Method for the assembly of a polynucleic acid sequence

Abstract

Provided herein are methods for the assembly of a polynucleic acid sequence that is at least partially carried out on a microfluidic device; methods for the preparation of a library of polynucleic acid sequences; microfluidic devices; methods for designing nucleic acid sequences; methods for planning the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences; systems comprising components for carrying out these methods; computer programs which, when run on a computer, implements these methods; and computer readable medium or carrier signals encoding such a computer program.

Claims

1. A method for the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences in which the polynucleic acid sequence is of a formula N.sub.n+1, in which N represents a nucleic acid sequence and where n is 1 or greater than 1 and each N may be the same or a different nucleic acid sequence, in which the method comprises: (i) providing a first nucleic acid sequence N1 which has an oligonucleotide linker sequence L1.sup.3′ attached at the 3′-end of the nucleic acid sequence; (ii) providing a second nucleic acid sequence N2 which optionally has an oligonucleotide linker sequence L2.sup.3′ attached at the 3′-end of the nucleic acid sequence and which has an oligonucleotide linker sequence L2.sup.5′ attached at the 5′-end of the nucleic acid sequence, wherein the 5′-end linker sequence L2.sup.5′ of nucleic acid sequence N2 is complementary to the 3′-end linker sequence L1.sup.3′ of nucleic acid sequence N1; (iii) optionally providing one or more additional nucleic acid sequences N, wherein nucleic acid sequence N2 has an oligonucleotide linker sequence L2.sup.3′ attached at the 3′-end of the nucleic acid sequence, and wherein said one or more additional nucleic acid sequences N comprises a terminal additional nucleic acid sequence NZ, and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence attached at its 3′-end, wherein said terminal additional nucleic acid sequence NZ optionally lacks an oligonucleotide linker sequence at its 3′-end and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence attached at its 5′-end, wherein for the first additional nucleic acid sequence N3 the 5′-end linker sequence L3.sup.5′ is complementary to the 3′-end linker sequence L2.sup.3′ of nucleic acid sequence N2 and for each second and subsequent additional nucleic acid sequence N the 5′-end linker sequence is complementary to the 3′-end linker sequence of the respective preceding additional nucleic acid sequence; (iv) ligating said nucleic acid sequences to form said polynucleic acid sequence, wherein said nucleic acid sequences are optionally purified immediately prior to said ligating; wherein at least step (iv) is carried out on a microfluidic device; and wherein the method does not require polymerase.

2. A method according to claim 1, wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1.sup.5′ attached at the 5′-end of the nucleic acid sequence; or wherein said second nucleic acid sequence N2 has an oligonucleotide linker sequence L2.sup.3′ attached at the 3′-end of the nucleic acid sequence; or wherein said terminal additional nucleic acid sequence NZ has an oligonucleotide linker sequence LZ.sup.3′ attached at the 3′-end of the nucleic acid sequence; or wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1.sup.5′ attached at the 5′-end of the nucleic acid sequence, and wherein said second nucleic acid sequence N2 has an oligonucleotide linker sequence L2.sup.3′ attached at the 3′-end of the nucleic acid sequence, and wherein the 5′-end linker sequence L1.sup.5′ of nucleic acid sequence N1 is complementary to the 3′-end linker sequence L2.sup.3′ of nucleic acid sequence N2; or wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1.sup.5′ attached at the 5′-end of the nucleic acid sequence, and wherein said terminal additional nucleic acid sequence NZ has an oligonucleotide linker sequence LZ.sup.3′ attached at the 3′-end of the nucleic acid sequence, and wherein the 5′-end linker sequence L1.sup.5′ of nucleic acid sequence N1 is complementary to the 3′-end linker sequence LZ.sup.3′ of nucleic acid sequence NZ.

3. A method according to claim 1, wherein each of said 3′-end linker sequences and said 5′-end linker sequences is partially double stranded; or wherein each said nucleic acid sequence has an overhang at each end.

4. A method according to claim 3, wherein said overhang at each end of said nucleic acid sequence is produced by digestion with one or more restriction enzymes; or wherein said overhang is 3 or 4 nucleotides in length; or wherein the overhang at the 3′-end of the nucleic acid sequence and/or at the 5′-end of the nucleic acid sequence is the same for each nucleic acid sequence.

5. A method according to claim 1, wherein each said nucleic acid sequence is attached to its said 3′-end linker sequence and to its said 5′-end linker sequence by ligation.

6. A method according to claim 1, wherein said nucleic acid sequences are purified on the microfluidic device; or purified using DNA purification spin columns or gel extraction.

7. A method according to claim 1, wherein each said nucleic acid sequence is a protein coding sequence or a regulatory or control element.

8. A method according to claim 1, wherein step (iv) is carried out using DNA ligase.

9. A method for the preparation of a library of polynucleic acid sequences, the method comprising simultaneously producing a plurality of different polynucleic acid sequences using the method according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as limiting on the invention. Reference is made to a number of Figures, in which:

(2) FIG. 1 is a schematic diagram of the method of the present invention. In phase 1, the parts and linkers are prepared. In phase 2, parts are ligated to appropriate linkers based on the desired pathway assemblies. In phase 3, all parts are ligated together. In this example, there are 3 parts being assembled: part A, part B and the plasmid backbone. Depending on the ligation method used, the assembly may leave a standard scar sequence between the parts (e.g. 3 bp).

(3) FIG. 2 is a schematic diagram of the part preparation phase of one embodiment of the invention. Parts are prepared to have overhangs and are stored with a set of oligos associated with the part. The overhang at the 3′-end of Part A (truncated) is a standard 3 bp sequence common to all parts in a library. The biotinylated oligo 5.sub.A can be used for purifying the part. The biotin is represented by the circle. Oligos 3.sub.A and 4.sub.A are stored for use during the assembly process.

(4) FIG. 3 is a schematic diagram of the part-linker fusion phase of one embodiment of the invention. In the part-linker fusion phase, part A is ligated with oligos for the next part B.

(5) FIG. 4 is a schematic diagram of the pathway assembly phase of one embodiment of the invention. In the pathway assembly phase, part-linker fusions are ligated together.

(6) FIG. 5 is a schematic diagram of the purification of the final assembly in one embodiment of the invention. The final assembly can be purified via biotinylated oligos (5.sub.A and 4.sub.D). The biotin is represented by the circle.

(7) FIG. 6 is a schematic diagram of a part-linker DNA assembly scheme using partially double-stranded oligonucleotide linkers.

(8) FIG. 7 is a schematic diagram of a part-linker DNA assembly scheme using partially double-stranded oligonucleotide linkers and truncated parts.

(9) FIG. 8 shows the expected flanking sequences (overhangs) on parts following digest with (A) EcoR1/Spe1 and (B) SapI/EarI. It can be seen that the parts prepared using EcoR1/Spe1 have standard 4-bp overhangs, whilst the parts prepared using SapI/EarI have standard 3-bp overhangs.

(10) FIG. 9 is a schematic diagram of the protocol used for 2-part assemblies in Example 1.

(11) FIG. 10 shows in more detail the protocol used for 2-part assemblies in Example 1. The letters in the last column of FIG. 10 refer to chip cycles shown in FIG. 28.

(12) FIG. 11 shows a purification approach that can be used in the part/linker pair purification step.

(13) FIG. 12 shows the 3D layout of the microfluidic device used in Example 1. The two input chambers have parallel fluid channels linking them to the central elliptical reaction chamber. The fluid channels of the two input chambers have two valves and a pump chamber. The storage chamber has a fluid channel linking it to the central elliptical reaction chamber. The fluid channel of the storage chamber also has two valves and a pump chamber. The two output chambers are linked by a fluid channel to the central elliptical reaction chamber. Control of the device is provided by 12 air channels, one temperature control and one magnetic part.

(14) FIG. 13 shows the dimensions of the microfluidic device used in Example 1.

(15) FIG. 14A shows the layout of another microfluidic device for use in the present invention. FIG. 14B shows the layout of asymmetric channels in this microfluidic device. FIG. 14C shows nozzled tip channels in this microfluidic device.

(16) FIG. 15 shows the layout of another microfluidic device for use in the present invention.

(17) FIG. 16: Packaging of microfluidic membrane using laser cutting and one layer of double-sided adhesive tape. The steps are as follows: 1. 3M Adhesive transfer tape 467 MP applied on the PMMA block. 2. Load the PMMA block into the Epilog tool. Load the CAD file and set the parameters on Corel Draw. 3. Laser rastering and vector cutting. Raster parameters PMMA+tape: 25/100. Vector cutting PMMA+tape: 5/100/5000. 4. Peel the protective layer 5. Apply the membrane on the adhesive layer and deframe it. 6. Chip ready with an excellent bonding

(18) FIG. 17: Cut-through packaging technique using two layers of double-sided adhesive layers. The steps are as follows: 1. Laser cutting of the 4 mm PMMA sheet, Microfluidic access, and edges of the chip. 2. Laser cut-through of the microfluidic channels in a 0.125 mm layer of PMMA covered on both faces with 50 μm adhesive layer and protective cover. 3. Release protective cover, align and bond the channels (2) onto the PMMA block (1). Release the second protective cover and place the membrane.

(19) FIG. 18 shows schematic views of the machining of hard magnets using (a) powder blasting and (b) milling or grinding using diamond coated tools; (c) shows some examples of possible machined structures on hard magnets.

(20) FIG. 19 shows the use of machined soft iron parts to focalise the magnetic field applied in order induce the same effect as for the machined magnets

(21) FIG. 20 shows the use of amorphous magnetic materials to cover the exposed or machined areas on the hard magnet.

(22) FIG. 21 shows a schematic view showing how the slight vibrations of the membrane can help during the mixing process.

(23) FIG. 22: Schematic views showing the layouts for the magnetic coils for (a) circular and rectangular geometries on PCB board, (b) rectangular and (c) semi-circular geometries for winding enamelled copper wire.

(24) FIG. 23: Schematic top and side views of the magnetic coil based system for separation and mixing of magnetic particles; (a) shows a configuration for which the magnetizing field is applied from under the chip, where the coils are applied, through a machined soft iron part, and (b) shows a configuration in which the magnetizing magnetic field is generated by a stack of hard magnets applied on opposite sides of the chip.

(25) FIG. 24: Isometric left and right views of the heating/cooling and electromagnet sub-assembly.

(26) FIG. 25: Isometric view of the mechanical assembly for a control unit for a microfluidic device as described herein, which also includes the heating/cooling and electromagnet sub-assembly.

(27) FIG. 26 is a diagram of the microfluidic functions and the different biological components involved in the first purification step of the plasmid pSB1C3 as described in Example 1.

(28) FIG. 27 demonstrates an example of how to operate a pump to flow liquid from one of the chambers into another chamber in a microfluidic device as described herein.

(29) FIG. 28 summarises a number of different fluidic steps that were used in Example 1.

(30) FIG. 29 is a flowchart of the method of the sixth aspect of the invention.

(31) FIG. 30 is a flowchart of the method of the seventh aspect of the invention.

(32) FIG. 31 shows exemplary inputs and outputs of the seventh aspect of the invention.

(33) FIG. 32 is a schematic of the bioinformatics aspects of the invention.

(34) FIGS. 33a and 33b are schematic illustrations of a microfluidic device according to one embodiment.

(35) FIG. 34 is a schematic illustration of a control platform.

(36) FIG. 35 is a schematic illustration, in perspective view, of the microfluidic device and the control platform.

(37) FIG. 36 is a schematic illustration of a microfluidic device and a control platform, which include alignment holes and pillars.

(38) FIGS. 37a and 37b are schematic illustrations a microfluidic device and a control platform in a further embodiment.

(39) FIGS. 38a to 38c are schematic illustrations of a microfluidic device and a control platform according to a further embodiment.

(40) FIG. 39 is a schematic illustration of a microfluidic device and a control platform according to another embodiment.

(41) FIGS. 40a and 40b are schematic illustrations of a microfluidic valve.

(42) FIGS. 41a and 41b are schematic illustrations of a microfluidic pump or mixer.

(43) FIG. 42 is a graph of pumping rate as a function of actuator operation frequency for the microfluidic pump of FIGS. 40a and 40b.

(44) FIG. 43: (A) Chemical structure of the indole-derivative, Violacein. (B) The biosynthesis of violacein from the precursor, L-tryptophan. Note that VioC and VioD have overlapping function and thus, only VioC was utilized in the assemblies.

(45) FIG. 44 shows the thermocycler conditions used for the linked ligation/digestion reactions in Example 4.

(46) FIG. 45 shows the results of gel based purification of the pathway assembly components in Example 4.

EXAMPLES

Example 1—2-Part Assembly on Chip

(47) Protocols for on-Chip Assembly

(48) The biology reactions consisted of two 2-part assemblies: RFP or GFP with a plasmid backbone pSB1C3 (pSB1C3 is also referred to herein as 1C3 or 1c3). pSB1C3 encodes resistance to the antibiotic chloramphenicol. A successful assembly of pSB1C3.GFP produces green cells and a successful assembly of pSB1C3.RFP produces red cells. The number of colonies (yield) and percent of colonies with correct phenotype (efficiency) was determined for test assemblies performed both on chip (using the microfluidic device) and off chip (in the conventional fashion with tubes and pipettes).

(49) The following parts (nucleic acid sequences), oligos (linkers) and reagents were used, as shown in FIGS. 9 and 10. Pre-prepared parts: pSB1C3 DNA pre-digested with EarI RFP DNA pre-digested with EarI GFP DNA pre-digested with EarI uncut pSB1C3.RFP as a positive control for transformation 2 pairs of part/linker oligos for the 2-part assemblies, P1, L1, P2, L2 matching biotinylated purification oligos for the above part/linker oligos, PP1, PL2, PP2, PL2 Buffers: Binding buffer: 10 mM Tris-HCl pH 7.5, 500 mM NaCl Elution buffer: 10 mM Tris-HCl pH 7.5 Salt solution: 4M NaCl Ligation mix: 15 ul mix is 10 ul T4 DNA ligase buffer, 4 ul T4 DNA ligase (New England Biolabs, MA catalogue #MO202S), 1 ul EarI (New England Biolabs, MA catalogue #R0528S) Magnetic beads: Streptavidin magnetic beads (New England Biolabs, MA catalogue # S1420S). These are 1 μm superparamagnetic particles covalently coupled to a highly pure form of streptavidin. The magnetic beads bind to the biotinylated part purification oligos.

(50) The protocol for the on-chip method is as follows and as described in more detail in FIG. 10:

(51) Ligations: 7.5 uL DNA parts, 0.5 uL of each oligo, 1.5 uL ligase mix

(52) Ligations carried out were pSB1c3 with oligos P1 and L2, RFP with oligos P2 and L1, GFP with oligos P2 and L1

(53) Mix and incubate for 2 hours at room temperature (RT)

(54) The biological and fluidic protocols used for two-step purification of RFP and pSB1C3 parts were as follows and as described in more detail in FIG. 10 (see part purification step 1 and part purification step 2). As described below, three different protocols were used and so in FIG. 10, the volume of each reagent is indicated as [Vx], x being a subscript corresponding to the particular reagent.

(55) Initial Protocol:

(56) 1. In a new or washed chip, 25 uL of beads are loaded in the reaction chamber and washed with 50 uL of binding buffer 2. Pump 2 uL of oligos with 10 uL binding buffer 3. 10 minutes wait at RT 4. Wash with 20 uL binding buffer 5. 15 uL ligation mix and 2 uL NaCl 6. About 10 minutes wait at RT 7. Wash with 20 uL elution buffer 8. Heat at 65 C then pump another 20 uL elution buffer 9. Collect 20 uL elution product e1 10. In a new or washed chip, 25 uL of beads are loaded in the reaction chamber and washed with 50 uL of binding buffer 11. Pump 2 uL oligos with 10 uL binding buffer 12. 10 minutes wait at RT 13. Wash with 20 uL binding buffer 14. 20 uL elution product e1 and 2.5 uL NaCl 15. 10 minutes wait at RT 16. Wash with 20 uL elution buffer 17. Heat at 65 C then pump another elution buffer 18. Collect 20 uL elution product e2
Updated Purification Protocol A 1. In a new or washed chip, 25 uL of beads are loaded in the reaction chamber and washed with 50 uL of binding buffer 2. Pump 5 uL of oligos with 10 uL binding buffer 3. 10 minutes wait at RT 4. Wash with 20 uL binding buffer 5. Pump 15 uL ligation mix and 2 uL NaCl 6. About 10 minutes wait at RT 7. Wash with 30 uL elution buffer 8. Heat at 65 C then pump another 20 uL elution buffer 9. Collect 20 uL elution product e1 10. In a new or washed chip, 25 uL of beads are loaded in the reaction chamber and washed with 50 uL of binding buffer 11. Pump 5 uL oligos with 10 uL binding buffer 12. 10 minutes wait at RT 13. Wash with 30 uL binding buffer 14. 20 uL elution product e1 and 2.5 uL NaCl 15. 10 minutes wait at RT 16. Wash with 20 uL elution buffer 17. Heat at 65 C then pump another 20 uL elution buffer 18. Collect 20 uL elution product e2
Updated Purification Protocol B 1. In a new or washed chip, 20 uL of beads are loaded in the reaction chamber and washed with 50 uL of binding buffer 2. Pump 5 uL of oligos with 10 uL binding buffer 3. 10 minutes wait at 20 C 4. Wash with 50 uL binding buffer 5. Pump 15 uL ligation mix and 2 uL NaCl 6. About 10 minutes wait at 20 C 7. Wash with 50 uL binding buffer 8. Pump 5 uL elution buffer, Heat at 65 C then pump another 10 uL elution buffer 9. Collect 10 uL elution product e1 10. In a new or washed chip, 20 uL of beads are loaded in the reaction chamber and washed with 50 uL of binding buffer 11. Pump 5 uL oligos with 10 uL binding buffer 12. 10 minutes wait at RT 13. Wash with 50 uL binding buffer 14. 10 uL elution product e1 and 2.5 uL NaCl 15. 10 minutes wait at RT 16. Wash with 20 uL elution buffer 17. Pump 5 uL elution buffer in the main chamber, heat at 65 C then pump another 10 uL elution buffer 18. Collect 10 uL elution product e2
Off-Chip/on-Chip Discrepancy: Second wash step only for the off-chip mixture
Pathway assembly: 1.5 uL ligase mix, 4.25 uL plasmid, 4.25 uL RFP or GFP

(57) Transformation was carried out in competent E. coli cells (New England Biolabs, MA catalogue # C30191) following the manufacturer's protocol. When purification protocol A was followed, the transformation protocol was: 5 uL ligation in 50 uL cells with 800/900 uLs SOC medium. When purification protocol B was followed, the transformation protocol was: 2 uL of ligation in 50 uL cells with 300 uL SOC medium. The transformed cells were plated out onto plates containing the antibiotic chloramphenicol and the outcome of the experiments determined by counting the number of colonies (yield) and percent of colonies with correct phenotype (efficiency). A successful assembly of pSB1C3.GFP produces green cells and a successful assembly of pSB1C3.RFP produces red cells.

(58) Microfluidic Device and System

(59) The method of the present invention is carried out on a microfluidic device. In the experiments described in this Example, the biological parts, liquids and reagents were first loaded onto the microfluidic chip. Then, an automated control unit platform, based on air and pneumatic valve actuation, allowed the user to perform all the biological manipulations and reactions on chip. At the end of the process, the biological products were then collected from the outputs.

(60) The microfluidic device used in this Example has a number of microfluidic functions. In addition to the basic microfluidic functions of pumping and mixing, the microfluidic device also has heating and magnetic control functions. The basic 3D layout of the microfluidic device used in this Example is shown in FIG. 12, and FIG. 13 shows the dimensions of the microfluidic device. The device contains two input chambers, two output chambers (for waste and/or products) and one storage chamber (temporary holding chamber or reservoir) with fluid channels linking each of these chambers to a central elliptical reaction chamber. Each “pump” unit for the two input chambers and the storage chamber feature two valves and one pump chamber, as shown in FIG. 13. The storage chamber allows the storage of product whilst flushing the reaction chamber. As can be seen from FIG. 13, the depth of all of the structures (chambers, channels etc) is 250 μm and the dimensions of the microfluic device are 70.1 mm×90.1 mm.

(61) As shown in FIG. 13, the inlet channel 1 comprises the first input chamber, together with its two valves and one pump chamber, the inlet channel 2 comprises the second input chamber, together with its two valves and one pump chamber, the intermediate product channel comprises the storage chamber (temporary holding chamber or reservoir), together with its two valves and one pump chamber, and each of the two waste and/or product chambers comprises an output chamber together with its valve.

(62) A total of 12 pneumatic valves had to be synchronously controlled for driving the liquid flow in the microfluidic chip to and from the reaction chamber, along with one temperature control for the reaction chamber and one magnetic part for trapping and mixing of the magnetic particles to be used in the biological reactions. The valves can be numbered as shown in FIG. 28.

(63) The microfluidic system used in this Example has three main parts: The mechanical assemblies The electronics The software interface
1. The Mechanical Assemblies

(64) The mechanical assemblies can be sub-divided into two separate sub-assemblies. The first one contains both a heating/cooling element and an electromagnet used to magnetize the magnetic particles in the reaction chamber. FIG. 24 shows isometric left and right views of this sub-assembly.

(65) The peltier device sits on a heat sink which in turn is mounted on top of a cooling fan. The peltier was positioned just underneath the reaction chamber, and was placed just on top of the soft iron part used to conduct the magnetic field lines to the reaction chamber. The cooling fan slides in a holder in which the base has an array of four compression springs. The springs are used to press the peltier element against the chip. All the different components are aligned and fixed using a set of four mounting brackets (as seen on the left image).

(66) In addition, the first sub-assembly contains an electromagnet which was used for the magnetic part of the device. The electromagnet also sits on a mounting bracket and has a soft iron bar connected to it. The soft iron bar is used as a magnetic canal to provide the chip with the required magnetic field in order to magnetize the magnetic particles.

(67) As an alternative to the above described heater/cooler module with electromagnet sub-assembly, another possible configuration would be to put the peltier element on top of the machined soft iron part, just underneath the reaction chamber, and replace the electromagnet by a hard magnet which would then allow for both temperature and magnetic control on chip.

(68) The second sub-assembly is the mechanical structure that holds all the different mechanical components, air connections, electronics, and on which the microfluidic device is mounted. The 3D design for this sub-assembly is shown in FIG. 25. The choice of plastic board allowed the base plate to be manufactured very quickly and efficiently. This plate sits in a three parts holder which allows both easy alignment of the microfluidic device and enough space for the air connections to come from the bottom of the plastic base.

(69) Finally, all the different mechanical parts sit on a thick plastic base plate, which has all the air connections coming from two opposite sides, and the other two opposites sides have two hook clamps fitted on them. These are used to press the microfluidic device against the plastic board through the two parts lid.

(70) In the absence of an air connection to the reaction chamber, the vibration of the membrane to promote mixing of the different bio-products could be induced using a vibrating piezoelectric film that can adhere to any physical support underneath, and which would generate slight transversal displacements of the membrane.

(71) 2. The Electronics

(72) The liquid flow in the microfluidic channels is driven by synchronized pumping sequences. These sequences are controlled from a LabVIEW interface through an electronic platform connected to an array of pneumatic valves. These valves provide compressed air, through sealed connections, to the chip thus deforming the elastic membrane covering the pumps and valves chambers. By using optimized pumping sequences, it is possible to have accurate control of the liquid flow in the microchannels. The electronic board that allows controlling each valve separately, or a set of valves in order to induce pumping sequences, is mainly a relay board made of an array of solid state relays, in which each relay is addressing one single pneumatic valve. This electronic board has two inputs connected to both the DAQ card (with a 25 way cable) and to a power supply, and two outputs connected to the solenoid valves manifolds by the mean of 15 way cables.

(73) To control the peltier device, two thermocouples were used. The first one was placed above the Peltier device while the second one was placed below the reaction chamber. The electronics behind the temperature control of the peltier device relies on the use of a programmable power supply unit, and a National Instruments DAQ card. The Peltier can be controlled and activated via the LabVIEW control interface described below. The temperature can also be monitored via the same interface.

(74) 3 LabVIEW Control Interface

(75) A semi-automated LabVIEW interface was used for the control of the microfluidic device platform.

(76) Operation of Microfluidic Device for on-Chip Assembly

(77) FIG. 26 summarizes how the biology interacts with the different microfluidic functions for a specific example: the first purification (part purification step 1) of the pSB1C3 ligation mix, as shown schematically in FIGS. 9 and 10 (NB in FIG. 26, pSB1C3 is referred to as 1C3). This step and all other purification steps require the entire range of functions present on the microfluidic system: pumping, mixing, magnetic capture and heating. These steps were extensively demonstrated on-chip (see below).

(78) As can be seen from FIG. 26, the products pumped in were the pSB1C3 DNA, the biotinylated purification oligo PP1 and streptavidin magnetic beads. These products were pumped through to the central reaction chamber where magnetic capture of the magnetic beads, mixing of the input products and heating took place. The output of these reactions was the product of pSB1C3-PP1 bound to the magnetic beads by means of the purification oligo PP1. The elution product in one of the output chambers was pSB1C3-PP1 whilst the other output chamber contained the waste, which was unligated pSB1C3 and PP1.

(79) Pumping to flow liquid from one of the input chambers into the reaction chamber was carried out as shown in FIG. 27. In this Example, only one pump between one input chamber and the reaction chamber was used at a time. Therefore, while one pump between one input chamber and the reaction chamber was activated, the other pump between the other input chamber and the reaction chamber remained closed at all times.

(80) FIG. 28 summarises a number of different fluidic steps that were used in this Example and are as described herein.

(81) Magnetic mixing was performed manually, i.e. by moving the magnet above the chamber by hand.

(82) Heating was carried out via a Peltier device placed below the chamber, in direct contact with the elastic membrane

(83) Results

(84) Results Obtained with the Updated Purification Protocol A

(85) Four transformations were performed. Table 1 shows the details of the part ligation and the results obtained. Experiments 3 and 4 represent full off-chip and full on-chip respectively.

(86) TABLE-US-00001 TABLE 1 Yield Efficiency (number (coloured/ Name of the ON/OFF CHIP of n.coloured Experiment transformations status colonies) cells) 1 RFP−.1C3+ PART ON-CHIP 42 33.3% 2 RFP+.1C3− PART ON-CHIP 8 37.5% 3 RFP−.1C3− OFF-CHIP 8 62.5% 4 RFP+.1C3+ ON-CHIP 102   52%

(87) On the edge on the plate (4) (full on-chip) a 100% efficiency was reached. This appeared to be due to a concentration effect and transformation protocol. One reason that explains the ring of cells on the edge was that the volume of the transformation mixture was too high (850 uL instead of 300 uL): only a small amount of liquid is needed to fill the plate and then the rest of it reaches the edges where it stays highly concentrated. In the subsequent experiment, only 300 uL of transformation mixture was used (see below).

(88) Results Obtained with the Updated Purification Protocol B

(89) Four transformations were performed. Table 2 shows the details of the part ligation and the results obtained. Experiments 3 and 4 represent full off-chip and full on-chip respectively.

(90) TABLE-US-00002 TABLE 2 Number of colonies Name of the ON/OFF Number with Experi- transfor- CHIP of phenotypic Efficiency ment mations status colonies change (%) 1 RFP−.1C3+ PART ON- 14 11 79% CHIP 2 RFP+.1C3− PART ON- 5 4 80% CHIP 3 RFP−.1C3− OFF-CHIP 12 10 83% 4 RFP+.1C3+ ON-CHIP 7 6 86%

(91) By doubling the washing steps the efficiency of the off-chip purifications was greatly improved. Washing steps to get rid of unwanted oligos seem to be important steps in the on-chip protocol. The transformation protocol in these experiments also used a smaller amount of transformation medium.

(92) In conclusion: The present inventors have shown that it is possible to carry out assembly of polynucleic acid sequences on-chip The results obtained on-chip are close to the off-chip results.

Example 2—Chip 3A

(93) A microfluidic device as shown in FIG. 14A was manufactured using cnc machining and tested by washing fluid through the device.

Example 3—5-Part Assembly on Chip

(94) A five-part assembly (RFP, GFP, KanR, AmpR and pSC101) with 0% contamination was demonstrated twice. Two identical experiments were carried out involving two on-chip tests and two off-chip tests. First of all the 5 parts, buffer and water were loaded into a chip well, The 5 parts and buffer were pumped (˜0.5 uL of each part) sequentially into the main channel, pushed into the output well and collected with a pipette to form the product P1. 1 uL of Water from the water well was pumped into the output channel and pipette off the chip to waste. More water and then pumped and formed product P2. P3 was formed by a 5 parts assembly and the addition of 1.5 uL composed equally of blue and yellow food dye solution and mineral oil. P4 was a conventional off-chip 5 part assembly.

(95) Table 3 summarizes the different products transformed and the results obtained.

(96) TABLE-US-00003 TABLE 3 Plate number Content Result P1 5 parts ON-CHIP Assembly Assembly successful P2 Water control No contamination P3 5 parts OFF-CHIP Assembly Assembly successful (with added dyes) P4 5 parts OFF-CHIP Assembly Assembly successful

(97) In conclusion, a 5 part assembly without subsequent contamination has been demonstrated (n=2) on chip.

Example 4—10 Part-Assembly on Chip

(98) Violacein Biosynthetic Pathway

(99) Violacein is an industrially-relevant, indole derivative possessing anti-tumoral, anti-ulcerogenic, antitumorigenic, antitrypanosomatid, and antiviral activities. FIG. 43A shows the chemical structure of Violacein. Biosynthesis of this product can be performed heterologously in E. coli and requires only 4 biosynthetic genes. FIG. 43B shows the biosynthesis of violacein from the precursor, L-tryptophan. The intensely colored violacein pigment can be used to diagnose successful assemblies, greatly aiding the assembly debugging process.

(100) 10-Part Assembly Design

(101) The 10-way assembly contained the following parts:

(102) TABLE-US-00004 TABLE 4 Part Name Part Description Part Number Kan kinase Provides cells with resistance to the 1076 antibiotic, kanamycin vioA Violacein biosynthetic enzyme 1217 vioB-alpha Violacein biosynthetic enzyme, first 1471 domain of vioB vioB-beta Violacein biosynthetic enzyme, second 1475 domain of vioB vioC Violacein biosynthetic enzyme 1223 vioE Violacein biosynthetic enzyme 1225 GFP Includes a promoter and RBS, yields Green 1097 Fluorescent Protein P15a origin Allows for plasmid replication by e. coli 1532 (medium copy) RFP Includes a promoter and RBS, yields Red 1104 Fluorescent Protein β- Provides cells with resistance to the 1109 lactamase antibiotic, ampicillin
Part Preparation for High-Order Assemblies

(103) The 10 parts were prepared and then assembled on-chip. Part preparation methods are described below.

(104) Linked Ligation/Digestion Reaction Conditions

(105) Each part Cloned vector was miniprepped from 4 mL of culture media and eluted in 50 uL EB buffer. Linked ligation-digestion reactions were setup, using a 40× dilution of T4 DNA ligase and EarI in 1×NEB buffer #2 supplemented with 1 mM ATP. Each reaction contained a 25-fold excess of the appropriate pre-annealed LOA and POA oligos. Reaction volumes were typically 90 uL. Reactions were run in a thermo cycler as shown in FIG. 44.

(106) Gel-Based Purification of Pathway Assembly Components

(107) 65 uL of each reaction was then run on a 1% agarose gel and the appropriate products were extracted from the gel using a Qiagen kit and eluted from the column using 50 uL EB. FIG. 45 shows the gel for fragments 1-7. The dots indicate the extracted band.

(108) Pathway Assembly Off-Chip and on-Chip

(109) Off-Chip Pathway Assembly Steps

(110) A control assembly was conducted off-chip in order to compare to the assembly conducted on chip. The method used was as follows: DNA concentration of each part was normalized and mixed in the presence of 1×NEBuffer #2. Assembly reactions were run for 20 minutes at room temperature. 3 uL of the assembly reaction was then used to transform chemically-competent NEB 10 B cells. Transformed cells were recovered in SOC for 1 hour at 37 C before plating on LB/agar/kanamycin plates.

(111) On-Chip Pathway Assembly Steps

(112) The assembly was conducted on-chip using a computer numerically controlled (CNC) machined chip.

(113) On-Chip Pathway Assembly Results

(114) Successful assemblies were verified by display of the correct colored colony phenotype and the results are shown in Table 5 below. The off-chip and on-chip showed essentially equivalent efficiency (88% correct transformants). The total yield was slightly lower on-chip, however the total number of colonies was well above that needed for ensuring a successful assembly. The water controls demonstrated that there was negligible contamination.

(115) TABLE-US-00005 TABLE 5 Plate number Description Results Yield P1 Water control before No contamination — assembly P2 10 parts on chip 88.1% of cells correctly Total 84 cells transformed P3 Water control No contamination — after assembly P4 10 parts off chip 88.2% of cells correctly Total 102 cells transformed