DNA assembly method based on in vivo recombination of engineering strains and application thereof

Abstract

The present application relates to the technical field of genetic engineering and synthetic biology, and specifically discloses a DNA assembly method based on in vivo recombination of an engineered strain and application thereof. The present application provides an engineered Escherichia coli strain with a stably integrated exogenous Lambda Red homologous recombination system in the genome, which can enhance the in vivo homologous recombination capability. Meanwhile, the present application provides a matching DNA preparation method, in which a circular DNA template is subjected to rolling circle amplification, and then treated with a nicking endonuclease to obtain a high-efficiency linear DNA monomer with a single-stranded homologous end. The linear DNA prepared by the method is transformed into the engineered strain, and the in vivo DNA assembly can be efficiently completed by using the strengthened recombination system of the strain itself. The present application integrates efficient DNA preparation and strengthened in vivo recombination capability, effectively improves the positive rate and the number of transformants of DNA assembly (especially long fragment and multi-fragment assembly), simplifies the operation process, and reduces the dependence on expensive in vitro recombination enzymes and super-high-efficiency competent cells.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and synthetic biology technology, specifically relating to a DNA assembly method based on in vivo recombination of engineered strains and its application. Background Technology

[0002] DNA in vitro assembly and manipulation technology is a core cornerstone of modern synthetic biology, genetic engineering, and metabolic engineering. The development of this technology aims to overcome the limitations of traditional cloning methods, achieving efficient and precise splicing of DNA fragments, especially long and multiple fragments. Its evolution is mainly reflected in the trend from site-specific ligation relying on restriction endonucleases, to seamless assembly based on homologous recombination, and then towards utilizing in vivo recombination systems.

[0003] 1. Traditional enzyme digestion-ligation method

[0004] Represented by T4 DNA ligase, this method achieves ligation by forming phosphodiester bonds at the ends of double-stranded DNA fragments. Although the technology is mature and widely used, it has inherent limitations: 1) It is highly dependent on pre-defined restriction endonuclease recognition sites, which usually introduces redundant base sequences into the assembly product, making it unsuitable for applications requiring seamless splicing; 2) When ligating large fragments exceeding 10 kb in length or assembling more than three fragments simultaneously, the efficiency drops significantly, making it difficult to meet the needs of constructing complex gene circuits or large gene clusters.

[0005] 2. Seamless in vitro assembly method

[0006] To overcome the limitations of traditional methods, seamless assembly technologies, such as Gibson Assembly and Golden Gate cloning, have been developed. Gibson Assembly utilizes the synergistic action of 5' exonuclease, DNA polymerase, and ligase to perform one-step in vitro recombination of multiple DNA fragments with terminal homologous sequences. Golden Gate cloning utilizes the characteristic of IIS-type restriction endonucleases to cut outside the recognition site, achieving directional and seamless assembly of multiple fragments. While these methods have improved the support for multi-fragment assembly, they also have the following shortcomings: 1) The reaction usually requires precise control of the molar ratio of each fragment and has high requirements for the purity of the DNA template; 2) The in vitro recombination efficiency is still limited when assembling very long or highly complex fragments; 3) They rely on commercially available enzyme mixtures or require the preparation of complex reaction systems, resulting in high costs.

[0007] 3. In vivo assembly strategy based on endogenous recombination system

[0008] In recent years, in vivo assembly utilizing the host cell's own DNA repair and recombination mechanisms has become a research hotspot. For example, introducing the Red homologous recombination system (Exo, Beta, Gam proteins) of λ phage into E. coli can enhance its homologous recombination ability with exogenous linear DNA. Theoretically, such strategies can transfer the assembly step to the cell, simplifying the operation. However, existing in vivo assembly protocols still have significant bottlenecks: 1) Directly transforming linear DNA fragments into conventional competent cells results in low transformation efficiency, especially for long DNA fragments, often leading to too few positive clones or even failure; 2) Most protocols still require pre-treatment with in vitro multi-fragment recombination reactions (such as the Gibson reaction) to form recombination substrates that can be recognized by cells, failing to completely eliminate dependence on complex in vitro biochemical reactions, and the process has not been truly simplified.

[0009] In summary, existing DNA assembly technologies, whether traditional in vitro enzymatic ligation or emerging in vitro / in vivo recombination strategies, generally suffer from one or more of the following problems when addressing the growing demand in synthetic biology for assembling long, multi-fragment, and highly complex DNA fragments: dependence on specific sequence sites, insufficient efficiency in assembling large / multi-fragment DNA, stringent requirements on the quality and proportion of input DNA, cumbersome procedures, high reagent costs, and limitations imposed by the transformation and recombination efficiency of host cells. Therefore, developing a novel integrated technology platform that effectively combines efficient in vivo recombination capabilities with convenient, high-yield DNA preparation processes is of great significance for lowering experimental barriers, improving assembly success rates, and advancing research in related fields. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a DNA assembly method based on in vivo recombination of engineered strains and its application. By integrating efficient DNA preparation with enhanced in vivo recombination capabilities, the positive rate and number of transformants for long-fragment and multi-fragment DNA assembly are improved, and the operation process is simplified.

[0011] To achieve this objective, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a DNA assembly method, comprising the following steps:

[0013] S1. A linear DNA monomer with single-stranded homologous ends is provided, wherein the linear DNA monomer is obtained by sequentially performing rolling circle amplification and nicking endonuclease treatment on a circular DNA template containing the target sequence;

[0014] S2. The linear DNA monomers obtained in step S1 are transformed into competent cells of an engineered strain of Escherichia coli, wherein the genome of the engineered strain of Escherichia coli contains expression units of the exogenous Lambda Red homologous recombination system.

[0015] S3. Culture the transformed cells and utilize the homologous recombination ability of the strain to complete DNA assembly in vivo.

[0016] Preferably, the expression unit of the Lambda Red homologous recombination system comprises the nucleotide sequence shown in SEQ ID NO.1.

[0017] Preferably, the expression unit is integrated into the attB site, araA site, or galK site of the strain genome.

[0018] Preferably, the host of the engineered Escherichia coli strain is Escherichia coli DH5α, T1, or TOP10.

[0019] Preferably, in step S1, the rolling circle amplification reaction uses phi29 DNA polymerase.

[0020] Preferably, in step S1, the nicking endonuclease is Nb.BbvCI or Nb.BtsI.

[0021] In a second aspect, the present invention provides an engineered strain of Escherichia coli, which is prepared by the method described in the first aspect, wherein the genome of the engineered strain has a stably integrated expression unit of an exogenous homologous recombination system.

[0022] Preferably, the homologous recombination system is the Lambda Red system or the RecET system.

[0023] Thirdly, the present invention provides the assembly method described in the first aspect, or the application of the engineered Escherichia coli strain described in the second aspect in the assembly of long-fragment and multi-fragment DNA.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This invention enhances the in vivo homologous recombination capacity of host cells by constructing a genome-integrating, highly recombinant engineered strain, significantly improving DNA assembly efficiency and success rate. Experiments show that for 2-5kb DNA fragments, the assembly positivity rate can reach 48.2% to 86.5% without in vitro Gibson reaction, effectively solving the technical challenge of low assembly efficiency for long and multi-fragment DNA.

[0026] 2. For the assembly of small and medium-sized fragments, linearized DNA fragments can be directly co-transformed into engineered strains, completely eliminating the expensive in vitro recombinase reaction step, reducing reagent costs and simplifying the operation process.

[0027] 3. The innovative "rolling circle amplification (RCA) - nicking endonuclease treatment" DNA preparation method of this invention can efficiently prepare linear DNA suitable for transformation from a very small amount (<1 ng) of template. The DNA treated by this method has single-stranded homologous ends, and its transformation potency is improved by an order of magnitude compared with conventional methods, effectively overcoming the bottleneck of insufficient transformants for long DNA fragments and greatly improving DNA transformation efficiency. Attached Figure Description

[0028] Figure 1 This is an electrophoresis diagram of the PCR amplification product of the DH5α integrated strain in Example 2.

[0029] Figure 2 This is a comparison of the DNA assembly positivity rates of DH5α and its integrated strain DH5α ΔattB:: lambda-Red in Example 3.

[0030] Figure 3 This is a comparison of transformation titers after linearization of DNA based on rolling circle amplification and nicking endonuclease in Example 4. Detailed Implementation

[0031] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0033] Example 1: Cloning and preparation of expression units for the Lambda Red homologous recombination system

[0034] The Lambda Red homologous recombination system, derived from λ phage, functions through the synergistic action of three proteins: Exo, Beta, and Gam. This system has been extensively validated for use in conjunction with the CRISPR-Cas system to achieve efficient gene editing and DNA repair in E. coli, and its technical reliability and host compatibility have been fully demonstrated.

[0035] To obtain the core expression unit of this system, the engineered E. coli plasmid pKDsgRNA containing CRISPR editing function was first used as a template, and polymerase chain reaction (PCR) amplification was performed using specific primer pairs F1 (SEQ ID NO. 13) and R1 (SEQ ID NO. 14). This PCR reaction amplified the complete optimized gene sequence of the Lambda Red homologous recombination system, which was named SEQ ID NO. 1.

[0036] Subsequently, the obtained PCR product (SEQ ID NO. 1) was purified by gel extraction and cloned into the pUC57Amp vector backbone linearized with the corresponding enzymes using enzyme digestion-ligation or homologous recombination methods to construct a recombinant plasmid. The constructed recombinant plasmid was verified by DNA sequencing. After confirming the correctness of the Lambda Red gene sequence and reading frame, the verified plasmid was transformed into competent E. coli cells, amplified, and stored at -20℃ as the gene source for subsequent experiments.

[0037] Example 2: Construction of an engineered strain of Escherichia coli with integrated genome

[0038] To achieve stable expression of the Lambda Red homologous recombination system in *E. coli*, this invention selects *E. coli* DH5α as the host strain and utilizes CRISPR-Cas9-mediated homologous recombination technology to precisely integrate the Lambda Red expression cassette into a specific site in its genome. The specific construction steps are as follows:

[0039] 2.1 Integration of Carrier Construction

[0040] 1) Obtaining homologous arm fragments: Using E. coli DH5α genomic DNA as a template, the sequences 200 bp upstream (SEQ ID NO.2) and 200 bp downstream (SEQ ID NO.3) of the attB site on chromosome were amplified by polymerase chain reaction, respectively, as the left and right arms of homologous recombination.

[0041] 2) Vector backbone preparation: The homologous arm fragments obtained by the above amplification are cloned into the corresponding multiple cloning site of the universal cloning vector pUC57Kan by Gibson Assembly or enzyme digestion-ligation method to construct an intermediate vector containing homologous arms. After sequencing verification, the vector is stored.

[0042] 3) Recombinase expression cassette insertion: Using the plasmid containing the optimized Lambda Red gene (SEQ ID NO. 1) obtained in Example 1 as a template, an expression cassette fragment containing the complete promoter, Lambda Red coding sequences (Exo, Beta, and Gam), and a terminator was amplified by PCR. This expression cassette fragment was then precisely inserted between the left and right homologous arms of the intermediate vector constructed in Step 2 using the Gibson Assembly method, thereby constructing the final genome integration vector.

[0043] 2.2 Construction of sgRNA expression vector

[0044] A guide RNA sequence (sgRNA, with its target sequence shown in SEQ ID NO.4) was designed and synthesized targeting the specific sequence of the attB site in the *E. coli* DH5α genome. This sgRNA sequence was cloned into the corresponding position in the expression vector pSynbio-sgRNA using enzyme digestion-ligation or homologous recombination methods to construct a CRISPR-sgRNA expression plasmid targeting the attB site. After sequencing verification, the plasmid was stored.

[0045] 2.3 Preparation of competent cells

[0046] 1) Bacterial culture: Single colonies of Escherichia coli DH5α were inoculated into LB liquid medium and cultured with shaking at 37℃ and 220 rpm until the OD600 value was 0.4-0.6.

[0047] 2) Preparation of electrocompetent cells: After cooling the culture in an ice bath, the cells were collected by centrifugation at 4°C. The cell pellet was washed twice each with pre-cooled sterile ultrapure water and 10% glycerol solution to remove ions and reduce electrolytic resistance. Finally, the cells were resuspended in an appropriate amount of pre-cooled 10% glycerol solution, aliquoted, and stored at -80°C for later use.

[0048] 2.4 CRISPR-Cas9-mediated genome integration

[0049] 1) Obtaining Cas9 protein expression strains: The plasmid pSynbio-Cas, expressing the Cas9 nuclease, was introduced into the DH5α competent cells prepared above via electroporation. After transformation, the cells were revived and cultured, plated on LB plates containing the appropriate antibiotics, and cultured at 37°C for 12-16 hours. Engineered strains containing the pSynbio-Cas plasmid were screened to obtain new competent cells.

[0050] 2) Genome integration: The constructed integration vector (linearized fragment) and pSynbio-sgRNA plasmid were co-electroporated into competent cells containing pSynbio-Cas plasmid. Immediately after electroporation, resuscitation medium was added, and the cells were cultured at 37°C and 150 rpm for 1 hour to allow the cells to recover and express the Cas9-sgRNA complex, cleaving the genomic target site. Simultaneously, the Lambda Red expression cassette on the integration vector was inserted into the cleavage site through homologous recombination.

[0051] 3) Screening for positive clones: Spread the revived bacterial culture onto LB plates containing specific antibiotics and incubate at 37°C for 12-16 hours to screen for potential positive clones.

[0052] 2.5 Validation of the integrated strain

[0053] 1) Preliminary PCR identification: Single colonies were picked from the screening plate and cultured in liquid. Using the culture as a template, colony PCR verification was performed using primer pairs located outside the integration site (upstream outer primer SEQ ID NO.5, downstream outer primer SEQ ID NO.6). If the Lambda Red expression cassette was successfully integrated, a specific band of approximately 3.8 kb would be amplified (results are shown in Figure 1). Figure 1 (As shown).

[0054] 2) Sequencing confirmation: The bands of PCR-positive products were recovered and sequenced to further confirm that the Lambda Red expression cassette had been accurately and completely integrated into the pre-defined attB site of the E. coli DH5α genome without frameshift or mutation.

[0055] 2.6 Nomenclature and Preservation of Engineered Strains

[0056] The verified strain was named *Escherichia coli* DH5α ΔattB:: lambda-Red and can be preserved long-term using conventional microbial preservation methods for future experimental use.

[0057] Example 3: Homologous recombination ability test of engineered strain DH5α ΔattB:: lambda-Red

[0058] To verify the effectiveness of the genome-integrating engineered strain DH5α ΔattB:: lambda-Red constructed in Example 2 as a DNA assembly platform, this example designed an experiment to test and compare the assembly and recombination capabilities of this strain and the original DH5α strain for DNA fragments of different lengths.

[0059] 3.1 Construction of the DNA Fragment for Testing

[0060] To evaluate the assembly efficiency of DNA fragments of different sizes, target DNA inserts of 2 kb, 5 kb, and 10 kb were constructed. First, each target fragment was cloned into the pUC57 vector using conventional in vitro cloning methods (such as Gibson Assembly) to construct test plasmids, named Plasmid-2, Plasmid-5, and Plasmid-10, respectively. After confirming correct cloning through sequencing, the plasmids were extracted, linearized using the corresponding restriction endonucleases, and purified by gel electrophoresis to obtain the linearized vector backbone and each target DNA insert fragment, which were then used as substrates for subsequent assembly tests.

[0061] 3.2 Preparation of competent cells

[0062] Competent cells were prepared using the original *Escherichia coli* DH5α strain and the DH5α ΔattB:: lambda-Red engineered strain constructed in Example 2, respectively, via calcium chloride (CaCl2) chemical transformation. The simplified steps are as follows: Single colonies were picked and inoculated into LB broth, and cultured at 37°C with shaking until mid-logarithmic growth (OD1). 600 (≈0.5); after cooling in an ice bath for 10 min, the cells were collected by centrifugation; the cells were washed and resuspended sequentially with pre-cooled 0.1 M CaCl2-MgCl2 solution and 0.1 M CaCl2 solution; finally, the cells were resuspended with pre-cooled 0.1 M CaCl2 solution, aliquoted, and stored at -80℃ for later use.

[0063] 3.3 Experimental Grouping and In Vitro Recombination Reaction Setup

[0064] To accurately assess the in vivo homologous recombination capacity of the engineered strain, two treatment groups were set up in this experiment:

[0065] In vitro recombinant treatment (HR group): As a control group, the linearized vector and insert fragment were subjected to in vitro Gibson Assembly reaction before transformation according to standard procedures. The reaction system (20 μL) contained: 10 μL Gibson AssemblyMaster Mix, 100 ng of linearized vector, and 2 molar amounts of insert fragment, with the volume made up to ddH2O. After mixing, the mixture was reacted at 50 °C for 45 minutes and then cooled on ice for later use.

[0066] No in vitro recombination treatment (NO-HR group): As the experimental group, no in vitro Gibson Assembly reaction was performed. Equal volumes of linearized vector and insert were directly mixed, and an equal volume of ddH2O was used instead of Gibson Assembly MasterMix. The mixture was placed on ice until transformation.

[0067] 3.4 Transformation and positive clone screening

[0068] Take 10 μL each of the DNA mixtures from the HR group and the NO-HR group, and add them to freshly thawed DH5α and DH5αΔattB:: lambda-Red competent cells, respectively. After incubating on ice for 30 minutes, heat shock at 42°C for 60 seconds, followed by incubation on ice for 5 minutes. Then add 900 μL of LB medium and incubate at 37°C and 150 rpm for 1 hour. Spread an appropriate amount of bacterial culture onto LB selection plates containing the corresponding antibiotics and incubate statically at 37°C for 12-16 hours.

[0069] 3.5 Recombination Efficiency Analysis and Evaluation

[0070] After incubation, the number of single colonies grown on each plate was counted. Single colonies were randomly selected from the plates of each treatment group and inoculated into LB broth for expansion. Using the culture medium as a template, colony PCR amplification was performed using primers specific to the target insert fragment. The PCR products were detected by agarose gel electrophoresis; colonies that amplified the expected band size were identified as positive clones.

[0071] 3.6 Results

[0072] Experimental results are as follows Figure 2 As shown. Comparative analysis shows that:

[0073] Positive rate comparison: In the NO-HR group, the DH5α ΔattB:: lambda-Red engineered strain showed a significantly higher assembly positive rate for 2 kb to 10 kb fragments than the original DH5α strain. Particularly for 2-5 kb fragments, the DH5α ΔattB:: lambda-Red strain still achieved an assembly positive rate of 48.2% to 86.5% without any in vitro recombination reaction (NO-HR), demonstrating its strong in vivo homologous recombination ability.

[0074] Technical advantages: The advantage of the DH5α ΔattB:: lambda-Red engineered strain is that for the assembly of smaller fragments (such as 2-5 kb), the expensive in vitro Gibson Assembly step can be completely omitted (taking the commercial reagents used in this experiment as an example, the cost of a single reaction is about 226.9 yuan). It can be completed efficiently by relying solely on the strain's own recombination system, which greatly simplifies the operation process and reduces experimental costs.

[0075] The results of this embodiment fully demonstrate that the DH5α ΔattB:: lambda-Red engineered strain, constructed by stably integrating the Lambda Red homologous recombination system into the genome, has significantly enhanced in vivo DNA homologous recombination ability, and is particularly suitable for the rapid assembly of small and medium fragments without in vitro enzymatic recombination.

[0076] Example 4: A Highly Efficient DNA Preparation Method Based on Rolling Circle Amplification and Cutter Endonuclease

[0077] Although the engineered strain DH5α ΔattB:: lambda-Red in Example 3 improved the positive rate of DNA assembly, the total number of transformants (clones) obtained on the plate was still limited when transforming long fragments, which limited its application in scenarios requiring large-scale clone screening. To improve transformation efficiency and simplify the DNA preparation process, this example developed a high-efficiency DNA preparation technique combining rolling circle amplification and nicking endonuclease treatment.

[0078] 4.1 Overview of Technical Principles and Processes

[0079] The core process of this method includes three main steps: 1) performing RCA on the initial circular DNA assembly to achieve exponential amplification of the DNA template in order to prepare high-yield DNA products in the microgram range; 2) treating the RCA product with a specific nicking endonuclease to cut it into linear DNA monomers with single-stranded homologous ends; 3) transforming the prepared linear DNA monomers into an engineered strain with enhanced homologous recombination ability, and using its in vivo recombination system to complete the precise assembly of linear DNA into circular plasmids.

[0080] 4.2 Preparation of RCA template

[0081] Using the vector plasmid pSynbio-YAC0 (SEQ ID NO. 7) as a template, a linear vector backbone was obtained by PCR amplification using primer pairs YAC0-F (SEQ ID NO. 8) and YAC0-R (SEQ ID NO. 9). Simultaneously, using the Plasmid-10 plasmid (SEQ ID NO. 10) constructed in Example 3 as a template, a 10 kb target insert fragment was obtained by PCR amplification using primer pairs Frag10-F (SEQ ID NO. 11) and Frag10-R (SEQ ID NO. 12). The PCR products were recovered and purified using a commercially available DNA purification kit (e.g., nucleic acid column method).

[0082] 4.3 Rolling ring amplification reaction

[0083] The purified linear vector backbone was ligated to the target insert using a standard in vitro assembly method (such as Gibson Assembly) to construct a circular RCA initial template. Less than 1 ng of this circular template DNA was mixed with 1×rCutsmart buffer, 100 µM of random hexameric primers modified with phosphothioester bonds, and an appropriate amount of ddH2O. The mixture was heated at 95°C for 3 minutes to denature it, and then immediately placed on ice for 5 minutes to cool.

[0084] To the denatured and cooled mixture described above, add dNTPs to a final concentration of 1 mM, supplement with 1× rCutsmart buffer, ddH2O, and 10 U of phi29 DNA polymerase to bring the final reaction volume to 20 µL. After gentle mixing, incubate the reaction system at 30°C for 16 hours. After the reaction is complete, heat at 65°C for 10 minutes to inactivate the phi29 polymerase, thus terminating the RCA reaction.

[0085] 4.4 Linearization treatment with nicking endonucleases

[0086] To obtain a linear DNA form with higher transformation efficiency, the RCA product was specifically cleaved using the nicking endonuclease Nb.BbvCI. The reaction mixture (50 µL) contained approximately 1 µg of RCA product, 10 U of Nb.BbvCI endonuclease, and 1×rCutsmart buffer, with the volume made up to ddH2O. The reaction mixture was incubated at 37°C for 1 hour to ensure complete digestion. Subsequently, it was heated at 80°C for 20 minutes to completely inactivate the Nb.BbvCI endonuclease. The digested product was then recovered and purified using a DNA purification kit to obtain a linear DNA product with single-stranded homologous ends.

[0087] 4.5 Conversion and Effect Verification

[0088] The purified linear DNA product was transformed into pre-prepared competent cells of the DH5α ΔattB:: lambda-Red engineered strain using the chemical transformation method described in Example 3. The transformed cells were plated on LB selection plates containing the appropriate antibiotics and incubated overnight at 37°C. The number of single colonies growing on the plates was then counted (transformant number / µg DNA, i.e., transformation titer).

[0089] 4.6 Results

[0090] Experimental results are as follows Figure 3 As shown, compared with the untreated conventional in vitro Gibson Assembly reaction products, the DNA prepared by RCA amplification exhibited a significantly improved transformation titer, mainly attributed to the exponential increase in DNA template quantity and quality optimization. More importantly, the linear DNA with single-stranded homologous ends generated after linearization treatment with the nicking endonuclease Nb.BbvCI showed a transformation titer nearly an order of magnitude higher than that of the conventional Gibson Assembly reaction products. These results demonstrate that the "RCA-nicking endonuclease" DNA preparation method provided by this invention, combined with highly recombinant engineered strains, can effectively overcome the technical bottleneck of low transformation efficiency for long DNA fragments, providing a complete solution for high-throughput, high-success-rate DNA assembly and transformation.

[0091] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A DNA assembly method, characterized in that, Includes the following steps: S1. A linear DNA monomer with single-stranded homologous ends is provided, wherein the linear DNA monomer is obtained by sequentially performing rolling circle amplification and nicking endonuclease treatment on a circular DNA template containing the target sequence; S2. The linear DNA monomers obtained in step S1 are transformed into competent cells of an engineered strain of Escherichia coli, wherein the genome of the engineered strain of Escherichia coli contains expression units of the exogenous Lambda Red homologous recombination system. S3. Culture the transformed cells and utilize the homologous recombination ability of the strain to complete DNA assembly in vivo.

2. The DNA assembly method according to claim 1, characterized in that, The expression unit of the Lambda Red homologous recombination system contains the nucleotide sequence shown in SEQ ID NO.

1.

3. The DNA assembly method according to claim 1, characterized in that, The expression unit is integrated into the attB, araA, or galK site of the strain genome.

4. The DNA assembly method according to claim 1, characterized in that, The host of the engineered Escherichia coli strain is Escherichia coli DH5α, T1, or TOP10.

5. The DNA assembly method according to claim 1, characterized in that, In step S1, the rolling circle amplification reaction uses phi29 DNA polymerase.

6. The DNA assembly method according to claim 1, characterized in that, In step S1, the nicking endonuclease is Nb.BbvCI or Nb.BtsI.

7. An engineered strain of *Escherichia coli* prepared by the method according to any one of claims 1 to 6, characterized in that, The genome of the engineered Escherichia coli strain contains expression units of an exogenous homologous recombination system that are stably integrated.

8. The engineered Escherichia coli strain according to claim 7, characterized in that, The homologous recombination system is either the LambdaRed system or the RecET system.

9. The assembly method according to any one of claims 1-6, or the application of the engineered Escherichia coli strain according to claim 7 or 8 in the assembly of long-fragment or multi-fragment DNA.

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