System for preparing target DNA, expression vector composition and application thereof

Abstract

The invention discloses a system for preparing target DNA, an expression vector composition and application thereof, and relates to the technical field of biology. The system for gene editing comprises: a bacteriophage activating protein; and a DRT2 system or a reorganized DRT2 system; the bacteriophage activating protein comprises at least one of single-stranded DNA annealing protein, single-stranded DNA binding protein and ribonucleotide reductase; the DRT2 system comprises a gene for coding reverse transcriptase and ncRNA; the recombinant DRT2 system is obtained by the following method: replacing the 45th to 104th basic groups of ncRNA in the DRT2 system with a target gene. According to the invention, the DRT2 system or the recombinant DRT2 system is activated by utilizing the phage activating protein, and then the target double-stranded DNA is generated through reverse transcription without depending on an exogenous infection signal, so that the system has relatively strong applicability in gene editing and genome engineering.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a system for preparing target DNA, an expression vector composition, and its applications. Background Technology

[0002] With the rapid development of bioinformatics and structural biology research technologies, the discovery and mechanistic study of anti-phage defense systems in archaea and bacteria have become increasingly comprehensive. The discovery of anti-phage defense systems in bacteria and archaea provides data for a comprehensive understanding of the interaction mechanisms between prokaryotes and bacteriophages. Furthermore, these studies have significant application value in the development of modern molecular biology tools, the development of clinical phage therapy, and understanding the similarities and differences between prokaryotic and eukaryotic immune defense systems.

[0003] The use of small-molecule antibiotics is an important means of clinical treatment for bacterial infections. However, the current heavy reliance on small-molecule antibiotics has led to increasingly serious drug resistance problems. Bacteriophages, as obligate bacterial parasitic viruses, are an important tool in combating drug-resistant bacteria. By genetically engineering bacteriophages, their lysis efficiency can be enhanced, their host range expanded, or their host specificity improved, thereby improving their therapeutic efficacy and safety.

[0004] Current gene editing technologies (such as the CRISPR-Cas system) typically require complex anti-selection steps when applied to phage genome editing, resulting in low efficiency and difficulty in achieving precise modifications at multiple sites.

[0005] Retrotrons are a class of reverse transcription systems found in bacteria, capable of synthesizing reverse-transcribed DNA (RT-DNA) to participate in bacterial immune defense, primarily used to recognize and respond to bacteriophage infections. In recent years, retrotrons have been introduced into the field of genome editing due to their unique functions, playing a crucial role as a potential tool. The RT-DNA generated by retrotrons can be used as an editing donor, guiding modifications to target sites in the genome through precise sequence matching. Compared to traditional gene editing methods, retrotrons offer a modular, efficient, and flexible solution, particularly suitable for genome engineering in bacteria and eukaryotes. However, the yield and editing efficiency of different retrotrons vary significantly, providing a research basis for optimizing editing tools. As more and more retrotron systems are validated, their application potential in synthetic biology, gene therapy, and biotechnology is becoming increasingly apparent. Currently, although several retrotron systems with anti-phage activity have been identified, only Retrotron-Eco1 has been extensively studied and optimized for prokaryotic genome editing; other retrotron systems have not yet been widely characterized, and their applications are mainly limited to prokaryotes. Furthermore, the editing efficiency of the Retron system depends on the function of endogenous proteins in the host cell (such as single-strand binding proteins and single-strand annealing proteins), and differences between different hosts may limit its universality. Retron systems from different sources also exhibit significant differences in the efficiency and quality of RT-DNA generation, further affecting their editing capabilities and stability.

[0006] The DRT (Defense-associated reverse transcriptase) system generates single-stranded RT-DNA (as an editing donor) through reverse transcription. Combined with the host cell's homologous recombination mechanism, it enables insertion, deletion, and mutation operations in the genome. The DRT system supports multiplexing and sequential editing without requiring an anti-selection step, and can be further combined with high-throughput screening or other editing tools, significantly expanding its application potential in genome engineering, functional gene research, and phage genome editing.

[0007] However, existing technologies mainly rely on phage infection to trigger the activation of the DRT system, which limits its application scope. Furthermore, traditional DRT systems typically produce single-stranded DNA, affecting their stability and efficiency in gene editing.

[0008] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the purpose of this invention is to provide a system for preparing target DNA, an expression vector composition and its application, in order to solve the problem that the existing DRT system mainly relies on phage infection to trigger activation, which limits its application scope.

[0010] The technical solution of the present invention is as follows: A first aspect of the present invention provides a system for preparing target DNA, wherein the system for preparing target DNA comprises: Phage activator protein; And, the DRT2 system or a reconfigured DRT2 system; The phage activation protein includes at least one of single-stranded DNA annealing protein (SSAP protein), single-stranded DNA binding protein (SSB protein), and ribonucleotide reductase (NrdA protein (ribonucleotide reductase α subunit) and / or NrdB protein (ribonucleotide reductase β subunit)). The DRT2 system is the defense-related reverse transcriptase 2 system, which contains a gene encoding reverse transcriptase and ncRNA (i.e., non-coding RNA). The recombinant DRT2 system was obtained through the following method: The 45th to 104th bases of the ncRNA in the DRT2 system were replaced with the target gene to obtain the recombinant DRT2 system.

[0011] In this invention, the DRT2 system or the recombinant DRT2 system can be activated using only phage activating proteins such as SSAP protein, SSB protein and NrdA / NrdB protein without relying on phage infection, thus having a wide range of applications.

[0012] This invention utilizes phage activating proteins to activate the DRT2 system or the recombinant DRT2 system, thereby reverse transcribing to generate a large amount of target double-stranded DNA (the length and sequence of which can be designed on demand and can be controllably replaced). The generation of target double-stranded DNA within the host cell helps improve its stability and enhances its applicability in gene editing and genome engineering. Furthermore, the target double-stranded DNA more readily binds to the host's homologous recombination mechanism, improving the efficiency and precision of gene editing. The large amount of target double-stranded DNA generated by the recombinant DRT2 system under the action of phage activating proteins can not only serve as a gene editing template but also be used for protein expression; the target double-stranded DNA can be transcribed and translated to generate the target protein.

[0013] Therefore, this invention effectively solves the problem that existing DRT systems, which mainly rely on phage infection to trigger activation, have limited application scope. Furthermore, this invention also addresses the issue that traditional DRT systems typically generate single-stranded DNA, affecting their stability and efficiency in gene editing.

[0014] In addition, the inventors discovered that the 45th to 104th bases of the ncRNA template region are editable regions. By replacing the target gene (whose length and sequence can be designed as needed) in this region, the target DNA or target protein can be generated.

[0015] Optionally, the DRT2 system is derived from Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae ).

[0016] Optionally, the SSAP protein is derived from T1 phage, the SSB protein is derived from T5 phage, and the NrdA protein and / or NrdB protein is derived from T4 phage.

[0017] A second aspect of the present invention provides an expression vector composition, wherein the expression vector composition comprises an expression vector carrying a DRT2 system gene and an expression vector carrying a phage activation protein gene; or, the expression vector composition comprises an expression vector carrying a recombinant DRT2 system gene and an expression vector carrying a phage activation protein gene. The phage activation protein gene includes at least one of the following: single-stranded DNA annealing protein gene (i.e., SSAP gene), single-stranded DNA binding protein gene (i.e., SSB gene), and ribonucleotide reductase gene (i.e., NrdA and / or NrdB genes; the NrdA and NrdB genes form a stable complex NrdAB, which can convert NTPs into dNTPs). The DRT2 system is the defense-related reverse transcriptase 2 system, which contains a gene encoding reverse transcriptase and ncRNA. The recombinant DRT2 system was obtained through the following method: The 45th to 104th bases of the ncRNA in the DRT2 system were replaced with the target gene to obtain the recombinant DRT2 system.

[0018] A third aspect of the present invention provides a cell, wherein the cell comprises the expression vector composition of the present invention as described above.

[0019] A fourth aspect of the present invention provides a system for preparing target DNA as described above, or the use of an expression vector composition as described above in the preparation of target DNA.

[0020] In some embodiments, the method of application is as follows: After replacing bases 45 to 104 of the ncRNA in the DRT2 system with the target gene, phage proteins are used for activation, and reverse transcription is used to generate the target DNA (specifically, the target double-stranded DNA) corresponding to the target gene; or, The recombinant DRT2 system is activated using a phage activator protein, and the target DNA (specifically, the target double-stranded DNA) corresponding to the target gene is generated by reverse transcription.

[0021] The system or expression vector composition provided by the present invention can generate target double-stranded DNA, which can be used as a gene editing template for gene editing (such as insertion, deletion, substitution or multi-site editing).

[0022] A fifth aspect of the present invention provides a system for preparing target DNA as described above, or the use of an expression vector composition as described above in the preparation of a target protein.

[0023] A sixth aspect of the present invention provides a method for preparing target DNA, wherein the method for preparing target DNA employs the system for preparing target DNA described above, and the method for preparing target DNA includes the following steps: The recombinant DRT2 system was constructed in a first plasmid to obtain a first expression vector; the target gene in the recombinant DRT2 system was designed based on the target DNA; The phage activation protein gene was constructed into a second plasmid to obtain a second expression vector; The first expression vector and the second expression vector are transferred into host cells or host strains, and after culturing, the target DNA (i.e., target double-stranded DNA) is obtained.

[0024] This invention obtains a recombinant DRT2 system by replacing the target gene (designed based on the target DNA) at positions 45 to 104 of the ncRNA in the DRT2 system. The recombinant DRT2 system and the phage activating protein gene are then constructed into plasmids and co-transformed into host cells or host strains for culture. The phage activating protein activates the recombinant DRT2 system to obtain the target double-stranded DNA.

[0025] A seventh aspect of the present invention provides a method for preparing a target protein, wherein the method for preparing the target protein employs the system for preparing target DNA described above, and the method for preparing the target protein includes the following steps: The recombinant DRT2 system was constructed in a third plasmid to obtain a third expression vector; the target gene in the recombinant DRT2 system was designed according to the target protein. The phage activation protein gene was constructed into a second plasmid to obtain a second expression vector; The third expression vector and the second expression vector are transferred into host cells or host strains, and after culturing, the target protein is obtained.

[0026] This invention obtains a recombinant DRT2 system by replacing the 45th to 104th bases of the ncRNA in the DRT2 system with the target gene (designed according to the target protein). The recombinant DRT2 system and the phage activating protein gene are constructed into plasmids respectively, and then co-transformed into host cells or host strains for culture. The phage activating protein activates the recombinant DRT2 system to obtain the target protein.

[0027] Optionally, the host cell is an Escherichia coli cell, and the host strain is an Escherichia coli strain. Beneficial Effects: In existing technologies, the DRT system typically generates only a small amount of DNA under physiological conditions when no phage activating protein is detected, and the DRT system is mainly activated by phage infection. However, this invention can activate the DRT2 system or recombinant DRT2 system to generate a large amount of double-stranded DNA without phage infection, without relying on exogenous infection signals. Therefore, it improves the stability and applicability of the system. Specifically, this invention utilizes the phage activating protein to activate the DRT2 system or recombinant DRT2 system. Under the activation of the phage activating protein, the DRT2 system or recombinant DRT2 system can reverse transcribe to produce a large amount of target DNA (double-stranded, the length and sequence of which can be designed as needed). The target DNA can be further transcribed and translated to generate target proteins, providing a novel platform and widely applicable technical means for synthetic biology, anti-phage research, and gene editing.

[0028] Specifically, the DRT2 system, or recombinant DRT2 system, relies on the regulation of phage activating proteins to make the generation of target DNA more efficient and controllable. The production of the target double-stranded DNA within the host cell helps improve its stability and enhances its applicability in gene editing and genome engineering. Furthermore, double-stranded DNA more readily binds to the host's homologous recombination mechanism, improving the efficiency and precision of gene editing. In other words, the obtained target double-stranded DNA can be used for homologous recombination, gene repair, and gene modification during gene editing, achieving precise genetic manipulation. In addition, the DRT2 system can be combined with existing gene editing technologies (such as CRISPR and TALEN) to expand its application potential in gene therapy, synthetic biology, and high-throughput screening.

[0029] This invention utilizes a specific design of the ncRNA template sequence of the DRT2 system. For example, gene substitutions of different lengths at bases 45 to 104 yield a recombinant DRT2 system. The recombinant DRT2 system then reverse-transcribes DNA of a specific length and sequence. Furthermore, gene substitutions of different lengths within the ncRNA template region of the DRT2 system do not affect its overall function. In other words, under the regulation of phage activating proteins, the DRT2 system can achieve controllable modification of the DNA sequence by altering the ncRNA template sequence, enabling autonomous and controllable DNA generation. Therefore, it allows for flexible design of target DNA fragments suitable for insertion, deletion, substitution, and multi-site editing, as well as desired target proteins, greatly expanding its application potential in complex genome manipulation. It demonstrates greater flexibility and applicability in gene editing, synthetic biology, and functional gene research. In addition, the large amount of target double-stranded DNA generated by this system under the action of phage activating proteins can not only serve as gene editing templates but also be used for protein expression. Through rational design of the ncRNA template sequence, DNA can be transcribed and translated into target proteins without the need for an additional vector system. This characteristic enables it to dynamically regulate protein expression under specific conditions, is applicable to a variety of hosts, and can be used for protein function screening, further expanding its application prospects in synthetic biology and biotechnology. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the experimental method flow in an embodiment of the present invention.

[0031] Figure 2 The diagram shows the resolved structures of the DRT2 system reverse transcriptase and ncRNA, as well as the DNA produced by reverse transcription. In the diagram, A is a schematic diagram of the DRT2 system domains, B is a schematic diagram of the DRT2 system cryo-electron microscopy structure, C is a schematic diagram of the secondary structure of the DRT2 system ncRNA, D is a diagram showing the detection results of single and double stranded cDNA synthesized by reverse transcription of the DRT2 system at different dNTP concentrations in vitro, and E is a schematic diagram of the sequencing of cDNA obtained by reverse transcription of the DRT2 system in vitro.

[0032] Figure 3 The image shows the screening results of phage mutants that escape the immune defense of the DRT2 system. In the image, A is a schematic diagram of phage evasion selected by the DRT2 system, and B is a diagram showing the key gene mutation sites verified by sequencing of the phage evasion strains obtained through screening.

[0033] Figure 4The diagram shows the activation verification results of phage activator protein. A represents the activation verification results of the DRT2 system and related genes co-expressed in vivo. B is a schematic diagram showing the activation of double-stranded DNA by reverse transcription of the DRT2 system after co-transformation with phage activator protein and the DRT2 system, and the transcription of mRNA from the double-stranded DNA. C shows the content of double-stranded DNA produced by reverse transcription of the DRT2 system after co-transformation with phage activator protein and the DRT2 system. D shows the content of mRNA transcribed from the double-stranded DNA after reverse transcription of the DRT2 system following co-transformation with phage activator protein and the DRT2 system.

[0034] Figure 5 The diagrams show the results of the analysis of the interaction mechanism between the DRT2 system and phage activating protein. A shows the results of in vivo co-expression of genes related to the DRT2 system and phage activating protein, validating their binding. B shows the results of an in vitro pull-down experiment of the DRT2 system and phage activating protein. C shows the dATP content in strains containing the DRT2 system after infection by wild-type T5 phage and NrdA mutant. D shows the dTTP content in strains containing the DRT2 system after infection by wild-type T5 phage and NrdA mutant. E shows the dCTP content in strains containing the DRT2 system after infection by wild-type T5 phage and NrdA mutant. F shows the dGTP content in strains containing the DRT2 system after infection by wild-type T5 phage and NrdA mutant.

[0035] Figure 6 The diagram shows the ncRNA region of the DRT2 system and the transcription and translation of double-stranded DNA generated after activation by phage activating proteins. A shows the ncRNA template sequence of the DRT2 system after bases 45-104 are replaced with 3xFlag (66 bp); B shows the double-stranded DNA obtained by reverse transcription after bases 45-104 of the DRT2 ncRNA template sequence are replaced with 3xFlag (66 bp), and the process of transcribing the double-stranded DNA into mRNA and translating it into protein; C shows the DRT2 system co-transformed with different phage activating proteins and the ncRNA template sequence replaced with the 66 bp 3xFlag sequence, and the resulting reverse transcription of the DRT2 system to produce double-stranded DNA, with the mRNA content obtained from the reverse transcription; D shows the DRT2 system after the ncRNA template sequence is replaced with the 66 bp 3xFlag sequence, activated by phage activating proteins, can produce double-stranded DNA through reverse transcription, which is then further transcribed and translated into proteins with the Flag tag. Detailed Implementation

[0036] This invention provides a system for preparing target DNA, an expression vector composition, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0038] The present invention will be further described below through specific embodiments.

[0039] In the following embodiments, unless otherwise specified, all materials and equipment used are commercially available products.

[0040] In the following embodiments, firstly, for the DRT2 system with phage defense capabilities, its reverse transcriptase and ncRNA complex were expressed and purified to resolve its structure. In vitro reaction conditions were optimized, and dNTPs (deoxynucleoside triphosphates) were added to obtain the reverse-transcribed DNA sequence, and its sequence characteristics were identified and analyzed. Then, by screening phage strains capable of escaping the DRT2 defense mechanism, related phage activation proteins were identified, and the mechanism by which phage activation proteins activate the DRT2 system was verified. Furthermore, based on the resolved DRT2 reverse transcriptase and ncRNA template structure and ncRNA sequence, editable ncRNA template regions were identified, and target sequence substitutions were performed to construct an engineered vector. Finally, the identified phage activation protein and the engineered vector were co-transformed into *E. coli* strain MG1655, and the expression of the phage activation protein was induced. The target double-stranded DNA, transcribed mRNA, and translated proteins generated intracellularly were identified and analyzed to evaluate the effectiveness of the modified DRT2 system. In short, as... Figure 1 As shown, the following steps are performed sequentially in the following embodiments: Structural analysis of S1 and DRT2 systems and determination of in vitro reverse-transcribed DNA double strands; S2. Screening and identification of phage activation proteins that can activate the DRT2 system; S3, verification of the mechanism by which phage activator protein activates DRT2 system; Modification and activation expression of S4 and DRT2 system ncRNAs; S5. Validation of the target mRNA and protein generated by the modified DRT2 system.

[0041] Example 1: DRT2 System Structure Analysis and In Vitro Reverse Transcription DNA Double-Strand Determination This embodiment focuses on the DRT2 system, which possesses phage defense capabilities. It expresses and purifies its reverse transcriptase and ncRNA complex to elucidate its structure, specifically to determine the key structural features of the DRT2 system. In vitro reaction conditions were optimized, and dNTPs were added to obtain the DNA sequence generated by reverse transcription. The sequence characteristics were then identified and analyzed (i.e., the purified protein was subjected to in vitro reverse transcription, and the resulting DNA was extracted for single / double strand and sequence feature analysis). The specific steps include the following: Based on Klebsiella pneumoniae ( Klebsiella pneumoniae The gene of the DRT2 system (its nucleotide sequence is shown in SEQ ID NO: 1, and its domain diagram is shown in...) Figure 2 The expression vector pRSFDuet-1 (a commercial plasmid) was constructed using isopropyl-β-D-1-thiogalactopyranoside (IPTG) to induce expression, carrying a Strep affinity chromatography tag, and transformed into the *E. coli* expression strain BL21 Star (DE3) for protein expression induction. Protein samples obtained through affinity chromatography were subjected to ion exchange chromatography and Superdex 200 gel chromatography to obtain proteins of suitable concentration, high purity, and uniform state. These were then prepared as cryo-electron microscopy samples for testing. Data were collected and structural analysis was performed to determine the ncRNA sequence (its nucleotide sequence is shown in SEQ ID NO: 2) and its binding mode with reverse transcriptase. The schematic diagrams of the cryo-electron microscopy structure analysis of the DRT2 system and the schematic diagrams of the secondary structure of the ncRNA in the DRT2 system are shown below. Figure 2 As shown in B and C.

[0042] Using purified protein, metal ions (Mg) were added in vitro. 2+ The DNA was reacted with sodium chloride (NaCl) and different concentrations of dNTPs (50 μM, 250 μM, 500 μM, and 1000 μM) at pH 8.0 at 37°C for 3 h. After treatment with RNase and protease, an equal volume of isopropanol was added to precipitate the DNA. The DNA precipitate was collected by centrifugation at 4°C, washed once with 75% ethanol, air-dried, and dissolved in ddH2O. Two equal volumes of the purified DNA were taken and digested with single-stranded and double-stranded enzymes, respectively, and then analyzed by agarose gel electrophoresis. The results are shown below. Figure 2 As shown in D, it can be seen that the reverse transcription of DRT2 in vitro produces double-stranded DNA.

[0043] The DNA obtained from the reaction with added dNTPs was sequenced, and the results are as follows: Figure 2 As shown in E, it was found to consist of 120bp repeat units, which originated from the rolling circle reverse transcription process of the ncRNA template region (positions 29 to 148).

[0044] Example 2: Screening and identification of phage activation proteins capable of activating the DRT2 system. This embodiment involves screening mutant strains of bacteriophages that the DRT2 system can defend against, identifying key bacteriophage proteins that may interact with the DRT2 system, and verifying their activation capabilities. Specifically, the steps include: The gene sequence of the DRT2 system (as shown in SEQ ID NO: 1) was cloned into the pACYC184 vector (commercial plasmid) to obtain the DRT2-pACYC184 expression vector. The DRT2-pACYC184 expression vector was transformed into Escherichia coli strain Mg1655. For example... Figure 3 As shown in Figure A, the resistance to different phages was verified using a serial dilution plate method. Phage plaques capable of escaping DRT2 defense were selected for culture to obtain escaped phages. The total genome of the escaped phage strains was extracted and sequenced, and the results are shown in Figure A. Figure 3 As shown in B, by comparing with the reference phage genome (wild-type phage that did not escape), it was found that the genes that mutated frequently only in escaped phage strains were the SSAP gene of T1 phage, the SSB gene of T5 phage, and the NrdA gene of T5 phage. That is, mutations in the SSAP gene of T1 phage, the SSB gene of T5 phage, and the NrdA gene of T5 phage were identified in multiple escaped strains, indicating that the proteins encoded by these genes may be related to the activation of the DRT2 phage defense system and are phage-associated proteins that can activate DRT2 defense capabilities.

[0045] Phage-related protein sequences were cloned and constructed into expression vectors induced by L-arabinose. Further verification was conducted to determine whether the screened phage proteins could activate the DRT2 system. Specifically, the three genes were cloned into pBAD vectors (commercial plasmids) to obtain vectors T1-SSAP-pBAD, T5-SSB-pBAD, and T4-NrdAB-pBAD. (Since NrdA and NrdB genes form a stable complex NrdAB, which can convert NTPs to dNTPs, NrdA and NrdB genes were cloned into the same multiple cloning site with a ribosome binding site inserted in between. Subsequent activation experiments used NrdAB for verification.) These three vectors were then co-transformed with the DRT2-pACYC184 expression vector into *E. coli* strain Mg1655. The bacterial culture was incubated overnight at 37°C in LB medium containing antibiotics (composed of carbenicillin and tetracycline; the concentration of tetracycline in LB medium was 10 μg / mL and the concentration of carbenicillin was 50 μg / mL) and 2 wt% glucose (glucose accounted for 2% of the total mass of antibiotics, glucose and LB medium, the same below). The next day, the culture was transferred to LB medium containing the same antibiotics and glucose components at a volume ratio of 1:100. When the OD600 reached 0.6, 1.5 mL of the bacterial culture was washed with antibiotic-free LB medium to remove glucose. Then, the culture was induced at 37°C for 4 h in LB medium containing the same antibiotics and 0.2 wt% L-arabinose (L-arabinose accounted for 2% of the total mass of antibiotics, L-arabinose and LB medium, the same below) to obtain the induced bacterial culture.

[0046] Take another 1.5 mL of bacterial culture and incubate it in LB medium containing 2 wt% glucose at 37°C for 4 h to obtain a non-induced bacterial culture.

[0047] The induced bacterial suspension and the non-induced bacterial suspension were serially diluted and dropped onto plates. The results are as follows: Figure 4 As shown in A, T1-SSAP-pBAD, T5-SSB-pBAD, T4-NrdAB-pBAD, and DRT2-pACYC184 co-transformation all induced cell death (i.e., the corresponding SSB at L-arabinose). T5 +DRT2、SSAP T1 +DRT2、NrdAB T4 The +DRT2 group, and the non-induction group, still survived (i.e., the corresponding SSB at glucose levels). T5 +DRT2、SSAP T1 +DRT2、NrdAB T4 (+DRT2 group) demonstrates that the SSAP protein of T1 phage, the SSB protein of T5 phage, and the NrdAB protein of T4 phage can activate the antiphage activity of the DRT2 system.

[0048] Example 3 verifies that the identified phage activator protein can activate the DRT2 system in vivo and produce a large amount of double-stranded DNA and post-transcriptional mRNA of double-stranded DNA. A schematic diagram of the process of co-transformation with phage activator protein and the DRT2 system to activate DRT2 for reverse transcription to produce double-stranded DNA and the mRNA transcribed from the double-stranded DNA is shown below. Figure 4 As shown in B. In this embodiment, the DRT2 system and the identified phage activation protein were co-transformed into *E. coli* strain MG1655 to induce the expression of the phage activation protein. Total DNA and total RNA were extracted. Total DNA was used for qPCR (quantitative real-time polymerase chain reaction) to detect the production of double-stranded DNA in vivo after induction, and total RNA was used for RT-qPCR (reverse transcription quantitative real-time polymerase chain reaction) to identify the mRNA level transcribed from the double-stranded DNA produced in vivo. The specific steps include the following: Following the method described in Example 2, induced and non-induced bacterial cultures were obtained. Cells were then collected and resuspended in TRIzol, followed by an equal volume of anhydrous ethanol. Total RNA was extracted using the Direct-zol™ RNA MiniPrep kit (ZYMO RESEARCH). The kit instructions were followed strictly to ensure the integrity and purity of the RNA extraction, meeting the requirements for subsequent cDNA synthesis and RT-qPCR analysis. Using 1 μg of total RNA as a starting template, cDNA was synthesized by reverse transcription using the HiFiScript All-in-one RTMaster Mix (CWBIO, CW3371M). The reverse transcription reaction was performed strictly according to the kit instructions to ensure high-fidelity cDNA synthesis and minimize potential degradation and secondary structure interference.

[0049] Bacterial cells were collected using the same method as for total RNA extraction for total DNA extraction. The TIANamp BacteriaDNA Kit (TIANGEN DP210831) was used, and the extraction procedure suitable for Gram-negative bacteria was followed. qPCR was performed using 20 ng of DNA as a template with a Superstar Universal SYBR Master Mix (CWBIO, CW3360M).

[0050] Amplification was performed on a QuantStudio3 real-time quantitative PCR system (Applied Biosystems). qPCR data were analyzed for relative quantification using the ΔΔCt method to standardize the double-stranded DNA content between different experimental groups. The *E. coli* Mg1655 reference gene *dxs* was used as an internal control gene to correct for genomic DNA differences between samples. Results are as follows: Figure 4As shown in C and D, after induction, the SSAP protein of T1 phage, the SSB protein of T5 phage, and the NrdAB protein of T4 phage activate the DRT2 system to produce a large amount of double-stranded DNA. The amount of mRNA transcribed from the double-stranded DNA proves that in the absence of phages, the SSAP protein of T1 phage, the SSB protein of T5 phage, and the NrdAB protein of T4 phage can activate the DRT2 system to produce a large amount of double-stranded DNA and transcribe it into mRNA.

[0051] Example 4: Investigation into the specific mechanism by which phage activator proteins activate the DRT2 system and generate large amounts of double-stranded DNA The binding of SSB protein from T5 phage and SSAP protein from T1 phage to single-stranded DNA synthesized by reverse transcription in the DRT2 system was verified using in vitro pull-down and in vivo Co-IP (co-immunoprecipitation) experiments. For NrdA protein from T4 phage, the increase in intracellular dNTP levels in cells containing the DRT2 system was detected after infection with wild-type and NrdA mutant phages. The specific steps included: The SSAP gene of T1 phage and the SSB gene of T5 phage were co-transformed into *E. coli* STAR strain with wild-type DRT2 (or the active mutant YCAA, i.e., the active mutant YCAA) for Co-IP experiments. Strains containing phage activating proteins and the DRT2 defense system (wild-type / mutant) were cultured in LB medium at 37°C until an OD600 of 0.4-0.6 was reached. After induction with an inducer, the cells were collected, sonicated, and then subjected to complex capture. The lysed samples were centrifuged at 20,000 g at 4°C, and the supernatant was collected and incubated overnight at 4°C with pre-washed Flag globulin (Smart Lifesciences) for immunoprecipitation. Subsequently, the magnetic beads were washed three times with lysis buffer, and the proteins were eluted with elution buffer A (0.3 M glycine, pH 3.5). Samples were heated to 95°C, electrophoresed by 4-20% SDS-PAGE, and then transferred to a 0.2 μm polyvinylidene fluoride membrane. Western blot analysis was performed using anti-Flag antibody, anti-HiS antibody, and HRP (horseradish peroxidase)-labeled goat anti-mouse IgG antibody (Sangon Biotech, 1:2000 / 3000 dilution), and development was performed using Immobilon Western chemiluminescent HRP substrate (Merck Millipore). Finally, the Western blot bands were imaged and analyzed using a ChempGel 7000 molecular imaging system (Sage Creation).

[0052] The results are as follows Figure 5 As shown in Figure A, it was found that the SSAP protein of T1 phage and the SSB protein of T5 phage can both form a complex with DRT2, but cannot form a complex with the YCAA active mutant. This verifies that the SSAP protein of T1 phage and the SSB protein of T5 phage bind to the DNA produced by reverse transcription of the DRT2 system.

[0053] The SSAP gene of T1 phage and the SSB gene of T5 phage were constructed into the pETDuet-1 vector with affinity chromatography tags (inconsistent with the affinity chromatography tags of the DRT2 system), followed by induced expression and protein purification. In vitro, the mixture was combined with purified DRT2 protein, and metal ions and a low concentration of dNTPs (less than 200 μm) were added to induce a small amount of double-stranded DNA in the DRT2 system. The mixture was then reacted at 37°C for 3 h at pH 8.0 and 150 mM NaCl. The complex was pulled out using the DRT2 affinity chromatography tag and detected by 4-20% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results are shown below. Figure 5 As shown in Figure B, it was found that the SSAP protein of T1 phage and the SSB protein of T5 phage could both be pulled out together with DRT2, while the DRT2-active mutant YCAA, which cannot reverse transcribe to produce DNA, could not bind to the SSAP protein of T1 and the SSB protein of T5. This indicates that the SSAP protein of T1 phage and the SSB protein of T5 phage bind to the DRT2 system to activate this phage defense system.

[0054] For the NrdA gene, strains containing the DRT2 system were infected with wild-type phages and NrdA mutant phages. The cells were collected, resuspended in 80% pre-cooled methanol, and lysed by sonication. The lysate was vacuum-dried and used for UPLC-MS analysis of dNTPs (dATP, dTTP, dCTP, and dGTP were deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate, respectively). The results are as follows: Figure 5 As shown in C, D, E, and F, wild-type phage infection resulted in a significant increase in intracellular dNTP levels, while NrdA-mutant phages showed no significant changes. The NrdA mutation in T5 phage prevented it from increasing dNTP levels to produce large amounts of double-stranded DNA, thus enabling it to evade DRT2 defense.

[0055] Example 5: Identifying the editable region of ncRNA and engineering a portion of the ncRNA template sequence of the DRT2 system. like Figure 6As shown in A, the ncRNA template region (bases 45 to 104) sequence from the DRT2 system was selected, and molecular cloning technology was used to replace it with a 3X Flag tag (66 bp in length, its nucleotide sequence is shown in SEQ ID NO: 3), and constructed in the pACYC184 vector. Figure 6 As shown in B, during the sequence substitution process, the DNA structure after reverse transcription from the DRT2 protein was ensured to contain repeating double-stranded DNA (dsDNA) with -10 and -35 elements to support transcriptional initiation by RNA polymerase. Simultaneously, the ribosome binding site (RBS) was retained to promote efficient translation. Furthermore, the stop codon was removed, allowing for continuous translation and generating Flag tag proteins of various lengths. The final nucleotide sequence of the modified pACYC-DRT2 expression vector is shown in SEQ ID NO: 4.

[0056] Using PCR and homologous recombination, phage activation protein genes (SSAP gene of T1 phage, SSB gene of T5 phage, and NrdAB gene of T4 phage) that can activate the DRT2 system to produce a large amount of double-stranded DNA were constructed into pBAD vectors, resulting in T1-SSAP-pBAD, T5-SSB-pBAD, and T4-NrdAB-pBAD expression vectors, respectively.

[0057] The modified pACYC-DRT2 expression vector was co-transformed into strain Mg1655 with T1-SSAP-pBAD, T5SSB-pBAD, and NrdAB-pBAD expression vectors, respectively. The culture was incubated overnight at 37°C in 3 mL of LB medium containing 10 μg / mL tetracycline, 50 μg / mL carbenicillin, and 2 wt% glucose. The next day, the culture was transferred to LB medium containing the same antibiotic and glucose at a volume ratio of 1:100. When the OD600 reached 0.6, 1.5 mL of the bacterial culture was washed with antibiotic-free LB medium to remove the 2 wt% glucose. The culture was then induced at 37°C for 1 h in medium containing the same antibiotic and 0.2 wt% L-arabinose.

[0058] Take another 1.5 mL of bacterial culture and incubate it at 37°C for 1 h in LB medium containing 2 wt% glucose.

[0059] Collected cells were resuspended in TRIzol and then an equal volume of anhydrous ethanol was added. Total RNA was extracted using the Direct-zol™ RNAMiniPrep kit (ZYMO RESEARCH). The kit instructions were followed strictly to ensure the integrity and purity of the extracted RNA, meeting the requirements for subsequent cDNA (complementary DNA) synthesis and RT-qPCR analysis. Using 1 μg of total RNA as a starting template, cDNA was synthesized via reverse transcription using the HiFiScript All-in-one RT Master Mix (CWBIO, CW3371M). The reverse transcription reaction was performed strictly according to the kit instructions to ensure high-fidelity cDNA synthesis and minimize potential degradation and secondary structure interference.

[0060] Amplification was performed on a QuantStudio 3 real-time quantitative PCR instrument (Applied Biosystems).

[0061] qPCR data were analyzed for relative quantification using the ΔΔCt method to standardize the double-stranded DNA content between different experimental groups. The *E. coli* Mg1655 reference gene *dxs* was used as an internal control gene to correct for genomic DNA differences between samples. Results are as follows: Figure 6 As shown in C, after induction, the SSAP protein of T1 phage, the SSB protein of T5 phage, and the NrdAB protein of T4 phage activate the DRT2 system to produce a large amount of target mRNA. This demonstrates that in the absence of phages, the SSAP protein of T1 phage, the SSB protein of T5 phage, and the NrdAB protein of T4 phage can activate the DRT2 system (i.e., the recombinant DRT2 system) after the ncRNA template sequence is replaced with a 66bp 3xFlag sequence, producing a large amount of target double-stranded DNA and transcribing it into mRNA.

[0062] We further used Western blot to verify whether the double-stranded DNA generated by reverse transcription could be further transcribed and expressed in cells to produce specific tag proteins.

[0063] First, the modified pACYC-DRT2 expression vector (nucleotide sequence shown in SEQ ID NO: 4) was co-transformed into the Mg1655 strain with T1-SSAP-pBAD, T5-T5SSB-pBAD, and T4-NrdAB-pBAD expression vectors (the construction of the four expression vectors is described above). The cells were cultured overnight in LB medium containing 10 μg / mL tetracycline, 50 μg / mL carbenicillin, and 2 wt% glucose. The next day, the cells were inoculated at a 1:100 volume ratio into 3 mL of glucose-free LB medium and cultured at 37°C until OD600 = 0.6. Subsequently, the cells were induced with 0.2 wt% L-arabinose for 60 minutes, and then harvested.

[0064] Meanwhile, the modified pACYC-DRT2 (nucleotide sequence as shown in SEQ ID NO: 4) expression vector was separately transformed into the Mg1655 strain. The strain was cultured in 3 mL LB medium containing 10 μg / mL tetracycline until OD600=0.6, then T1 phage and T5 phage were added for infection for 30 min, MOI (Multiplicity of infection)=5, and then the cells were collected.

[0065] Collected cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl). Samples were heated to 95°C and transferred to a 0.2 μm polyvinylidene fluoride membrane after 4–20% SDS-PAGE electrophoresis. Western blot analysis was performed using anti-Flag antibody and HRP (horseradish peroxidase)-labeled goat anti-mouse IgG antibody (Sangon Biotech, 1:2000 / 3000 dilution), and development was performed using Immobilon Western chemiluminescent HRP substrate (Merck Millipore). Finally, the Western blot bands were imaged and analyzed using a ChempGel 7000 molecular imaging system (SageCreation).

[0066] The results are as follows Figure 6 As shown in Figure D, the engineered DRT2 system, in the absence of phage infection, can be activated by SSAP, SSB, and NrdAB proteins, subsequently generating target double-stranded DNA via reverse transcription, and then producing the target protein through transcription and translation. Therefore, this invention, through specific modification of the ncRNA template sequence in DRT2 and the use of phage activating proteins, can generate abundant target double-stranded DNA sequences in vivo, possessing high application value.

[0067] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A system for preparing target DNA, characterized in that, The system for preparing the target DNA includes: Phage activator protein; And, the DRT2 system or a reconfigured DRT2 system; The phage activation protein includes at least one of single-stranded DNA annealing protein, single-stranded DNA binding protein, and ribonucleotide reductase. The DRT2 system is the defense-related reverse transcriptase 2 system, which contains a gene encoding reverse transcriptase and ncRNA. The recombinant DRT2 system was obtained through the following method: The 45th to 104th bases of the ncRNA in the DRT2 system were replaced with the target gene to obtain the recombinant DRT2 system.

2. The system for preparing target DNA according to claim 1, characterized in that, The DRT2 system is derived from Klebsiella pneumoniae.

3. The system for preparing target DNA according to claim 1, characterized in that, The single-stranded DNA annealing protein is derived from T1 phage, the single-stranded DNA binding protein is derived from T5 phage, and the ribonucleotide reductase is derived from T4 phage.

4. An expression vector composition, characterized in that, The expression vector composition comprises an expression vector carrying the DRT2 system gene and an expression vector carrying the phage activation protein gene; or, the expression vector composition comprises an expression vector carrying the recombinant DRT2 system gene and an expression vector carrying the phage activation protein gene. The phage activation protein gene includes at least one of the following: single-stranded DNA annealing protein gene, single-stranded DNA binding protein gene, and ribonucleotide reductase gene. The DRT2 system is the defense-related reverse transcriptase 2 system, which contains a gene encoding reverse transcriptase and ncRNA. The recombinant DRT2 system was obtained through the following method: The 45th to 104th bases of the ncRNA in the DRT2 system were replaced with the target gene to obtain the recombinant DRT2 system.

5. A cell, characterized in that, The cells comprise the expression vector composition of claim 4.

6. The use of a system for preparing target DNA according to any one of claims 1-3 or the use of an expression vector composition according to claim 4 in the preparation of target DNA.

7. The use of a system for preparing target DNA according to any one of claims 1-3 or the expression vector composition according to claim 4 in the preparation of target protein.

8. A method for preparing target DNA, characterized in that, The method for preparing the target DNA uses the system for preparing target DNA as described in any one of claims 1-3, and the method for preparing the target DNA includes the following steps: The recombinant DRT2 system was constructed in a first plasmid to obtain a first expression vector; the target gene in the recombinant DRT2 system was designed based on the target DNA; The phage activation protein gene was constructed into a second plasmid to obtain a second expression vector; The first and second expression vectors are transferred into host cells or host strains, and after culturing, the target DNA is obtained.

9. A method for preparing a target protein, characterized in that, The method for preparing the target protein employs the system for preparing target DNA as described in any one of claims 1-3, and the method for preparing the target protein includes the following steps: The recombinant DRT2 system was constructed in a third plasmid to obtain a third expression vector; the target gene in the recombinant DRT2 system was designed according to the target protein. The phage activation protein gene was constructed into a second plasmid to obtain a second expression vector; The third expression vector and the second expression vector are transferred into host cells or host strains, and after culturing, the target protein is obtained.

10. The preparation method according to claim 8 or 9, characterized in that, The host cell is an Escherichia coli cell, and the host strain is an Escherichia coli strain.

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