High-throughput automatic gene splicing and assembling method

Abstract

The invention relates to a high-throughput automatic gene splicing and assembling method. On the basis of the high-throughput gene construction automation device and system, a high-throughput automatic gene splicing and assembling method is further designed so as to realize overall collaboration, and an operation process capable of improving efficiency, reducing cost, improving automation degree and completing process standardization in a laboratory / production workshop is developed; the working efficiency and the applicability are remarkably improved, and high-throughput automatic construction of genes from 300bp to Kb can be met.

Description

Technical Field

[0001] The invention belongs to the technical field of gene synthesis and relates to a method for high-throughput automated splicing and assembling genes. Background Art

[0002] DNA synthesis and assembly are core components in the field of synthetic biology. Traditional DNA synthesis technology mainly relies on chemical synthesis methods, especially solid-phase phosphoramidite triester synthesis, which can efficiently synthesize oligonucleotides less than 200 bp in length. However, the length of de novo DNA synthesis is limited by single-stranded DNA synthesis technology, so it is necessary to assemble longer genes or genomes from DNA fragments. DNA assembly is the process of assembling segmented synthesized oligonucleotide fragments into long DNA fragments, which is crucial for synthesizing gene-length or even genome-length DNA sequences. Existing assembly technologies include DNA polymerase-based assembly methods (such as polymerase cycle assembly technology and overlap extension PCR technology), and nuclease-based assembly methods (such as Gibson assembly technology and SLIC assembly technology). The assembly of longer DNA fragments is usually carried out in vivo, usually using organisms such as Escherichia coli, Bacillus subtilis and yeast.

[0003] In the field of synthetic biology, large-scale synthetic bioengineering experiments face huge challenges that are beyond the capabilities of traditional labor-intensive research methods. Although the existing automated gene splicing instruments on the market can perform gene splicing and construction, they cannot customize different splicing / assembly schemes very well, and some have problems such as insufficient throughput (less than 96 genes), lack of electrophoresis gel detection function, or lack of support for the simultaneous construction of different types of plasmids. The application limitations are relatively large, and it is difficult to achieve multiple uses of one machine. Therefore, the development of a high-throughput gene splicing and construction scheme and its automated device / system that can improve efficiency, reduce costs, increase the degree of automation, complete process standardization operations in laboratories / production workshops, and flexibly adapt to different research and industry needs is of great significance for promoting cross-application and development in multiple fields such as biology, medicine, agriculture, and industry. Summary of the invention

[0004] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for high-throughput automated splicing and assembly of genes, and develops a high-throughput automated gene construction solution with high efficiency, low cost, high degree of automation, standardization and wide adaptability based on gene automated construction equipment.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for high-throughput automated splicing and assembly of genes, the method comprising the following steps:

[0007] (S1) splitting the target gene sequence and designing corresponding primers according to the split sequence;

[0008] (S2) synthesizing the primers, mixing the primers of a single target gene, using the mixed primer pool to perform gene splicing and assembly, and then using amplification primers to perform amplification to obtain the target gene;

[0009] The reaction processes involved in steps (S1) to (S2) are performed on a gene automation construction device, which includes:

[0010] A frame (1) is provided on the frame (1): a sliding system (2) and an enzyme-labeled concentration detection system (3) capable of docking with the sliding system (2), an electrophoresis system (4), a filtration purification system (5), a storage system (6) capable of storing a preset solvent, a centrifugal system (7), a constant temperature oscillation system (8), a film sealing system (9) and a PCR instrument (10); the frame (1) is also provided with a gel cutting system (20) and a monitoring device; the gel cutting system (20) can dock with the storage system (6); the monitoring device can be electrically connected to the above structures respectively, and configure primers, configure gene splicing reaction liquid, run the splicing reaction, configure gene product amplification reaction liquid, run the amplification reaction, perform agarose gel electrophoresis, cut target gene bands, purify electrophoretic PCR products, perform enzyme-labeled quantification and concentration uniformity, configure enzyme-linked reaction liquid and run the enzyme-linked reaction according to preset information; the sliding system (2) is equipped with a liquid pipette head (26) for taking and placing solvents.

[0011] In the present invention, based on the high-throughput gene construction automation equipment, a high-throughput automated gene construction scheme is further designed to achieve overall synergy, which can meet the automated high-throughput construction of 300bp to Kb-level genes.

[0012] Preferably, step (S1) specifically includes:

[0013] (S1-1) Importing the target gene sequence data into the sequence analysis software and performing sequence splitting operations;

[0014] (S1-2) Design corresponding primers and select the first and last primers of the gene as amplification primers.

[0015] Preferably, step (S2) specifically includes:

[0016] (S2-1) starting the automated equipment for high-throughput splicing and assembly;

[0017] (S2-2) selecting and executing an automated splicing program according to the splitting scheme of step (S1);

[0018] (S2-3) Importing the relevant parameter table into the gene automated construction equipment, and placing the required reagents and consumables in the designated locations;

[0019] (S2-4) starting the automated splicing program and continuing to run until the reaction process is completed;

[0020] (S2-5) The product of step (S2-4) is transferred and amplified using amplification primers to obtain the target gene.

[0021] Preferably, the high-throughput automated gene construction method further comprises a purification step, including electrophoretic separation of the amplified product, followed by gel image acquisition, and the system will automatically identify and determine the correct product bands and perform gel cutting and recovery operations.

[0022] Preferably, the high-throughput automated gene construction method further comprises the step of constructing a plasmid, including assembling the target gene into a target plasmid vector.

[0023] Preferably, assembling the target gene into the target plasmid vector includes: transferring the target gene, performing sample quantification, the system automatically calculating and executing a sample concentration homogenization program, and then entering a transfer program of the target plasmid vector to complete the construction of the plasmid.

[0024] In the present invention, the calculation of sample concentration homogenization refers to the sample being subjected to enzyme-labeled quantitative analysis. Based on the obtained concentration value, the software automatically calculates the amount of pure water that needs to be added, and automatically instructs the pipetting module to move the required amount of water into the sample to dilute it to the concentration required for the next reaction procedure. The above mentioned sample concentration homogenization uses the same formula: W = ((C*E) / (X / V))-E, and the meaning of each symbol in the formula is as follows:

[0025] E is the volume after purification and recovery (μL);

[0026] C is the concentration after purification and recovery (ng / μL);

[0027] X is the required amount of DNA (ng) fixed;

[0028] V is the required DNA volume (μL) fixed;

[0029] Y is the maximum total concentration (ng / μL) to be adjusted;

[0030] W is the water (μL) to be added in the end.

[0031] Preferably, the high-throughput automated gene construction method further comprises the steps of transforming the plasmid into competent cells, screening positive clones and sequencing.

[0032] Preferably, the automated splicing procedure comprises:

[0033] Pre-denaturation: 95-98°C, 3-5min;

[0034] Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 45-50°C, 13-16s, extension: 70-73°C, 15-25s, 18-25 cycles;

[0035] Thorough extension: 70-73℃, 3-5min.

[0036] Specifically, the automated splicing system can be:

[0037] Components concentration volume Gene Primer Pool 0.1~2pmol / primer / μL 1μL DNA polymerase reaction solution 2× 12.5μL Sterilized water / Make up to 25 μL

[0038] Preferably, the amplification procedure comprises:

[0039] Pre-denaturation: 95-98°C, 3-5min;

[0040] Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 50-55°C, 13-16s, extension: 70-73°C, 30-60s, 25-30 cycles;

[0041] Thorough extension: 70-73℃, 3-5min.

[0042] Specifically, the amplification system can be:

[0043] Components concentration volume Splicing products / 2.5μL DNA polymerase reaction solution 2× 12.5μL Forward primer 8-12 pmol / μL 1.25μL Reverse primer 8~12pmol / μL 1.25μL Sterilized water / Make up to 25 μL

[0044] In the present invention, an automated splicing program and amplification program and system are designed to improve the applicability of the overall process, and more target genes can be automatically constructed, which is conducive to achieving high-throughput construction.

[0045] In the present invention, the spliced ​​and assembled gene sequences can be the sequences shown in SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO.7, the primer pool sequences can be the sequences shown in SEQ ID NO.10-SEQ ID NO.21, SEQ ID NO.22-SEQ ID NO.35 and SEQ ID NO.36-SEQ ID NO.59, and the specific amplification primers can be the sequences shown in SEQ ID NO.2-SEQ IDNO.3, SEQ ID NO.5-SEQ ID NO.6, SEQ ID NO.8-SEQ ID NO.9.

[0046] Preferably, the steps in the method for high-throughput automated splicing and assembly of genes are performed on a gene automated construction device, specifically including:

[0047] (S1-1) Importing the target gene sequence data into the sequence analysis software and performing sequence splitting operations;

[0048] (S1-2) Designing amplification primers and primers corresponding to the splitting sequences;

[0049] (S2-1) starting the automated equipment for high-throughput splicing and assembly;

[0050] (S2-2) selecting and executing an automated splicing program according to the splicing scheme of step (S1);

[0051] (S2-3) Importing the relevant parameter table into the gene automated construction equipment, and placing the required reagents and consumables in the designated positions; the sliding system (2) is docked with the storage system (6) to configure the gene splicing reaction solution; the sliding system (2) is docked with the sealing system (9) and the PCR instrument (10) to run the splicing reaction;

[0052] (S2-4) entering the automated splicing program and continuing to run until the reaction process is completed;

[0053] (S2-5) transferring the product of step (S2-4), performing an amplification procedure using amplification primers, and obtaining the target gene; the sliding system (2) is docked with the storage system (6) to configure a gene product amplification reaction solution; the sliding system (2) is docked with the sealing system (9) and the PCR instrument (10) to run the amplification reaction;

[0054] (S3) transfer the target gene, perform sample quantification, the system automatically calculates and executes the sample concentration homogenization procedure, and then enters the target plasmid vector transfer procedure to complete the construction of the plasmid; the sliding system (2) is respectively docked with the electrophoresis system (4) and the storage system (6) to perform agarose gel electrophoresis, and the gel cutting system (20) is docked with the storage system (6) to cut the target gene band; the sliding system (2) is respectively docked with the storage system (6), the constant temperature oscillation system (8), the filtration purification system (5) and the centrifugal system (7) to purify the electrophoresis PCR product; the sliding system (2) is docked with the enzyme label concentration detection system (3) to perform enzyme label quantification and make the concentration uniform; the sliding system (2) is docked with the storage system (6) to configure the enzyme linkage reaction solution; the sliding system (2) is respectively docked with the sealing system (9) and the PCR instrument (10) to run the enzyme linkage reaction;

[0055] (S4) Transform the target plasmid into competent cells, screen positive clones and perform sequencing.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] Based on high-throughput gene construction automation equipment, the present invention further designs a method for high-throughput automated splicing and assembly of genes to achieve overall synergy, and develops an operating procedure that can improve efficiency, reduce costs, increase the degree of automation, and complete process standardization in the laboratory / production workshop, significantly improving work efficiency and applicability, and can meet the automated high-throughput construction of 300bp to Kb-level genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the method for high-throughput automated splicing and assembly of genes.

[0059] Figure 2 This is the electrophoresis result of splicing 782bp gene.

[0060] Figure 3 This is the electrophoresis result of splicing 910bp gene.

[0061] Figure 4 This is the electrophoresis result of splicing 1623bp gene.

[0062] Figure 5 Schematic diagram of automated apparatus and system for high-throughput gene sequencing.

[0063] Figure 6 Layout of structures on the substrate for high-throughput gene production automation devices and systems;

[0064] Figure 7A schematic diagram of a third mobile device and a support frame for high-throughput gene construction automation device and system;

[0065] Figure 8 Schematic diagram of the consumables system for high-throughput genetic construction automation devices and systems;

[0066] In the figure, 1-frame; 11-base plate; 12-support frame; 13-support plate; 14-third moving device; 141-belt transmission device; 1411-driving wheel; 1412-driven wheel; 1413-belt; 142-guide device; 1421-guide rail; 1422-slider; 143-driving motor; 15-fan filter system;

[0067] 2-sliding system; 21-first moving device; 22-second moving device; 23-96-channel liquid transfer device; 24-8-channel liquid transfer device; 25-carrying clamp; 26-liquid transfer head; 27-liquid transfer head box;

[0068] 3-Enzyme label concentration detection system; 31-384 enzyme label plate;

[0069] 4-electrophoresis system; 41-peristaltic pump; 42-electrophoresis apparatus;

[0070] 5- filtration purification system; 51- filtration plate;

[0071] 6-storage system; 61-liquid storage tank; 62-PCR plate; 63-deep well plate; 64-waste liquid tank;

[0072] 7-centrifugal system; 71-centrifuge; 72-first centrifugal plate; 73-second centrifugal plate;

[0073] 8- Constant temperature oscillation system;

[0074] 9-Film sealing system;

[0075] 10-PCR instrument;

[0076] 20- glue cutting system; 201- glue cutting head; 202- visual detection device; 203- glue cutting head box;

[0077] 30-consumable system; 310-consumable stack; 3101-consumable rack; 3102-installation cabin; 3103-consumable motor; 320-consumable extraction mechanism; 3201-driving element; 32011-first driving element; 32012-second driving element; 3202-clamping claw; 3203-lifting mechanism;

[0078] 40- Panoramic camera;

[0079] 50-Abandoned barrel.

[0080] Fig. 9This is the electrophoresis result of high-throughput gene splicing. DETAILED DESCRIPTION

[0081] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0082] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0083] In the present invention, based on the high-throughput gene construction automation equipment, a high-throughput automated splicing and assembly gene method is further designed to achieve overall synergy, significantly improve work efficiency and applicability, and reduce costs. Specifically, the process of the high-throughput automated splicing and assembly gene method is as follows Figure 1 As shown, it mainly includes the following steps:

[0084] (1) Pretreatment

[0085] 1) Import the gene sequence data into a sequence analysis tool (such as AnalysisTool (Software Registration Numbers 2023SR1470468 and 2023SR1386947)) and perform sequence splitting operations;

[0086] 2) Use sequence splitting software to optimize the codons of the imported gene sequence and accurately evaluate the complex sequence to determine the appropriate splicing scheme;

[0087] 3) According to the primer sequence table of the split template, the sequence is split, and the corresponding primers are synthesized according to the specified well positions, and then the split primers corresponding to the single gene are accurately mixed in the preset well positions through the oligonucleotide automated pooling device;

[0088] (2) Splicing

[0089] 1) Start high-throughput splicing and assembly automation equipment to prepare for efficient sequence splicing operations;

[0090] 2) Select and execute the correct automated splicing program based on the optimal splicing solution determined in the pre-processing stage;

[0091] 3) Follow the system prompts to import the relevant parameter table into the system and place the required reagents and consumables in the designated locations;

[0092] 4) Start the plate automation splicing program, continue running until all reaction processes are completed, enter the product purification automation program, and then transfer to the automation process of the product purification stage.

[0093] (3) Purification

[0094] 1) In the automated process, electrophoresis separation is performed first, followed by gel image acquisition. The system will automatically identify and determine the correct product bands and perform precise gel excision and recovery operations.

[0095] 2) Next, the process enters the negative pressure filtration purification step, through which the purified splicing product is efficiently obtained.

[0096] 3) The purified product will automatically enter the automated procedure for plasmid construction.

[0097] (4) Plasmid construction (optional)

[0098] 1) First, perform sample quantification. The system will automatically calculate and execute the sample concentration homogenization procedure to ensure sample consistency;

[0099] 2) Subsequently, the splicing product group will automatically enter the transfer program of the target plasmid vector to complete the construction of the plasmid gene.

[0100] (5) Manual transfer (optional)

[0101] 1) Manually transfer samples to automated high-throughput competent cell transformation equipment;

[0102] 2) Then, the transformed bacterial solution is manually transferred to an automated high-throughput bacterial solution coating device;

[0103] Finally, the coated plates are manually sent to an automated high-throughput colony picking device, which is specifically used for clone selection and subsequent sequencing.

[0104] Example 1

[0105] This example performs gene splitting sequence design and determines the biochemical conditions for high-throughput splicing and assembly.

[0106] 1. Design of multiple gene template primer pools

[0107] The sequence splitting software AnalysisTool was used to split the gene sequences of three lengths of 782bp, 910bp, and 1623bp to obtain the corresponding primer pools. According to the gene length, each primer pool contains 12 to 26 sets of primers with a length range of 70 to 90nt and an overlap control of 15 to 22bp. According to the above design, the first and last primers of the corresponding gene were selected for subsequent gene amplification.

[0108] The complete sequence of the 782 bp gene and the forward and reverse primers used for amplification are shown in Table 1 .

[0109] Table 1

[0110]

[0111] The complete sequence of the 910 bp gene and the forward and reverse primers used for amplification are shown in Table 2 .

[0112] Table 2

[0113]

[0114]

[0115] The complete sequence of the 1623 bp gene and the forward and reverse primers used for amplification are shown in Table 3 .

[0116] Table 3

[0117]

[0118] The 782 bp gene splicing primer pool is shown in Table 4.

[0119] Table 4

[0120]

[0121]

[0122] The 910 bp gene splicing primer pool is shown in Table 5.

[0123] Table 5

[0124]

[0125]

[0126] The 1623 bp gene splicing primer pool is shown in Table 6.

[0127] Table 6

[0128]

[0129]

[0130]

[0131] 2. Determine the conditions and steps for gene synthesis reaction, which is divided into two steps: splicing and amplification.

[0132] The PCA system in the 782bp, 910bp, and 1623bp gene synthesis steps includes those shown in Table 7.

[0133] Table 7

[0134] Components concentration volume Gene premixed primer pool 0.2pmol / primer / μL 5μL Gloria Nova HS2×Master Mix 2× 10μL Sterilized water / Make up to 20 μL

[0135] The PCA reaction conditions in the 782 bp, 910 bp, and 1623 bp gene synthesis steps are shown in Table 8.

[0136] Table 8

[0137]

[0138] The amplification (PCR) system in the 782bp, 910bp, and 1623bp gene synthesis steps is shown in Table 9.

[0139] Table 9

[0140]

[0141]

[0142] The reaction conditions of the amplification (PCR) process in the 782bp and 910bp gene synthesis steps are shown in Table 10.

[0143] Table 10

[0144]

[0145] The reaction conditions of the amplification (PCR) process in the 1623 bp gene synthesis step are shown in Table 11.

[0146] Table 11

[0147]

[0148] 3. Purify and recover the synthesized product, and perform first-generation sequencing after continuous transfer. The gene splicing effect is determined based on the sequencing results.

[0149] The electrophoresis results of the 782bp gene synthesis product are as follows Figure 2 As shown, the length is in line with expectations. The first-generation sequencing data results are shown in Table 12, and the correct gene can be obtained. The phenomenon of incomplete correct cloning usually originates from base errors in the template DNA itself, or in the PCA / PCR amplification process, due to the natural error rate of the polymerase, the gene sequence is successfully spliced ​​and assembled, but there are errors in some bases, which are not errors introduced by the scheme of the present invention.

[0150] Table 12

[0151]

[0152]

[0153] The electrophoresis results of the 910bp gene synthesis product are as follows Figure 3 As shown, the length is in line with expectations. The first-generation sequencing data results are shown in Table 13, and the correct gene can be obtained. The phenomenon of incomplete correct cloning usually originates from base errors in the template DNA itself, or in the PCA / PCR amplification process, due to the natural error rate of the polymerase, the gene sequence is successfully spliced ​​and assembled, but there are errors in some bases, which are not errors introduced by the scheme of the present invention.

[0154] Table 13

[0155] Sample name Number of sequencing samples Exactly the right sample amount Incorrect sample size SEQ910 10 6 4

[0156] The electrophoresis results of the 1623bp gene synthesis product are as follows Figure 4 As shown, the length is in line with expectations. The first-generation sequencing data results are shown in Table 14, and the correct gene can be obtained. The phenomenon of incomplete correct cloning usually originates from base errors in the template DNA itself, or in the PCA / PCR amplification process, due to the natural error rate of the polymerase, the gene sequence is successfully spliced ​​and assembled, but there are errors in some bases, which are not errors introduced by the scheme of the present invention.

[0157] Table 14

[0158] Sample name Number of sequencing samples Exactly the right sample amount Incorrect sample size SEQ1623 10 4 6

[0159] Example 2

[0160] This example further performs automated high-throughput gene splicing and assembly on automated equipment based on the conditions for gene splicing and assembly designed in Example 1.

[0161] The schematic diagram of the automation equipment is as follows Figure 5As shown, it includes a frame 1 and a sliding system 2, an enzyme label concentration detection system 3, an electrophoresis system 4, a suction filtration purification system 5, a storage system 6 capable of storing solvents, a centrifugal system 7, a constant temperature oscillation system 8, a film sealing system 9, a PCR instrument 10, a gel cutting system 20 and a monitoring device arranged on the frame 1. Among them, the monitoring device is electrically connected to the sliding system 2, the enzyme label concentration detection system 3, the electrophoresis system 4, the suction filtration purification system 5, the storage system 6 capable of storing preset solvents, the centrifugal system 7, the constant temperature oscillation system 8, the film sealing system 9, the PCR instrument 10 and the gel cutting system 20, respectively, so that the sliding system 2 can be docked with the enzyme label concentration detection system 3, the electrophoresis system 4, the suction filtration purification system 5, the storage system 6 capable of storing solvents, the centrifugal system 7, the constant temperature oscillation system 8, the film sealing system 9 and the PCR instrument 10, and the gel cutting system 20 can be docked with the storage system 6. Among them, the sliding system 2 is equipped with a liquid pipette head 26 to take and put the solvent. The monitoring device configures primers according to preset information, configures gene splicing reaction solution, runs the splicing reaction, configures gene product amplification reaction solution, runs the amplification reaction, performs agarose gel electrophoresis, cuts target gene bands, purifies electrophoretic PCR products, performs enzyme labeling quantification and concentration uniformity, configures enzyme-linked reaction solution, and runs the enzyme-linked reaction.

[0162] The structural layout on the substrate is shown in the figure Figure 4 As shown, the schematic diagram of the third moving device and the support frame is as shown Figure 5 The schematic diagram of the consumable system is shown in Figure 6 shown.

[0163] The sliding system 2 includes a first moving device 21, a second moving device 22, a 96-channel pipetting device 23, an 8-channel pipetting device 24 and a carrying clamp 25, all of which are electrically connected to the monitoring device. The 96-channel pipetting device 23 is connected to the end of the first moving device 21 to achieve horizontal movement, and the 8-channel pipetting device 24 and the carrying clamp 25 are connected to the end of the second moving device 22 to achieve horizontal movement, and the 96-channel pipetting device 23, the 8-channel pipetting device 24 and the carrying clamp 25 can each move in the vertical direction, so that the 96-channel pipetting device 23, the 8-channel pipetting device 24 and the carrying clamp 25 can be independently controlled to work. The 96-channel pipetting device 23 and the 8-channel pipetting device 24 are both equipped with a pipette head 26. The sliding system 2 also includes a pipette head box 27, which is used to store the pipette head 26, and the pipette head 26 can be installed on the sliding system 2.

[0164] The rubber cutting system 20 includes a rubber cutting head 201, a blue light lamp and a visual detection device 202. A rubber cutting head box 203 is provided on the frame 1. A plurality of rubber cutting heads 201 are provided in the rubber cutting head box 203. The first movable device 21 can be docked with the rubber cutting head box 203 so that the rubber cutting head 201 can be replaceably arranged at the end of the first movable device 21. The rubber cutting head 201 and the visual detection device 202 are both arranged at the end of the first movable device 21 to realize horizontal movement, and the rubber cutting head 201 can realize vertical movement; the blue light lamp is provided on the frame 1 and can illuminate the storage system 6, the visual detection device 202 is used to take pictures of the storage system 6, and the rubber cutting head 201 is docked with the storage system 6 for rubber cutting.

[0165] The suction filtration purification system 5 includes a suction filtration plate 51 and a recovery plate, the centrifugal system 7 includes a centrifuge 71, a first centrifugal plate 72 and a second centrifugal plate 73, and the sliding system 2 can transport the suction filtration plate 51 to the recovery plate and the centrifuge 71. The electrophoresis system 4 includes an electrophoresis instrument 42 and an agarose gel plate for accommodating agarose gel. The sealing system 9 includes a sealing instrument.

[0166] The frame 1 is provided with a base plate 11, the sliding system 2 is provided above the base plate 11, the base plate 11 is provided with a support frame 12, a support plate 13 and a third moving device 14, the support frame 12 is slidably connected to the base plate 11 and connected to the output end of the third moving device 14, the support plate 13 is provided above the support frame 12, and the third moving device 14 is electrically connected to the monitoring device. A part of the liquid pipetting head box 27, the rubber cutting head box 203, the enzyme label concentration detection system 3, the electrophoresis system 4, the suction filtration purification system 5, the storage system 6, the centrifugal system 7, the constant temperature oscillation system 8, the film sealing system 9 and the PCR instrument 10 are provided on the base plate 11, and the other part is provided on the support plate 13.

[0167] The third moving device 14 includes a driving motor 143, two belt transmission devices 141 and two guide devices 142. The belt transmission device 141 includes a driving wheel 1411, a driven wheel 1412 and a belt 1413. The belt 1413 is wound around the driving wheel 1411 and the driven wheel 1412. The driving motor 143 is connected to the driving wheel 1411 of the belt transmission device 141. The guide device 142 includes a guide rail 1421 and a slider 1422 slidably connected to the guide rail 1421. The guide rail 1421 is connected to the base plate 11. The slider 1422 is connected to the support frame 12. The support frame 12 and the support plate 13 form a gate shape. The two ends of the support frame 12 are correspondingly connected to the belts 1413 of the two belt transmission devices 141 and the sliders 1422 of the two guide devices 142. The third moving device 14 is arranged below the base plate 11. The support frame 12 passes through the base plate 11, which further makes the structure more compact in the vertical direction.

[0168] The base plate 11 is divided into 44 working positions, of which T1-T10 positions are all located on the support plate 13, and the remaining positions are all located on the base plate 11. The positions and corresponding functions are as follows: T1, T3-8, T10 are room temperature PCR plates, T2 is a 384 ELISA plate, T9 and T35 are centrifugal plates, T11 is a 96-channel agarose gel electrophoresis tank, T12-T15 are gel cutting head boxes, T16 is a waste liquid tank, and T17-19 are room temperature shaking tanks. Liquid storage tank, T20-T28 is the 4℃ reaction liquid configuration working area, which can be used to place liquid storage tank, deep well plate, liquid adding tank and PCR plate, T29-T34, T45-46 are pipette head boxes, T36 is a waste bucket, T37 is a film sealing instrument, T38 is a 96-channel constant temperature oscillator, T39-40 is a 96-channel PCR instrument, T41 is the consumable system docking point, T42-43 are filtration plates, and T44 is a special well plate centrifuge for automation equipment.

[0169] The consumable system 30 includes a consumable stack 310 and a consumable extraction mechanism 320, both of which are electrically connected to the monitoring device. The consumable stack 310 includes a consumable rack 3101 and a consumable motor 3103. The consumable rack 3101 is provided with a plurality of installation cabins 3102, and the installation cabins 3102 are used to place the liquid pipetting head box 27. The consumable motor 3103 drives the consumable rack 3101 to rotate so that the corresponding installation cabin 3102 rotates to a preset position to dock with the consumable extraction mechanism 320. The driving member 3201 may include a first driving member 32011 and a second driving member 32012, which respectively realize the movement of the clamping jaw 3202 in the horizontal and vertical directions.

[0170] 1. Prepare primer pools for 86 genes (numbered GV2402-1 to GV2402-86) respectively, with the final concentration of each primer pool being 0.2 pmol / primer / μL.

[0171] 2. Start high-throughput gene synthesis with the primer pool as a template, including two steps of splicing (PCA) and amplification (PCR). After the high-throughput splicing and assembly program is started, the gene synthesis process will be automatically carried out, and the preset parameters are input into the monitoring component to perform the following steps: the sliding system is docked with the storage system to configure the primers; the sliding system is docked with the storage system to configure the gene splicing reaction solution; the sliding system is docked with the sealing system and the PCR instrument respectively to run the splicing reaction; the sliding system is docked with the storage system to configure the gene product amplification reaction solution; the sliding system is docked with the sealing system and the PCR instrument respectively to run the amplification reaction; the The sliding system is docked with the electrophoresis system and the storage system respectively to perform agarose gel electrophoresis; the gel cutting system is docked with the storage system to cut the target gene band; the sliding system is docked with the storage system, the constant temperature oscillation system, the filtration purification system and the centrifugal system respectively to purify the electrophoresis PCR product; the sliding system is docked with the enzyme label concentration detection system to perform enzyme label quantification and make the concentration uniform; the sliding system is docked with the storage system to configure the enzyme-linked reaction solution; the sliding system is docked with the sealing system and the PCR instrument respectively to run the enzyme-linked reaction. This process covers the splicing and assembly of the GV2402-1 to GV2402-86 genes, such as Fig. 9 As shown, the band marked with a red frame is the desired target band, indicating a successfully spliced ​​and assembled gene fragment.

[0172] 3. After gel excision and recovery from GV2402-1 to GV2402-86, sample quantification is performed. The system will automatically calculate and execute the sample concentration homogenization procedure to ensure sample consistency. Subsequently, the splicing product group will automatically enter the transfer procedure of the target plasmid vector to complete the construction of the plasmid gene. The sample quantification results after gel excision and recovery from GV2402-1 to GV2402-86 are shown in Table 15.

[0173] Table 15

[0174]

[0175]

[0176] 4. The sample is manually transferred to the automated high-throughput competent cell transformation device. After the transformation is completed, the transformed bacterial solution is manually transferred to the automated high-throughput bacterial solution coating device. After the bacterial solution coating is completed, the plate is manually sent to the automated high-throughput colony picking device for clone selection and subsequent sequencing. The first-generation sequencing result data is shown in Table 16. Most genes can be correctly spliced ​​and assembled. The phenomenon of incomplete correct cloning is usually due to base errors in the template DNA itself, or during the PCA / PCR amplification process, due to the natural error rate of the polymerase, the gene sequence is successfully spliced ​​and assembled, but some bases have errors, which are not errors introduced by the scheme of the present invention.

[0177] Table 16

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] In summary, the present invention is based on high-throughput gene construction automation equipment, and further designs a high-throughput automated splicing and assembly gene scheme to achieve overall synergy, and develops an operating procedure that can improve efficiency, reduce costs, increase the degree of automation, and complete process standardization in the laboratory / production workshop, which significantly improves work efficiency and applicability, and can meet the automated high-throughput construction of 300bp to Kb-level genes.

[0185] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for high-throughput automated gene splicing and assembly, characterized in that: The method for high-throughput automated splicing and assembly of genes comprises the following steps: (S1) splitting the target gene sequence and designing corresponding primers according to the split sequence; (S2) synthesizing the primers, mixing the primers of a single target gene, using the mixed primer pool to perform gene splicing and assembly, and then using amplification primers to perform amplification to obtain the target gene; The reaction processes involved in steps (S1) to (S2) are performed on a gene automation construction device, which includes: A frame (1) is provided on the frame (1): a sliding system (2) and an enzyme-labeled concentration detection system (3) capable of docking with the sliding system (2), an electrophoresis system (4), a filtration purification system (5), a storage system (6) capable of storing a preset solvent, a centrifugal system (7), a constant temperature oscillation system (8), a film sealing system (9) and a PCR instrument (10); the frame (1) is also provided with a gel cutting system (20) and a monitoring device; the gel cutting system (20) can dock with the storage system (6); the monitoring device can be electrically connected to the above structures respectively, and configure primers, configure gene splicing reaction liquid, run the splicing reaction, configure gene product amplification reaction liquid, run the amplification reaction, perform agarose gel electrophoresis, cut target gene bands, purify electrophoretic PCR products, perform enzyme-labeled quantification and concentration uniformity, configure enzyme-linked reaction liquid and run the enzyme-linked reaction according to preset information; the sliding system (2) is equipped with a liquid pipette head (26) for taking and placing solvents.

2. The method for high-throughput automated gene splicing and assembly according to claim 1, characterized in that: Step (S1) specifically includes: (S1-1) Importing the target gene sequence data into the sequence analysis software and performing sequence splitting operations; (S1-2) Design corresponding primers and select the first and last primers of the gene as amplification primers.

3. The method for high-throughput automated gene splicing and assembly according to claim 1 or 2, characterized in that: Step (S2) specifically includes: (S2-1) starting the automated equipment for high-throughput splicing and assembly; (S2-2) selecting and executing an automated splicing program according to the splitting scheme of step (S1); (S2-3) Importing the relevant parameter table into the gene automated construction equipment, and placing the required reagents and consumables in the designated locations; (S2-4) starting the automated splicing program and continuing the process until the reaction process is completed; (S2-5) The product of step (S2-4) is transferred and amplified using amplification primers to obtain the target gene.

4. The method for high-throughput automated gene splicing and assembly according to any one of claims 1 to 3, characterized in that: The high-throughput automated gene construction method also includes a purification step, including electrophoretic separation of the amplified product, followed by gel image collection. The system will automatically identify and determine the correct product bands and perform gel cutting and recovery operations.

5. The method for high-throughput automated gene splicing and assembly according to any one of claims 1 to 4, characterized in that: The high-throughput automated gene construction method also includes a plasmid construction step, including assembling the target gene into a target plasmid vector.

6. The method for high-throughput automated gene splicing and assembly according to claim 5, characterized in that: Assembling the target gene into the target plasmid vector includes: transferring the target gene, performing sample quantification, the system automatically calculates and executes the sample concentration homogenization program, and then enters the target plasmid vector transfer program to complete the construction of the plasmid.

7. The method for high-throughput automated gene splicing and assembly according to claim 5 or 6, characterized in that: The high-throughput automated gene construction method also includes the steps of transforming the plasmid into competent cells, screening positive clones and sequencing.

8. The method for high-throughput automated gene splicing and assembly according to claim 3, characterized in that: The automated splicing procedure includes: Pre-denaturation: 95-98°C, 3-5min; Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 45-50°C, 13-16s, extension: 70-73°C, 15-25s, 18-25 cycles; Thorough extension: 70-73℃, 3-5min.

9. The method for high-throughput automated gene splicing and assembly according to claim 3, characterized in that: The amplification procedure includes: Pre-denaturation: 95-98°C, 3-5min; Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 55-60°C, 13-16s, extension: 70-73°C, 30-60s, 25-30 cycles; Thorough extension: 70-73℃, 3-5min.

10. The method for high-throughput automated gene splicing and assembly according to any one of claims 1 to 9, characterized in that: The steps in the high-throughput automated gene construction method are performed on an automated gene construction device, specifically including: (S1-1) Importing the target gene sequence data into the sequence analysis software and performing sequence splitting operations; (S1-2) Designing amplification primers and primers corresponding to the splitting sequences; (S2-1) starting the automated equipment for high-throughput splicing and assembly; (S2-2) selecting and executing an automated splicing program according to the splicing scheme of step (S1); (S2-3) Importing the relevant parameter table into the gene automated construction equipment, and placing the required reagents and consumables in the designated positions; the sliding system (2) is docked with the storage system (6) to configure the gene splicing reaction solution; the sliding system (2) is docked with the sealing system (9) and the PCR instrument (10) to run the splicing reaction; (S2-4) entering the automated splicing program and continuing to run until the reaction process is completed; (S2-5) transferring the product of step (S2-4), performing an amplification procedure using amplification primers, and obtaining the target gene; the sliding system (2) is docked with the storage system (6) to configure a gene product amplification reaction solution; the sliding system (2) is docked with the sealing system (9) and the PCR instrument (10) to run the amplification reaction; (S3) transfer the target gene, perform sample quantification, the system automatically calculates and executes the sample concentration homogenization procedure, and then enters the target plasmid vector transfer procedure to complete the construction of the plasmid; the sliding system (2) is respectively docked with the electrophoresis system (4) and the storage system (6) to perform agarose gel electrophoresis, and the gel cutting system (20) is docked with the storage system (6) to cut the target gene band; the sliding system (2) is respectively docked with the storage system (6), the constant temperature oscillation system (8), the filtration purification system (5) and the centrifugal system (7) to purify the electrophoresis PCR product; the sliding system (2) is docked with the enzyme label concentration detection system (3) to perform enzyme label quantification and make the concentration uniform; the sliding system (2) is docked with the storage system (6) to configure the enzyme linkage reaction solution; the sliding system (2) is respectively docked with the sealing system (9) and the PCR instrument (10) to run the enzyme linkage reaction; (S4) Transform the target plasmid into competent cells, screen positive clones and perform sequencing.

Images