining high accuracy and low cross-contamination. This method could also be automated.

In an embodiment, the method further comprises: flushing a wash fluid through a plurality of wash channels controlled by a second plurality of controllable valve channels, each wash channel arranged in fluidic connection with a sample channel or air channel, the outlet and the wash fluid; and flushing the sample channels, the air channel and the outlet with the wash fluid. The separate wash channels minimize cross-contamination between wash fluid and samples. This is particularly important when handling small volumes in the microliter range. The valve channels may control each wash channel independently or any combination of channels simultaneously.

In an embodiment, the method further comprises: rinsing the sample channels with water; and flushing the sample channels, the air channel and the outlet.

Each washing step may be followed by purging with air to further reduce the likelihood of cross-contamination.

In an embodiment, each of the samples comprise a replication origin, an antibiotic resistance gene, or a target gene.

The same sample can be used repeatedly to create different recombinant plasmids. This eliminates the need to make multiple mixes of each DNA fragment, thus saving time and costs.

Brief Description of the Figures

Figure 1 shows a microfluidic device fabrication workflow: positive resist and negative resist were used for flow layer mold and control layer mold, respectively.

Figure 2 shows a microfluidic setup for sample (a) with a large volume (> lOOpl), and (b) with a small volume (5 pl - 100 pl). Figure 3 shows (a) Schematic diagram of DNA assembly process, (b) Chip layout.

Figure 4 shows a schematic diagram of the demonstration of microvalve functionality.

Figure 5 shows the chip operation workflow, (a-d) Sample dispensing and collection workflow, (e-h) Channel washing workflow.

Figure 6 shows a graph of collection volume versus collection time under different injection pressure conditions.

Figure 7 shows the high-throughput combinatorial mixing function of the microfluidic chip using dyes, (a) Dyes used to test combinatorial mixing capability, (b) All possible combinations of dyes.

Figure 8 shows the sequential combinatorial mixing process, (a) Chip layout, (b) Chip before sample dispensing. (C) Twelve sequential combinatorial mixing processes.

Figure 9 shows a schematic diagram of the high-throughput combinatorial DNA assembly hierarchy based on the input fragments/variants listed in Table 1. The gene maps of the whole library are provided in Figures 12 and 13.

Figure 10 shows examples of colonies growing on agar plates with antibiotics after cell transformation. Each plate comprises colonies with a particular construct/plasmid, which was assembled using the microfluidic chip.

Figure 11 shows the gel electrophoresis result, verifying the colony-PCR products for random 16 colonies from each origin of replication from different plates.

Figures 12 and 13 show gene maps of the plasmid constructs assembled using the microfluidic chip.

Figure 14 shows examples of the sequencing data of the constructs assembled using the microfluidic chip.

Figure 15 shows additional gene sequencing data. Detailed Description of the Invention

To tackle the problems in existing platforms for DNA assembly, many proposed microfluidic approaches. Microfluidics has shown great potential for the development of high throughput platforms with minimal reagent consumption and the capability to perform multiple reactions in a single platform. Many microfluidic-based technologies have been developed for DNA assembly recently. The most commonly adapted technique for combinatorial DNA assembly was digital microfluidics (DMF) [Shih, S. C. C. et al., ACS Synthetic Biology 4(10): 1151-1164 (2015); Husser, M. C. et al., ACS Synthetic Biology 7(3): 933-944 (2018); Gach, P. C. et al., ACS Synthetic Biology 5(5):426-433 (2016); Ben Yehezkel, T. et al., Nucleic Acids Research 44(4):e35-e35 (2016); Khilko, Y. et al., BMC Biotechnology 18(1):37 (2018)]. DMF is an emerging liquid-handling technology that enables precise control over individual droplets on an open array of electrodes. These picoliter- to microliter-sized droplets serve as an isolated chamber for reaction; and these droplets can be dispensed, moved, mixed and incubated on a set of electrodes. The advantage of DMF is that it does not require external pump to drive the microfluidic flow, and the entire droplet manipulation process is often automated. However, DMF-based approach suffers from a low throughput. Currently, the most efficient DMF setup can assemble 16-24 constructs [Shih et al., ACS Synthetic Biology 4(10):l 151-1164 (2015)]. This limitation in throughput is due to the use of many electrodes and complex algorithm to realize series of droplet manipulation, such as relocation, addition, and division. The electrode layout and wiring options are constrained on a two-dimensional (2D) plane, thus there is a limitation in the maximum number of electrodes placing in a relatively small microfluidic chip. DMF reaches its bottleneck in achieving large scale assembly. In addition, the fabrication of DMF device is rather sophisticated and may have difficulty in manufacturing in large batches.

An alternative microfluidic approach employed 2D microvalve technology to create microscale wells for combinatorial DNA assembly [Linshiz, G. et al., Journal of Biological Engineering 10 (2016); Linshiz, G. et al., ACS Synthetic Biology 3(8):515- 524 (2014)]. Linshiz et al. performed DNA construction with programmable and automated control of the microvavles. However, due to the limited inlets and the constrains of the 2D layout, this device suffers from a low throughput which only allowed the assembly of up to 8 variants in parallel. Pincer valve has also been used for combinatorial DNA synthesis [Tangen, U. et al., Biomicrojluidics 9(4):44103 (2015)], but it also has limited throughput due to similar reasons. To avoid the constraints of limited space in the current valve systems, droplet-based microfluidics was demonstrated with a much higher throughput in DNA assembly [Unger, M. A. et al., Science 288(5463) (2000)]. However, droplet carriers are rather difficult to manipulate precisely in a continuous flow setup and this would lead to a huge challenge in automating the system without a skilled personnel operating. In addition, droplets are very sensitive to the pressure inside the chip, and an optical system is essential to monitor the droplets. An optical system, such as a microscope is often too complex and costly to be integrated for commercial usage.

At this current stage of described technology, mechanical three-way valves were used to demonstrate the capability of our system. Thus a minimal manual input was necessary. However, if the mechanical valves are replaced with pneumatic valves, full automation can be achieved.

In this work, the inventors have developed a microfluidic system for high-throughput combinatorial DNA assembly. The system provides a high-throughput assembly capability, enabling the assembly of up to 100 combinations of DNA fragments per run. The recombinant DNA fragments may be recombinant plasmid constructs. This microfluidic DNA assembly platform consumes minimal reagents and has the potential to automate the assembly process.

The invention comprises a microvalve microfluidic platform for the automated and hassle-free DNA assembly to replace the laborious and low-throughput conventional pipetting technique. The proposed microfluidic device can be loaded with user-defined DNA fragments to perform mixing without time-consuming liquid handling. Through controlling the microvalves to allow the reagents to flow through the microchannel, the desired fragments can be mixed and collected in a high-throughput fashion (< 30s per combination) with a wide volume dispensing range (0.5-20 pl). Here we demonstrated a 16 microchannels to build 65 different plasmid constructs in less than an hour with high success rate verified by sequencing. This platform can be easily automated and scaled to accommodate up to 1000 3-fragment constructs with 30 channels. The proposed technique offers potential for automation of synthetic biology procedures with significantly reduced cost and time.

Examples Example 1

Methodology - Device Fabrication

Microfluidic chip was fabricated with conventional photolithography: Flow layer mold was fabricated with positive resist AZ P4620 (MicroChemicals) and control layer mold was fabricated with negative resist SU-8 2025 (MicroChem).

Example 2

Microfluidic Device Fabrication Workflow

Fabrication workflow of the microfluidic device is provided in Figure 1.

The master mould for the sample layer (comprising sample channels) was fabricated on 4” silicon wafers using positive resist AZ P4620 (MicroChemicals). AZ P4620 was spin-coated onto the wafer at 500 rpm for 10 seconds and then 1000 rpm for 30 seconds. The resulting film thickness was 15 um, calibrated by profilometer. The substrate was baked at 110°C for 3 mins, and then rehydrated at room temperature for 1 hour before exposure. UV exposure of the resist was done with the Karl Suss MJB4 mask aligner (exposure power: 22.8 mW/cm²; exposure cycles: 5 cycle; exposure time: 10s per cycle; interval waiting time: 30s). The resist was developed in AZ 400K developer (MicroChemicals) with 1 :4 developer to water ratio for 90 seconds. After development, the mould was rinsed with DI water and blow-dried with nitrogen gas. Finally, a hard bake was performed at 135°C for 2 minutes to reflow the positive resist, thereby rendering the channel cross-section profile to be rounded. The width of the sample channels is 200 pm at the inlet region and 50 pm at the centre outlet region. The master mould for the valve layer (comprising valve channels) was fabricated using negative resist SU-8 2025 (MiroChem). SU-8 2025 was spin-coated onto a 4” silicon wafer at 500 rpm for 10 seconds and 3000 rpm for 30 seconds, resulting in a 25pm- thick film. Soft bake was performed at 65°C for 1 minute and 95°C for 5 minutes. Exposure was done with Karl Suss MJB4 mask aligner (exposure energy: 180 mJ/cm²). After exposure, the wafer was post baked at 65°C for 1 minute, 95°C for 5 minutes. After cooling down, the resist was then developed in SU-8 developer for 3 minutes with gentle agitation, then rinsed thoroughly with propanol and dried with nitrogen. A hard bake step was performed at 200°C for 10 minutes to improve the mechanical properties and thermal performance of SU-8. The width of the valve channels is 300 pm.

Microfluidic channels were fabricated using multilayer PDMS soft-lithography [Unger, M. A. et al., Science 288(5463): 113 (2000)]. Prior to PDMS (poly dimethyl siloxane) moulding, all master wafer was rendered hydrophobic by vapour deposition of trichloro(lH,lH,2H,2H-perfluorooctyl) silane (Sigma- Aldrich). Sylgard 184 silicone elastomer kit (Dow Coming) was used for soft-lithography. The layer comprising sample channels was made by mixing 10: 1 base and curing agent, and the layer comprising valve channels was made by mixing 20:1 base and curing agent. Both mixers were first degassed for 30 mins before further process. For sample layer fabrication, the degassed PDMS mixture (10:1 base to curing agent ratio) was poured onto the sample layer master wafer and cured at 70°C for 1 hour. After curing, the PDMS layer was peeled-off from the wafer. Inlet and outlet holes for the sample channels were drilled by a biopsy punch. For valve layer fabrication, PDMS (20: 1 base to curing agent ratio) were spun onto the valve layer master wafer at 500 rpm for 10 seconds and then 1200 rpm for 30s, resulting in a thin PDMS layer. This layer was cured on a 100°C hot plate for 10 minutes. The PDMS membrane is thick and stable enough to fully separate the channels in different layers, but still thin enough to allow the sample channels to be pinched shut under positive pressure in the valve channel underneath.

The alignment and bonding of the control layer and flow layer were done manually. The channels are designed in such a way that a precise alignment between different layers is not required. A simple alignment by eye is enough, and there is no need for a special equipment or microscope for the alignment process. Therefore, the fabrication cost related to the alignment process can be significantly reduced. More importantly, the yield for successful chip fabrication can be significantly improved.

Before bonding, the surfaces of PDMS samples were activated by oxygen plasma. Once the sample channels and valve channels were aligned, the two PDMS pieces were brought into conformal contact to form permanent bonding. The alignment and bonding process could be done within 1 minute. After bonding, the sample was further cured on a 100°C hot plate for 15 minutes to strengthen the bonding between PDMS layers. After baking, the PDMS was then peeled off from the valve layer master wafer. Inlet holes for the valve channels were drilled by a biopsy punch. Finally, the PDMS sample and a glass substrate was activated by oxygen plasma and bonded together permanently to close the channels in the valve layer. The final device was again cured in a 70°C oven for 1 hour to improve the bonding strength between PDMS and glass. Example 3

Microfluidic Experiment Setup

The fabricated device and the experiment setup have been provided in Figure 2. We have developed two fluidic setups to accommodate for different desired volumes of input samples. Based on the volume of sample required, user can choose either setup to achieve an effective operation.

For loading input samples with large volume (>100 pl), the fabricated device was connected to sample reservoirs via Tygon tubings (1/16” OD; 0.51 mm ID) and 23G stainless steel couplers (0.025” OD; 0.013” ID). The sample reservoirs were connected to a pressure control system (Fluigent MFCS-EZ) to control the fluidic flow in the microfluidic device. Two pressure channels of the pressure control system were used as two independent pressure sources for the chip: one pressure source for driving the fluidic flow in the sample channels, while the other one for providing pressures in the valve channels. The two pressure sources can be controlled independently, thereby providing two different pressures. Each pressure source was connected to an eighteen- channel pressure manifold with eighteen 3-way valves, so that all the eighteen channels in the same layer (either the sample layer or the valve layer) could share a common pressure. The supplying of pressure to each channel can be independently controlled by the 3 -way valve.

For loading input samples with small volume (5-100 pl), the fabricated device was connected to 23 G needles, which were used directly as on-chip sample reservoirs. In this way, no tubing was used and the dead volume (10-20 pl) in the tubing was eliminated. The reduction in dead volume is critical in the situation where the input sample has a comparative volume, or the sample is precious or scarce. In this setup, the dead volume was typically less than 5 pl. Therefore, this setup can be used for any sample volume in between 5 pl to 100 pl. The needle reservoirs were then connected to a pressure control system (Fluigent MFCS-EZ) to control the fluidic flow in the microfluidic device. The pneumatic setup for the pressure control system was the same as described above.

After the device was connected to the pressure sources, the valve channels were loaded with water and pressurized at 1000 mbar to deplete any air inside the channels. After all the valve channels were filled with water, samples were injected into the sample channels of the device under a constant injection pressure (100-200 mbar). A tubing was connected at the outlet to transfer the output sample from the chip into a collection tube. The combinatorial sample dispensing and mixing process could be performed according to the workflow as described in the Chip operation workflow section.

Example 4

Preparation ofDNA Fragments for Assembly Test on the Chip

A library of plasmid variants was designed comprising configurations of three DNA parts including a replication origin (REP), an antibiotic resistance (AbR), and a target gene-of-interest (GOI). Bio-parts are linked by a random-sequence- 18bp spacer. For the GOI, we placed either a GFP (green fluorescence protein), RFP (red fluorescence protein), and sfGFP (super folding GFP) reporter gene under the control of a constitutive promoter (J23119, J23101, J23106) from the Anderson promoter collection and RBS0034 (IGEM), while REP and AbR were varied (Table 1). Primers for amplification of fragments were designed based on junction sequence between spacers and bio-parts. The illustrations and maps of the plasmids were prepared using SnapGene Ver 5.2.3 (GSL Biotech). The fragments were designed to be compatible with our own DNA assembly technology - SENAX™. In particular, we created fragments with 18bp overlapping region by PCR and primers for PCR amplification were designed based on the sequence of 18bp-overlapping region and the 5’- sequence of the target parts (Primers were listed in Table 2). Fragments were produced by PCR amplification using KOD One PCR Master Mix (TOYOBO) according to the manufacturer’s protocol, followed by Dpnl (NEB) treatment and purification on the 1% agarose gel with QIAquick gel extraction kit(QIAGEN). Solutions of purified DNA (10/20 ng/uL) were used as input DNA fragments for microfluidic chip tests with desired volume per usage. The assembly enzyme mix (SENAX) and buffer were applied onto the chip to catalyse the assembly reaction. All assembled constructs were chemically transformed into either E. coll Stellar (Takara) or E.coli lOBeta (NEB), and grew up on antibiotic (either of Ampicillin, Kanamycin, Chloramphenicol, Spectinomycin) selective-agar plates.

Table 1 : Output library after combinatorial assembly.

Table 2: Primers used in this study

Example 5

Evaluation of Chip Process by Screening of Positive Colonies

The E. coli transformants derived from the transformation of assembly mix were grown and screened on antibiotic screening LB plates and the fluorescent colonies were visualized with a trans-illuminator (GeneDireX, Inc). To evaluate the accuracy of the method, the positive fluorescent colonies were further examined by colony-PCR by KOD One PCR Master Mix (TOYOBO) and the PCR products were visualized on 1% agarose gel. To confirm the junction sequence of assembled construct, cells were cultivated from the fluorescent colonies and the plasmids extracted from each designed plasmid configuration were sent for sequencing (Ist-BASE, Axil Scientific Pte Ltd). The alignment of obtained sequences against design constructs was carried out using Snapgene Ver 5.2.3 (GSL Biotech). Example 6

Chip Operation Workflow

The chip operation workflow is presented in Figure 5. There are mainly two steps: 1) sample dispensing and collection; 2) channel washing. Figure 5(a-d) demonstrates the workflow for sample dispensing and collection. Firstly, all the microvalves on the chip are closed by pressurizing all the valves channels (Figure 5a). At this point, there is no flow in any channel of the chip. Next, the microvalves for the samples to be dispensed are opened by depressurizing corresponding valve channels. For example, samples in channels S2, Se and S12 are dispensed by opening the microvalves for the respective channels, by depressurizing the valve channel V2, Ve and V12 (Figure 5b). The different samples converge at the outlet area (the centre of the chip) and mix with each other when flowing through the outlet. When a desired volume of sample mixture has been dispensed, the valves for the dispensing channels are closed. In this example, the valves channel V2, Ve and V12 are pressurized to stop the sample dispensing in S2, Se and S12, respectively. The air channel is then opened to purge out the sample for collection, via depressurizing the valve channel V_a (Figure 5c). Once the air channel is opened, air is purged into the device to push samples out of the chip and outlet tubing. At the same time, the air purging process also leads to some turbulence and vortex flows at the outlet region, facilitating the sample mixing process. The sample mixtures are collected in collection tubes and ready for the downstream process. After sample collection, all valves are closed again by pressurizing all the valve channels (Figure 5d). At this point, the chip is ready for the next step - channel washing.

The workflow for channel washing is presented in Figure 5(e-h). First of all, the two main washing channels (alternatively called “wash channel”; the ‘Air/OiF channels in Figure 5e) are opened by depressurizing the valve channel Vol and V02. Either air or oil can be used as the washing buffer in these washing channels for cleaning the chip. When these two main washing channels are opened, the washing buffer will enter each sample channel via the side channel, thereby pushing out any liquid residues left in the sample channels (Figure 5e). The ‘Air/Oil’ washing channels are opened for 10-15 seconds to ensure a complete removal of residues and a thorough clean-up of the chip. After this step, all valves are closed again to stop any flow in the chip (e.g., pressurizing Vol and V02 to close the main washing channels). Next, the water washing channel is opened by depressurizing the valve channel Vw (Figure 5f). Fresh water is thus dispensed to the chip. This step is to further clean the outlet region and the tubing to remove any residue that may have attached to the surface of the chip or tubing. After washing with water for 10-15 seconds, the water channel is closed. Next, the main washing channels are opened again for 10-15 seconds to remove any water from the device and the outlet tubing, by depressurizing the valve channel Vol and V02 (Figure 5g). After removing water from the chip, all the valve channels are re-pressurized to close all the microvalves on the chip (Figure 5h). Now, the chip is ready for another run of sample dispensing and mixing. The sample dispensing step and channel washing step are repeated until all the possibilities of input sample combinations are carried out. Figure 8 shows a sequential twelve repeats of the workflow to obtain different input combinations, demonstrating the feasibility, reliability and repeatability of the chip and the workflow described above.

The sample dispensing and mixing step requires 20-30 seconds to finish and the channel washing step needs 30-60 seconds to complete. In total, the construction of one combination of input samples takes about 1 minute to 1.5 minutes to complete. It should be noted that the time required for constructing one combination/construct can be further reduced by automating the system and optimising the software control. The proposed chip can be easily automated because of the standardized operation workflow. Therefore, this technique provides great potential for automating the DNA assembly process and speeding up the design and test cycle for synthetic biology.

Results

DNA Assembly

DNA parts (20-40 ng/pl) were prepared as described in Example 1. Assembly enzyme and buffer were used for the DNA assembly. Each DNA part was first mixed with 10% enzyme mix, and then loaded into each sample channel. Table 3 presents an example of loading 14 DNA parts into the microfluidic chip for combinatorial DNA assembly. However, the sample loading sequencing is not limited to the one listed in the table. As the chip provides great flexibility in terms of sample loading, users can load the fragments into the chip by any order and quantity.

Table 3: An example of DNA fragment loading sequence for the chip-based DNA assembly process.

Channel SI . S2. S3. S4. S5. S6. S7. S8. S9. S10. S I I S 12 S 13. S 14.

DNA parts ^RSF P^ucP^{BR322 15A ColE1} RFP sfGFP101GFP106GFP119GFPAmp Cm Km Spc

On-demand DNA fragments dispensing was achieved by controlling corresponding valve channels. After dispensing, the DNA parts to be assembled met at the outlet and mixed with each other at the outlet region and in the outlet tubing. The output sample (i.e., the mixture of different DNA parts) was then pushed out from the outlet tubing and collected in collection tubes. After all the combination of DNA parts were conducted, the samples were incubated at 37°C for 15 mins to form full plasmids, finishing the assembly process. Table 1 summarises all the output combinations/constructs obtained from the chip. Afterwards, the assembled plasmids were transformed in E. coli cells for downstream screening and analysis.

Demonstration of chip functionality and capability

The microfluidic chip described in this work allows for liquid dispensing and sample collection in a controllable manner. The sample volume collected can be easily controlled by either the collection time or the injection pressure at the sample channels. Figure 6 shows a calibration curve showing the collection volume versus collection time. Different injection pressures at the sample channels are presented. The higher the injection pressure, the higher volume can be collected in the same time period. The longer the collection time, the more sample can be collected. For example, it takes 30 seconds to collect 7 pl of sample under 150 mbar injection pressure, while at 200 mbar, collecting 7 pl of sample takes 20 seconds. Users can have high controllability and flexibility in terms of the collection volume. Using a lower injection pressure (e.g. 50 mbar or less), it is also possible to collect samples with volume in sub-microlitre range. Working in low volume range can greatly reduce the consumption in reagent, thereby significantly decreasing the cost for synthetic biology processes. In summary, the proposed microfluidic chip allows for a wide range of dispensing volume, from <lul to >10ul, which can cover most situations for synthetic biology.

To demonstrate the high-throughput combinatorial mixing capability of the microfluidic chip, dyes were first used to examine the functionality of the chip and also for the purpose of easy visualization (Figure 7). Red (R), yellow (Y), blue (B) and green (G) dyes are used. (Dyes appear in greyscale in the drawings.) The dyes were diluted two times to get Rl, Yl, Bl and Gl, and ten times to get R2, Y2, B2 and G2. Water (H) and purple dye (P) were added to increase the variants. To mimic 3-fragment DNA assembly, the fourteen colours were grouped into 3 groups. The first group contained R, Y, B, G and H (5 variants); the second group contained Rl, Yl, Bl, Gl and P (5 variants); and the third group contained R2, Y2, B2, G2 (4 variants). If one colour was chosen from each of the three groups to combine to a new colour, there would be 100 (i.e., 5x5x4) possible combinations.

The microfluidic chip was used to perform high-throughput combinatorial sample dispensing and mixing. The dyes were loaded into the each of the sample channels respectively, as shown in Figure 7a. The on-chip combinatorial mixing process was performed according to the workflow described in the previous section. The output dye mixtures were collected in a 96-well plate. The table in Figure 7b lists all the possible combinations of the input colours, while the picture shows the output ‘synthetic’ colours collected from the chip, with each colour corresponding to one combination in the table. There was no visible contamination between each combinations, demonstrating the good functionality of the chip. The dye experiment also demonstrates the high- throughput capability of the chip. As the dye experiment mimics the 3-fragment assembly process, it also demonstrates that the chip is capable of constructing an output library of up to 100 variants per experiment for 3-fragment assembly process.

Combinatorial DNA assembly Following the testing of the chip using colour dyes, we next evaluated and demonstrated the chip’s capability in performing combinatorial DNA assembly. As a case study, using the chip, we built a large combinatorial library of constructs based on 3 -fragment assembly using homology based method. Figure 9 presents an example of how a combinatorial library is constructed, based on 3-fragment assembly. Three DNA fragments are involved, including the fluorescence gene, the antibiotics-resistance gene, and the origin of replication. For each DNA fragment, there are multiple variants. For example, there are five fluorescence gene variants (e.g., RFG, sfGFP, 101GFP, 106GFP and 119GFP), four antibiotic-resistance gene variants (e.g., Amp, Km, Cm and Spc), and five origins of replication (e.g. RSF, pUC, pBR322, 15A and ColEl) for the coding sequences that were used to generate the combinatorial library. In total, there are 100 possible combinations to assemble all the three DNA parts into a plasmid.

DNA parts (10-20 ng/pl) were prepared as described in Methodology. Assembly enzyme and buffer were used for the DNA assembly. Each DNA part was first mixed with 10% enzyme mix, and then loaded into each sample channel. Table 3 presents an example of loading 14 DNA parts into the microfluidic chip for combinatorial DNA assembly. However, the sample loading sequencing is not limited to the one listed in the table.

On-demand DNA fragments dispensing was achieved by controlling corresponding valve channels. After dispensing, the DNA parts to be assembled met at the outlet and mixed with each other at the outlet region and in the outlet tubing. The output sample (i.e., the mixture of different DNA parts) was then pushed out from the outlet tubing and collected in collection tubes. After all the combination of DNA parts were conducted, the samples were incubated at 37°C for 15 mins. After which, the plasmids were transformed into E.coli cells for downstream screening and analysis.

The gene maps for all the constructs are provided in Figures 12 and 13.

Evaluation of the method

To demonstrate the functionality of our DNA assembly chip, 3 fragments DNA assembly was performed and evaluated. Fragments and enzyme mix were mixed up by the chip and the resulting mix was incubated at 37°C for 15 mins, followed by transformation. The accuracy of the method was evaluated by examination of 16 colonies per each origin of replication involved. To this end, the colony-PCR products for random 16 colonies from each origin of replication from different plates were verified on agarose gel. As the result shown in Figure 11, a single band corresponding to 765-565-752-860-750-1713 bp fragment appeared in all wells (16/16) for configuration of RSF, fl, pUC19, pBR322, 15A, pSClOl origin of replication, respectively. This demonstrated that our assembly method is highly reliable; any of the randomly picked colonies appeared on the plate were assembled correctly. Because the size of amplified fragments appeared to be correct on the gel and the primers were designed to specifically bind to the target sequence, there was no sign of cross contamination among different origins that were used, regardless of fragment positioning in the chip. Depending on the origin of replication in the configuration and aliquot brought down to transformation, dozens to hundreds fluorescent colonies were achieved with different batches of experiment (Figure 10). This number is comparable with that of the conventional assembly with benchtop pipetting. Plasmid derived from each configuration (65) were sent out for Sanger sequencing.

Figure 14 presents the sequencing results of twelve variants assembled using the microfluidic chip. The sequencing data for the rest of achieved constructs in the library are provided in Figure 15. Based on the sequencing results, among whole 65 constructs, junction sequence (18bp and relative area) between fragments in obtained construct were identical to the design. This implies that the inventors’ on-chip mix assembly method was able to precisely concatenate the fragment without running errors (or mutations) into constructs. It is important that every construct obtained from this system is highly reliable, reducing the efforts of downstream verification. The inventors successfully created a mid-size library of combinatorial constructs that differ from each other either by GOI, AbR or REP. This is the first time such high number of variants was obtained using a microfluidic chip.

Conclusions

A microfluidic device has been developed for large-scale combinatorial DNA assembly with low reagent consumption. The device was designed with low complexity in fabrication and simple external flow control system, thereby significantly reducing the cost of the hardware for automated DNA assembly. The device is capable of assembling up to 100 constructs per run for 3-fragment DNA assembly process. The device also provides promising scalability, since more channels can be easily added to get more constructs per run and achieve an even higher throughput. The current design can accommodate up to fourteen different input samples, providing great flexibility for users to select inputs, load samples, and assemble constructs. A wide range of collection volume can be achieved, ranging from sub-microliter range to tens or hundreds of microliters, which can suit most of synthetic biology scenarios. The device provides great potential for automating the DNA assembly process and speeding up the design and test cycle for synthetic biology. In summary, the microfluidic device proposed in this work provides great potential for synthetic biology study.

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Claims

29 Claims

1. A microfluidic device for creating recombinant plasmid constructs, comprising: a first layer comprising a plurality of sample channels and an air channel arranged to converge with an outlet; a second layer comprising a first plurality of controllable valve channels arranged to control each sample channel, wherein the first layer is adjacent to the second layer, each sample channel is in fluidic connection with the outlet and a sample reservoir; the air channel is in fluidic connection with the outlet and an air inlet.

2. The device according to claim 1, wherein the first layer further comprises a plurality of wash channels; and the second layer further comprises a second plurality of controllable valve channels arranged to control each wash channel, wherein each wash channel is in fluidic connection with a sample channel or air channel, the outlet and a wash fluid.

3. The device according to claim 2, wherein the wash fluid comprises air, oil or water.

4. The device according to any one of claims 1 to 3, wherein the sample reservoir is arranged to comprise a DNA fragment selected from the group consisting of: a replication origin, an antibiotic resistance gene, and a target gene.

5. A method of creating recombinant plasmid constructs, comprising the steps of: a) providing a microfluidic device comprising a first layer comprising a plurality of sample channels and an air channel arranged to converge with an outlet; a second layer comprising a first plurality of controllable valve channels arranged to control each sample channel, wherein the first layer is adjacent to the second layer, each sample channel is in fluidic connection with the outlet and a sample reservoir; the air channel is in fluidic connection with the outlet and an air inlet; 30 b) releasing at least two samples from the sample reservoirs through the sample channels into the outlet; c) stopping sample flow in the sample channels; d) purging the sample channels and the outlet; and e) collecting a sample mixture from the outlet. The method according to claim 5, further comprising: f) flushing a wash fluid through a plurality of wash channels controlled by a second plurality of controllable valve channels, each wash channel arranged in fluidic connection with a sample channel or air channel, the outlet and the wash fluid; g) flushing the sample channels, the air channel and the outlet with the wash fluid. The method according to claim 5 or 6, further comprising: h) rinsing the sample channels with water; and i) flushing the sample channels, the air channel and the outlet with the wash fluid. The method according to any one of claims 5 to 7, wherein each of the samples comprises a DNA fragment selected from the group consisting of: a replication origin, an antibiotic resistance gene, and a target gene. The method according to any one of claims 6 to 8, wherein the wash fluid comprises air, oil or water.