Efficient large DNA fragment synthesis and assembly method based on novel programmable nuclease argonaute

Abstract

An efficient large DNA fragment synthesis and assembly method based on a novel programmable nuclease Argonaute. The method specifically comprises: constructing a resistance gene reconstruction vector and using Argonaute to perform linearization processing on the resistance gene reconstruction vector, fragmenting a target DNA into a plurality of small DNA fragments and synthesizing the small DNA fragments, loading the small DNA fragments into the resistance gene reconstruction vector, and using SLIC cloning and resistance gene reconstruction to implement assembly of the target DNA. SLIC cloning and a resistance gene reconstruction strategy are integrated, and 5-6 small fragments can be assembled by reconstructing a resistance gene once, thereby achieving higher efficiency and shorter cycle; and the frequency of reconstructing the resistance gene can be flexibly selected on the basis of the length of fragments of a DNA, mutation caused by PCR is not introduced, and the reconstructed large fragment does not need to undergo secondary sequencing, thereby saving time and reducing costs, and providing new ideas for the synthesis and assembly technology of large DNA fragments.

Description

Efficient synthesis and assembly of large DNA fragments based on the novel programmable nuclease Argonaute Technical Field

[0001] The present invention belongs to the technical field of large-fragment DNA synthesis and assembly, and specifically relates to a method for efficiently synthesizing and assembling large-fragment DNA based on a novel programmable nuclease Argonaute (Ago for short). Background Art

[0002] DNA synthesis is playing an increasingly important role in various fields, including genetic engineering, clinical diagnosis and treatment, and forensic medicine. Large-scale, low-cost DNA synthesis promises rapid development in areas such as engineering biology, therapeutics, data storage, and nanotechnology. While DNA synthesis technology has made significant progress in recent years, challenges remain in terms of throughput, cost control, and, in particular, the synthesis of large DNA fragments. The assembly of large DNA fragments still relies on time-consuming and expensive cloning techniques. The development of synthetic biology currently faces a bottleneck in DNA supply. Writing technologies, primarily based on DNA synthesis, have significant room for improvement in terms of synthesis length, accuracy, and cost. While the assembly and transfer technologies for small DNA fragments are relatively mature, the large molecular weight and susceptibility of large DNA fragments to fragmentation make in vitro manipulation cumbersome and inefficient. Therefore, there is an urgent need to develop low-cost, high-efficiency methods for DNA synthesis and assembly of large DNA fragments to improve the ability to "write DNA" and further expand the scope of DNA applications.

[0003] Currently, DNA assembly technologies primarily include enzyme-dependent assembly, non-enzyme-dependent assembly, and in vivo homologous recombination-based assembly methods. Enzyme-dependent assembly includes DNA polymerase-based assembly. Polymerase Cycling Assembly (PCA), based on PCR technology, relies on high-fidelity proofreading PCR enzymes and is unsuitable for the synthesis of long DNA fragments. Furthermore, as DNA length increases, the mutation rate increases, making it unsuitable for the synthesis of specialized DNA sequences such as those with high GC content and repetitive sequences. Restriction enzyme-based methods also include Bio-Brick and Golden Gate. However, the main limitations of these two methods are that the element sequence cannot contain restriction site sequences in the prefix and suffix, and scar sequences are formed at the junctions. These scar sequences may affect the function of the biological element and prevent seamless assembly. Large DNA fragment assembly methods based on Cas programmable enzymes, however, all suffer from scarring effects, as Cas proteins require PAM sequences for their enzymatic cleavage, preventing strict seamless assembly. Furthermore, Cas guide nucleic acids are all relatively long RNAs, which are unstable and expensive, resulting in high assembly costs and low efficiency.

[0004] In summary, current DNA synthesis, especially the assembly of large DNA fragments, suffers from low efficiency and high cost. Furthermore, there are currently no efficient methods for synthesizing specialized DNA fragments, such as those with high GC content and repetitive sequences. This has significantly hindered the development and progress of synthetic biology. Therefore, there is an urgent need to develop efficient and low-cost DNA synthesis and large DNA assembly technologies to promote the development of synthetic biology.

[0005] Summary of the Invention

[0006] In response to the problems existing in the prior art of large-fragment DNA assembly, the present invention provides a method for efficient synthesis and assembly of large-fragment DNA based on a novel programmable nuclease Argonaute.

[0007] The technical method of the present invention is specifically as follows:

[0008] A method for efficiently synthesizing and assembling large DNA fragments based on the novel programmable nuclease Argonaute comprises the following steps:

[0009] S1. Construction of resistance gene reconstruction vector pNEW (Amp r ) plasmid, the pNEW (Amp r ) The plasmid contains a stuffer segment and a resistance gene segment, and there is a homologous sequence of m (bp) between the resistance gene segments;

[0010] S2. Use Argonaute and gDNA to remove pNEW (Amp r ) plasmid to obtain a linear vector with a 3' end protruding m (nt);

[0011] S3, dividing the target DNA into n small DNA fragments of about 500 bp, synthesizing the small DNA fragments and introducing m (nt) sequences that are homologous and complementary to the linear vector;

[0012] S4, the linear vector obtained in S2 was mixed with the small DNA fragment synthesized in S3 and transformed into E. coli competent cells to obtain the recombinant plasmid pNEW (Amp r )1,2,3,……,n;

[0013] S5, based on the reconstruction of Argonaute and resistance genes, the recombinant plasmid pNEW (Amp r )1,2,3,……,n for assembly.

[0014] In the method of the present invention, the nuclease Argonaute used needs to cut the plasmid into a linear vector, but not all Argonaute or its mutants that have the function of cutting double-stranded DNA can achieve the present invention; the inventors found that the nuclease Argonaute used has the ability to cut the plasmid into a linear vector. 2+The method of the present invention can be well implemented only when the activity can efficiently cut double-stranded DNA and plasmid DNA under the conditions, especially when it can cut double-stranded DNA with a GC content ≥ 50 (such as a GC content of up to 70%).

[0015] Preferably, in the above method, pNEW(Amp r ) The resistance genes in the plasmid include but are not limited to Kan r 、Chl r 、Gen r and Spc r , and the number of resistance genes can be set according to actual assembly requirements.

[0016] Preferably, in the above method, pNEW(Amp r ) The filler fragment in the plasmid is the ccdb lethal gene (sequence shown in SEQ ID NO.3); by introducing the ccdb lethal gene for reverse screening, the background is extremely low, which reduces the workload of later screening of recombinant colonies.

[0017] In a specific embodiment of the present invention, there is provided pNEW (Amp r ) plasmid also contains Kan r 、Chl r 、Gen r and Spc r Resistance genes, and the ccdb lethal gene was used as a filler fragment. There were 10bp homologous sequences between the resistance fragments 5Kana / 3Kana, 5Chl / 3Chl, 5Gen / 3Gen and 5Spc / 3Spc.

[0018] Preferably, in the above method, the nuclease Argonaute is ApfAgo and its amino acid sequence is shown in SEQ ID NO.1; more preferably, the gene sequence encoding the ApfAgo is shown in SEQ ID NO.2, and the nuclease can cut 80%-90% of double-stranded DNA with a 70% GC content within 15 minutes.

[0019] Preferably, in the above method, step S2 specifically comprises: designing 16-18nt gDNA based on the homologous sequence and performing 5'-end phosphorylation treatment, incubating the 5'-P gDNA with Argonaute and then mixing it with the target plasmid for reaction, and recovering it for later use after the reaction is completed by agarose gel detection.

[0020] In a specific embodiment of the present invention, ApfAgo is used to linearize the target plasmid, and the reaction conditions are as follows: 5'-P gDNA is first incubated with ApfAgo at 70°C for 5 minutes, then mixed with the target plasmid and reacted at 92°C for 10 minutes.

[0021] Preferably, in the above method, the method for synthesizing small DNA fragments in step S3 is: designing a seamless oligonucleotide primer combination with overlapping 17-20 nt for the small DNA fragments, wherein the length of the oligonucleotide primers is 50-59 nt, and the 5' ends of the first and last oligonucleotide primers respectively introduce m (nt) sequences that are homologous and complementary to the linear vector; placing the oligonucleotide primer combination and LA Taq Buffer in a reaction tube for annealing to synthesize small DNA fragments.

[0022] More preferably, the annealing procedure is: 94°C, 5 min, 94°C-37°C slope 20 min, 37°C, 7 min.

[0023] Preferably, in the above method, step S5 is specifically: the recombinant plasmid pNEW (Amp r )1,2,3,……,n are divided into x groups, with 2 to 6 recombinant plasmids in each group. The first and last two recombinant plasmids of each group are treated with Argonaute enzyme to obtain fragments with the target sequence and resistance gene unit. The other intermediate plasmids are treated with Argonaute enzyme to obtain the target fragment with only m (nt) protruding from the 3' end. Since the reconstructed resistance gene is used for forward genetic screening, there is no need to worry about background plasmid contamination without enzyme digestion. Therefore, the enzyme digestion products do not need to be recovered. The enzyme digestion products can be directly mixed and transformed into Escherichia coli competent cells without F plasmid (F-). X recombinants are screened and the above steps are repeated until all small DNA fragments are assembled into the target DNA.

[0024] It should be noted that, in addition to plate coating, the recombinant screening method in step S5 can also be performed by directly using solution culture with antibiotics containing corresponding resistance genes. This method further shortens the cycle and reduces costs.

[0025] In the method of the present invention, starting from a small DNA fragment of 450-500bp, if 6 fragments are assembled at a time and the resistance gene is reconstructed once, a fragment of 3kb in length can be obtained, which can meet more than 99% of gene synthesis; reconstructing twice can assemble to 18kb, which can meet more than 99% of the synthesis of large DNA fragments; reconstructing three times can reach 30kb.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention divides large DNA fragments into small fragment units of 450-500 bp for resynthesis. The small fragment units are directly annealed through a seamless oligonucleotide primer combination, and hydrogen bonds between bases are used for complementary pairing. The small fragment units are then annealed with a vector with sticky ends to form a circularized plasmid with a nick. E. coli is transformed and synthesis is performed using the E. coli's own repair system. No PCR amplification step is introduced, so mutations introduced by PCR amplification will not be introduced, which greatly improves the accuracy of synthesis.

[0028] (2) The present invention introduces the ccdb lethal gene into the resistance gene reconstruction vector, and utilizes the principle of ccdb lethality to transform Escherichia coli competent cells that do not contain F plasmid (F-). This can make the background plasmid without the target fragment inserted unable to grow due to the presence of the ccdb lethal gene, thereby greatly reducing the background, improving the transformation efficiency and recombination efficiency, and reducing the workload of screening recombinants.

[0029] (3) The present invention utilizes a combination of SLIC cloning and resistance gene reconstruction strategies to assemble small fragments. Small fragment DNA units are obtained by Argonaute digestion. The digested products are directly transformed into Escherichia coli without PCR operation. The resistance reconstruction gene is used for genetic screening, and mutations caused by PCR amplification are not introduced. Therefore, the mutation rate is extremely low, even zero, and no secondary sequencing is required, which is low in cost. Combined with SLIC cloning, multiple fragments can be assembled in one reconstruction, which improves efficiency and shortens the synthesis cycle. Reconstruction is done once a day. That is, starting from a correctly sequenced 450-500bp target fragment plasmid, it can be assembled to 30kb in 3 days.

[0030] (4) The operating system of the present invention is simple. Only one Ago enzyme is used to digest the plasmid. The digestion product does not need to be purified or ligated and can be directly transformed into Escherichia coli.

[0031] (5) The present invention utilizes the principle of resistance gene reconstruction. While reconstructing the resistance gene, multiple target fragments with mutually complementary homologous sequences are assembled together. Forward genetic screening is performed using the corresponding antibiotics to which the resistance is reconstructed. After the addition of the corresponding antibiotic, only plasmids with a complete corresponding resistance gene reconstructed can grow. Some plasmids that have not been digested by enzymes or products with incorrect reconstructions cannot grow in the medium containing antibiotics because they cannot obtain a complete resistance gene. This method has high accuracy and is simple to operate.

[0032] (6) Special DNA sequences can be synthesized, such as highly repetitive sequences, Poly A and other Poly sequences, and some complex structures or some regulatory DNA sequences can be synthesized for research. Since this method starts from a plasmid and the fragments are obtained by enzyme digestion, PCR is not required, and highly repetitive sequences that cannot be synthesized by PCR can be synthesized.

[0033] (7) Since the antibiotic resistance gene is used for positive screening, there is no fear of interference from background plasmids. The recombinant plasmid transformants after reconstruction can be cultured directly in the antibiotic liquid culture medium corresponding to the reconstructed resistance gene without the need for plate coating. That is, the screening operation is simple and solution operation can be realized, which is conducive to the realization of automated operation and further shortens the cycle.

[0034] (8) It is conducive to the construction of mutant libraries, and is very efficient for site-directed mutagenesis and directed evolution. The screening workload can be reduced to 2-3 orders of magnitude compared with the existing methods for constructing mutant libraries. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 is a diagram showing the SDS-PAGE analysis results of the nucleic acid programming enzyme Argonaute (APfAgo) used in an embodiment of the present invention.

[0036] FIG2 is a diagram of the resistance gene reconstruction vector pNEW (Amp r ) Schematic diagram of the plasmid structure.

[0037] FIG3 is a diagram of pNEW (Amp r ) Agarose gel electrophoresis detection results of the products obtained by ApfAgo at different cutting times of the plasmid, OC is an open circular plasmid (one chain of the plasmid is broken), LIN is a linearized plasmid (double-stranded plasmid is broken), SC is a supercoiled plasmid, and the red arrows indicate the enzyme cutting products.

[0038] FIG4 is a graph showing the agarose gel electrophoresis detection results of the products obtained by cleaving double-stranded DNA with different GC contents by APfAgo used in an embodiment of the present invention, where product bands represent cleavage products.

[0039] FIG5 is a schematic diagram of the structure of a recombinant plasmid formed after the introduction of a small DNA fragment in an embodiment of the present invention.

[0040] FIG6 is a graph showing the results of agarose gel electrophoresis detection of enzyme digestion of plasmids 1-16 in the first round of reconstructed plasmids in Example 5 of the present invention.

[0041] FIG7 is a diagram of the first round of reconstruction of Kan in Example 5 of the present invention. r Peak diagram of sequencing results at the junction of the recombinant plasmid after resistance.

[0042] FIG8 is a flow chart of assembling large DNA fragments based on Argonaute, SLIC, and resistance gene reconstruction strategies in an embodiment of the present invention (assembling and reconstructing two plasmids at a time).

[0043] Figure 9 is a schematic diagram of the first step (first SLIC and reconstruction of Kan resistance gene) of assembling large DNA fragments (6 plasmids reconstructed at one time) based on Argonaute, SLIC and resistance gene reconstruction strategy in an embodiment of the present invention.

[0044] Figure 10 is a schematic diagram of the second step (second SLIC and reconstruction of Chl resistance gene) of assembling large DNA fragments (6 plasmids reconstructed at one time) based on Argonaute, SLIC and resistance gene reconstruction strategy in an embodiment of the present invention.

[0045] Figure 11 is a schematic diagram of the third step (third reconstruction of the Gen resistance gene) of assembling large DNA fragments (6 plasmids reconstructed at one time) based on Argonaute, SLIC and resistance gene reconstruction strategies in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms "include" and "have" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.

[0048] In order to solve the problems of low efficiency, high cost, and difficulty in synthesizing special DNAs such as high GC content and repetitive sequences in existing large-fragment DNA assembly technologies, the present invention provides a large-fragment DNA synthesis and assembly method based on the programmable nuclease Argonaute.

[0049] The method of the present invention mainly comprises the following steps:

[0050] (1) Preparation of resistance gene reconstruction vector for assembling DNA fragments.

[0051] The resistance gene reconstruction vector of the present invention contains a stuffing segment and a resistance gene segment, and homologous sequences for SLIC cloning are arranged between the resistance gene segments.

[0052] In one embodiment of the present invention, plasmid pET-23a vector was selected as the starting vector for transformation, and Kan r Resistance, Chl r Resistance, Gen r Resistance and Spc r Resistance plasmid as template for amplification Kan r 、Chl r、Gen r and Spc r Resistance gene, and finally constructed into pNEW (Amp r ) plasmid; the plasmid uses the ccdb lethal gene as a stuffer fragment, and the homologous sequence length is 10bp.

[0053] (2) Preparation of linear vectors.

[0054] The resistance gene reconstruction vector that has been sequenced correctly is digested with Ago enzyme. By designing a suitable guide DNA, the filler fragment (ccdb lethal gene) is cut out, and the cut linear vector is allowed to protrude 10nt at each of its 3' ends. The enzyme digestion product is recovered by gel and stored at -20℃ for later use.

[0055] (3) Synthesis of small DNA fragments.

[0056] Select the target DNA, divide it into small DNA fragments, and then synthesize each small DNA fragment separately.

[0057] Taking a small DNA fragment of 450-500 bp as an example, oligonucleotide primer combinations with overlapping 17-20 nt and no gaps were designed. For each 450-500 bp fragment, 16 oligonucleotide primers of 50-59 nt in length were synthesized, and the 5' ends of the first and last primers were introduced with sequences complementary to the linear vector. Each of the 16 synthesized oligonucleotide sequences was diluted to a final concentration of 10 M. Then, 0.5 μL of each was transferred to a PCR tube, and 2 μL of 10× Buffer (500 mM KAc, 200 mM Tris-Ac, 100 mM MgAc, 1 mg / mL BSA) was added. Finally, double-distilled water was added to a final volume of 20 μL and mixed thoroughly. Annealing was performed using a stepwise cooling program: 94°C for 5 minutes, a 94°C-37°C slope for 20 minutes, and 37°C for 7 minutes.

[0058] (4) SLIC cloning to prepare recombinant plasmid.

[0059] After mixing the prepared linear vector with the annealed small fragment DNA, directly transform the competent E. coli cells without F factor (such as DH5a), apply ampicillin resistance plates, screen the recombinants, and name the recombinant plasmids with correct sequencing as pNEW (Amp r )1,2,3........

[0060] (5) Assemble large fragments using SLIC cloning and resistance gene reconstruction strategies.

[0061] While reconstructing the resistance gene, multiple target fragments with homologous and complementary sequences were assembled together through SLIC cloning.

[0062] Taking the synthesized DNA fragment of 30kb as an example, first divide it into 60 fragments of about 500bp, refer to the above steps, and use pNEW (Amp r ) plasmids to produce 60 recombinant plasmids. The 60 recombinant plasmids were divided into 10 groups, each with 6 plasmids, and the first reconstruction was carried out. Specifically, the recombinant plasmids containing the first and last fragments in each group were digested with Ago enzyme to cut out the fragments with the target sequence and the resistance gene unit. The plasmids containing the middle fragments in each group were also digested with Ago enzyme to cut out only the target fragment with the homologous sequence protruding from the 3' end. The digested products were then mixed and directly transformed into Escherichia coli, and coated with Kan r Resistance plates were used for screening to obtain 10 new recombinant plasmids (i.e., 1 in each group). The target DNA fragment size of each recombinant plasmid was about 3 kb.

[0063] The 10 recombinant plasmids obtained in the first reconstruction were divided into 2 groups, with 5 plasmids in each group. The second reconstruction of Chl r Resistance gene, you can get two new recombinant plasmids, and the target DNA fragment size of each recombinant plasmid is about 15kb. Then the two plasmids obtained by the second reconstruction are cut into two by two enzymes, and the third reconstruction is carried out. r resistance gene, and a plasmid was obtained, which contained a 30kb target DNA fragment.

[0064] The method of the present invention starts from the plasmid, does not pass through PCR, and will not introduce mutations introduced by PCR. The large fragments after assembly do not require secondary sequencing, and the cost is low. Moreover, the method of the present invention performs forward genetic screening by reconstructing the antibiotic resistance gene, and at the same time utilizes the difference between the resistance of the recombinant after reconstructing the resistance and the resistance of the starting plasmid, and the transformed recombinant is a recombinant, which can realize solution operation, simplify the operation steps, reduce costs, and is easier to realize automated operation. In addition, the enzyme cutting system has only one Ago enzyme, the system is simple, and the screening is carried out by utilizing the different resistance of the recombinant and the background plasmid, without fear of interference from the background plasmid. The enzyme-cut fragments do not need to be recovered and purified, and no enzyme-linked operation is required. Direct conversion is performed, and no special expensive instruments such as PCR instruments are required, and the operation is simple.

[0065] In the method of the present invention, the nuclease Argonaute or its mutant can achieve the present invention as long as it meets the following conditions:

[0066] With Mg 2+ The cleavage activity of double-stranded DNA and plasmid DNA can be highly efficient under certain conditions, and it can also cleave double-stranded DNA with a high GC content (such as a GC content of 70%).

[0067] Compared with Cas programmed enzymes, Argonaute nucleases do not require PAM recognition sequences when cleaving DNA, and the cleavage is not restricted by any sequence. It only requires a 16-18nt DNA guide to cleave any site, which is conducive to seamless assembly without avoiding any cleavage sites. It has the advantages of simple experimental design and simple operation.

[0068] The following are some specific examples. It should be noted that the examples described below are illustrative and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the examples, they were performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions. Reagents or instruments used, for which the manufacturer is not specified, are all commercially available conventional products.

[0069] Example 1

[0070] Taking the synthesized DNA fragment of 30kb as an example, this example realizes the synthesis and assembly of large DNA fragments through the following steps:

[0071] (1) Expression and purification of Argonaute.

[0072] The programmable nuclease Argonaute used in this example is a PfAgo mutant (abbreviated as APfAgo), whose amino acid sequence is shown in SEQ ID NO. 1. The specific preparation method is as follows:

[0073] The APfAgo gene sequence (as shown in SEQ ID NO. 2) was connected to pET28a to obtain the pET28a-APfAgo plasmid, which was then transformed into Escherichia coli BL21 (DE3). A single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured on a shaker at 37°C and 220 rpm. When the bacterial OD 600 When the pH reached 0.8, the cells were transferred to an 18°C ​​shaker and induced overnight with IPTG. The cells were harvested by centrifugation at 6000 rpm for 10 minutes and washed with Buffer A (20 mM HEPES pH 7.5, 250 mM NaCl, 1 mM DTT). The cells were then resuspended in Buffer A and supplemented with PMSF to a final concentration of 1 mM. The cells were then disrupted by high pressure and centrifuged at 18000 rpm for 30 minutes. The supernatant was collected.

[0074] After filtering the supernatant, Ni-NTA purification was performed. 10 column volumes of 20mM and 50mM imidazole were each washed (added three times), and 3 column volumes of 100mM, 200mM, and 300mM were each washed. Samples were taken for SDS-PAGE detection. The eluted fractions containing the highly pure target protein were collected and ultrafiltered into Buffer A. After diluting the NaCl concentration to 125mM with 20mM HEPES (pH 7.5), purification was performed on a heparin column (HiTrap Heparin HP, GE Healthcare). The heparin column was pre-equilibrated with Buffer B (20mM HEPES pH 7.5, 125mM NaCl), and APfAgo was eluted by increasing the NaCl concentration. The purified protein was collected, the purity was identified by SDS-polyacrylamide gel, and the activity was verified. The protein was then divided into small portions, snap-frozen in liquid nitrogen, and stored at -80°C.

[0075] The purified target protein was concentrated using Millipore 50-kDa ultrafiltration tubes at 4°C and 4000 rpm, and the solution was exchanged to remove imidazole. The enzyme concentration was quantitatively determined using a BCA assay kit according to the manufacturer's instructions. Using BSA as the standard, a standard solution was prepared and a standard curve was plotted to calculate the protein concentration of the purified APfAgo.

[0076] The results of SDS-PAGE identification analysis are shown in Figure 1. According to calculations at http: / / www.expasy.org / , the expected size of APfAgo is 87 kDa.

[0077] (2) Preparation of resistance gene reconstruction vector.

[0078] In this case, the plasmid pET-23a vector was selected as the starting vector for transformation, and the resistance gene reconstruction vector pNEW (Amp r ) plasmid, the specific operation is as follows:

[0079] PCR amplification of Kan was performed using four resistant plasmids pET-28a, pASK-IBA7C, pDONR223, and pMP2463 as templates. r 、Chl r 、Gen r and Spc r Resistance gene, on this basis, through several rounds of overlap extension PCR, all fragments were finally fused into four entries of fragments, and the pure target fragment was obtained by gel recovery. The four fragments were cloned by T5 exonuclease at a molar ratio of 1:1:1:1. The positive clones were initially screened by colony PCR and sent to a biological company for sequencing. The plasmid of the correctly sequenced colony was extracted and named pNEW (Amp r), and stored at -20°C until use.

[0080] In pNEW(Amp r ) plasmid, a 10 bp homologous sequence was reserved between each resistance fragment 5Kana / 3Kana, 5Chl / 3Chl, 5Gen / 3Gen, and 5Spc / 3Spc for subsequent gDNA design.

[0081] Figure 1 shows the pNEW(Amp r ), in which the ccdb lethal gene serves as a filler fragment for subsequent cloning of small DNA fragments.

[0082] (3) ApfAgo to pNEW(Amp r ) Plasmid cutting activity verification and linearization treatment.

[0083] Since APfAgo targets ssDNA through guide DNA (gDNA), two pairs of 16nt gDNA were designed in this example to cut double-stranded DNA (i.e., pNEW (Amp r ) plasmid), and the gDNA was 5' end phosphorylated.

[0084] The cleavage and detection process was as follows: 5'-PgDNA was first incubated with APfAgo protein at 70°C for 5 minutes, then mixed with the target plasmid and reacted at 92°C for 1, 3, and 5 minutes, respectively. 2 μL of 6× DNA loading was added, and the mixture was detected by electrophoresis on a 1.0% agarose gel. The incubation and reaction systems used in this process are shown in Tables 1 and 2, respectively.

[0085] The detection results are shown in Figure 3: APfAgo can use a pair of 5'P-gDNA to target one chain of the plasmid respectively, cut the supercoiled plasmid, and generate linearized plasmid DNA; and as the cutting time increases, the content of linearized plasmid in the product increases.

[0086] Table 1 Incubation system of gDNA and ApfAgo

[0087] Table 2 Reaction system of APfAgo enzyme digestion plasmid

[0088] Referring to the above enzyme digestion conditions, APfAgo was used to digest pNEW (Amp r ) plasmid was digested for 10 min to obtain a linear vector backbone with a 10 nt overhang at the 3' end, which was recovered by agarose gel detection and stored at -20°C for future use.

[0089] (4) ApfAgo cleavage activity in regions with different GC contents

[0090] In order to investigate the activity of APfAgo in cutting high GC content regions of plasmids, the pNEW (Amp r Three sets of gDNA with varying GC contents (50%, 60%, and 70%) were designed using plasmids. Target bands were amplified by PCR, and the resulting linear double-stranded DNA (dsDNA) was cleaved. The cleavage products were analyzed on a 1.0% agarose gel. The results demonstrated that APfAgo can cleave regions with GC contents up to 70%, whereas wild-type PfAgo has no cleavage activity against double-stranded DNA with GC contents of 50% or higher, as shown in Figure 4.

[0091] (5) Small fragment pNEW(Amp r ) Synthesis of plasmids.

[0092] The target DNA was divided into n small fragments of 500 bp, and the small fragment DNA was synthesized by the following process: oligonucleotide primer sequence pairs with 17-20 nt overlap and no gaps were designed. For each 500 bp fragment, 16 oligonucleotide primers with a length of approximately 50-59 nt were synthesized, and the 5' ends of the first and last primers respectively introduced 10 nt of sequences homologous to the linear vector. The 16 synthesized oligonucleotide sequences were then diluted to a final concentration of 10 M. 0.5 μL of each was then transferred to a PCR tube, 2 μL of 10× Buffer (500 mM KAc, 200 mM Tris-Ac, 100 mM MgAc, 1 mg / mL BSA) was added, and finally double-distilled water was added to a final volume of 20 μL to mix. The mixture was annealed according to a stepwise cooling program of 94°C for 5 minutes, 94°C-37°C slope for 20 minutes, and 37°C for 7 minutes to obtain small DNA fragments.

[0093] Prepare the pNEW(Amp r ) linear vector was mixed with the small fragment DNA formed by annealing, and then transformed into Escherichia coli DH5a competent cells without F factor after being placed at 37℃ for 30min. The cells were coated with ampicillin resistance plates. Since the background plasmid contained the ccdb lethal gene, all the recombinants that grew were recombinants. The recombinant plasmids that were sequenced correctly were named pNEW (Amp r )1,2,3.......n. The structure of the obtained recombinant plasmid is shown in Figure 5.

[0094] (6) Assembly of large DNA fragments.

[0095] When the present invention utilizes the resistance gene reconstruction strategy for assembly, 2-6 plasmids can be reconstructed each time. The following examples illustrate the specific operation:

[0096] 1. The target DNA was prepared according to step (5) to obtain 16 recombinant plasmids, which were divided into 8 groups, with two plasmids in each group.

[0097] First, Kan r Resistance reconstruction, parallel 8 groups of recombinant plasmids pNEW (Amp r ) The corresponding gDNA was selected to cut 16 plasmids for 10 minutes (the conditions were the same as in step (3)). The obtained enzyme digestion products were detected by 0.7% agarose gel, as shown in Figure 6. 2 μL of each enzyme digestion product was mixed and directly transformed into Escherichia coli. It was directly cultured with LB liquid medium with 50 μg / mL kanamycin. The plasmids were extracted and further sent to the company for sequencing. The sequencing results (Figure 7) showed that there was no increase or decrease in the cutting position and no scar was generated at the junction. The 8 pNEW (Kan r ) plasmids were divided into 4 groups, with two plasmids in each group, and Chl r Resistance reconstruction, resulting in four plasmids pNEW (Chl r ). Then the four plasmids were divided into two groups, with two plasmids in each group, and the r Resistance reconstruction, resulting in two plasmids pNEW (Gen r Finally, the two plasmids pNEW (Gen r ) for Spe r The resistance reconstruction obtained the plasmid pNEW (Spe r ), the DNA assembly is completed, and the assembly process is shown in Figure 8.

[0098] 2. Prepare 60 plasmids by target DNA according to step (5), and divide the plasmids into 10 groups, with 6 plasmids in each group. Each group contains the recombinant plasmid pNEW (Amp r ) were digested with APfAgo for 10 min (same conditions as step (3)) to cut out the fragments with the target sequence and resistance gene unit, and the plasmids of the middle fragments were digested with nuclease Ago for 10 min (same conditions as step (3)) to cut out the target fragments with only 10 nt protruding from the 3' end. Then 2 μL of the digested products were mixed and directly transformed into Escherichia coli, and directly cultured with LB liquid medium supplemented with 50 μg / mL kanamycin, thus obtaining 10 recombinant plasmids pNEW (Kanamycin). r ), the target DNA fragment size of each recombinant plasmid is about 3kb. r ) plasmids were divided into 2 groups, each with 5 plasmids. Each group of 5 plasmids was used to reconstruct the chloramphenicol resistance gene and SLIC clone, and 2 plasmids pNEW (Chl r), the target DNA fragment size of each recombinant plasmid is about 15kb. Then two plasmids pNEW (Chl r ) were digested in pairs and the gentamicin resistance gene was reconstructed to obtain a plasmid pNEW (Gen r ) to obtain a 30kb target DNA fragment, and the assembly process is shown in Figures 9-11.

[0099] Since the starting plasmid is a plasmid that has been verified to be correct by sequencing, the present invention assembles large fragments by reconstructing the plasmid starting from the correctly sequenced plasmid. No PCR is performed in the entire process, only enzyme digestion is performed, and no mutations caused by PCR are introduced. Therefore, the recombinants screened by positive genetic factors such as resistance genes are correct, that is, the reconstructed large fragments do not need to be sequenced again, saving time and cost.

[0100] In summary, the present invention divides the large DNA fragment to be assembled into several small DNA fragments of 450bp-500bp, and then loads the small DNA fragments into the resistance gene reconstruction vector pNEW (Amp r ) plasmid, adjacent small fragments have 10 bp of homology sequence at the beginning and end for SLIC cloning. SLIC cloning and resistance gene reconstruction can then be used to assemble large DNA fragments. The number of resistance gene reconstructions can be determined based on the length of the target DNA fragment. Furthermore, the present invention utilizes Argonaute, which has high enzymatic activity against plasmid DNA, to linearize the target plasmid. After the cleaved target fragments are mixed, their cohesive ends with homology arms can anneal to form a nicked circular DNA molecule. The nick is then repaired using the in vivo repair mechanism of E. coli to obtain a recombinant plasmid, making the method simple and efficient.

[0101] The efficient and low-cost large-fragment DNA assembly technology developed by the present invention can provide a powerful supplement for the development of the underlying biotechnology toolkit for synthetic biology, and provide new ideas for the development of large-fragment DNA synthesis and assembly technology on the market, and has strong application value.

[0102] The above description is a specific implementation method of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for efficient synthesis and assembly of large DNA fragments based on a novel programmable nuclease Argonaute, characterized in that: The following steps are involved: S1. Construction of antibiotic resistance gene reconstruction vector pNEW (Amp r ) plasmid, the pNEW (Amp r ) The plasmid contains a stuffer fragment and a resistance gene fragment, and there is a homologous sequence of m (bp) between the resistance gene fragments; S2. Using Argonaute and gDNA to remove pNEW (Amp r ) plasmid to obtain a linear vector with a 3' end protruding m (nt); S3, dividing the target DNA into n small DNA fragments of 450-500 bp, synthesizing the small DNA fragments and introducing m (nt) sequences that are homologous and complementary to the linear vector; S4, the linear vector obtained in S2 was mixed with the small DNA fragment synthesized in S3 and transformed into competent E. coli cells to obtain the recombinant plasmid pNEW (Amp r )1,2,3,……,n; S5, based on Argonaute and resistance gene reconstruction, recombinant plasmid pNEW (Amp r )1,2,3,……,n for assembly.

2. The method for efficient synthesis and assembly of large DNA fragments according to claim 1, characterized in that: In pNEW(Amp r ) plasmid, the resistance gene is Kan r , Chl r , Gen r and Spc r One or more of .

3. The method for efficient synthesis and assembly of large DNA fragments according to claim 2, characterized in that: In pNEW(Amp r ) plasmid, the resistance gene is Kan r , Chl r , Gen r and Spc r , and there are 10 (bp) homologous sequences between the resistance fragments 5Kana / 3Kana, 5Chl / 3Chl, 5Gen / 3Gen and 5Spc / 3Spc.

4. The method for efficient synthesis and assembly of large DNA fragments according to claim 1, characterized in that: The filler fragment is the ccdb lethal gene.

5. The method for efficient synthesis and assembly of large DNA fragments according to claim 1, characterized in that: The Argonaute is able to 2+ The Argonaute can effectively cut double-stranded linear DNA and plasmid DNA under the conditions, and the Argonaute can cut double-stranded DNA with a GC content of ≥50%.

6. The method for efficient synthesis and assembly of large DNA fragments according to claim 5, characterized in that: The amino acid sequence of Argonaute is shown in SEQ ID NO.

1.

7. The method for efficient synthesis and assembly of large DNA fragments according to claim 1, characterized in that: Step S2 specifically includes: designing 16-18nt gDNA for the homologous sequence and performing 5'-end phosphorylation treatment, incubating the 5'-P gDNA with Argonaute and then mixing it with the target plasmid for reaction, and recovering it for later use after the reaction is completed by agarose gel detection.

8. The method for efficient synthesis and assembly of large DNA fragments according to claim 1, characterized in that: The method for synthesizing small DNA fragments in step S3 is as follows: designing a seamless oligonucleotide primer combination with 17-20 nt overlap for the small DNA fragments, wherein the length of the oligonucleotide primers is 50-59 nt, and the 5' ends of the first and last oligonucleotide primers respectively introduce m (nt) sequences that are homologous and complementary to the linear vector; placing the oligonucleotide primer combination and buffer in a reaction tube for annealing to synthesize small DNA fragments.

9. The method for efficient synthesis and assembly of large DNA fragments according to claim 8, characterized in that: The annealing procedure is: 94°C, 5 min, 94°C-37°C slope for 20 min, 37°C, 7 min.

10. The method for efficient synthesis and assembly of large DNA fragments according to claim 1, characterized in that: Step S5 specifically comprises: recombinant plasmid pNEW (Amp r )1,2,3,...,n are divided into x groups, each group has 2 to 6 recombinant plasmids, the first and last two recombinant plasmids of each group are treated with Argonaute enzyme to obtain fragments with target sequence and resistance gene unit, the other intermediate plasmids are treated with Argonaute enzyme to obtain target fragments with only 3' end protruding m (nt), the enzyme digestion products are directly taken and mixed and transformed into Escherichia coli, and x new recombinant plasmids are screened; the above steps are repeated until all small DNA fragments are assembled into target DNA.

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