M13 bacteriophage preparation and DNA delivery method based on helper plasmid
By constructing helper plasmids and donor plasmids of specific nucleotide sequences and inducible promoters, the problem of helper phage residues in M13 phage preparation is solved, and efficient M13 phage purity and DNA delivery are achieved, simplifying the operation process and reducing costs.
Patent Information
- Application Number
- CN202510553762.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-08-08
AI Technical Summary
The residual problem of commercially-assisted phage M13KO7 during the preparation of M13 phages results in low phage purity and affecting DNA delivery efficiency.
Helper plasmids and donor plasmids are constructed, containing specific nucleotide sequences and inducible promoters, and efficient packaging and delivery of M13 phages are achieved by transforming into host cells and inducing phage-related proteins in liquid culture medium.
It improves the purity and DNA delivery efficiency of M13 phages, reduces the residue of auxiliary plasmids, simplifies the operation process, and reduces costs.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing M13 phage and delivering DNA based on a helper plasmid. Background Art
[0002] There are three pathways for bacterial horizontal gene transfer, including transformation, conjugation, and phage-mediated transduction. Electroporation is currently the most commonly used library construction method, with an efficiency of about 10 9 -10 10 / μg DNA. For example, multiplexed automated genome engineering (MAGE) involves repeatedly introducing modifications into the E. coli genome through multiple rounds of electroporation of single-stranded DNA into E. coli. However, these library construction methods, which rely on electroporation, require automated equipment or continuous manual operation to complete dozens of rounds of electroporation for competent cell preparation and DNA introduction, which is costly, time-consuming, and labor-intensive. Therefore, a method for in situ DNA introduction into cells without requiring additional tedious manipulations during the culture process is urgently needed.
[0003] Unlike commonly used transformation methods (chemical transformation, electroporation transformation and protoplast transformation), phage infection can be carried out spontaneously in situ during the liquid culture of the recipient bacteria. The M13 phage is a filamentous phage that can deliver plasmid DNA by infecting Escherichia coli containing the F' plasmid. The currently commonly used method for preparing artificial M13 phage is to use the M13KO7 helper phage from New England Biolabs in the United States. The replication-related sequences of the helper phage are partially destroyed, resulting in a decrease in the replication frequency, but a certain proportion of helper phage contamination will still be detected in the prepared phage suspension, resulting in the efficiency of continuous library construction or editing using artificial M13 phage being affected.
[0004] Therefore, developing a method for preparing artificial M13 phage that is stable, easy to operate, and has high phage purity is expected to provide a new method for DNA delivery. Summary of the Invention
[0005] The main problem to be solved by the present invention is to overcome the problem of residual commercial helper phage M13KO7 in the existing preparation process of M13 phage.
[0006] In order to solve the above problems, the present invention provides a method for preparing M13 phage based on a helper plasmid. The method for preparing M13 phage based on a helper plasmid of the present invention comprises the following steps:
[0007] 1) constructing a helper plasmid, wherein the helper plasmid contains a partial sequence of the M13 phage genome, a pSC101 replicon, an arabinose-inducible promoter, a rhamnose-inducible promoter, or an anhydrotetracycline-inducible promoter, an araC gene encoding an arabinose-inducible promoter regulatory protein, or a rhamnose-inducible promoter activator protein encoding a rhaRS gene, and an ampicillin resistance gene; the partial sequence of the M13 phage genome does not contain the remaining sequences after the replicon and packaging signal of the M13 phage genome;
[0008] 2) constructing a donor plasmid containing an M13 phage replicon and packaging signal, a p15A replicon or an R6K replicon, a kanamycin resistance gene, and a target donor sequence;
[0009] 3) Transforming the helper plasmid described in step 1) and the donor plasmid described in step 2) into a recipient strain to induce the production of the phage.
[0010] In the above method, in step 1), the partial sequence in the M13 phage genome is a molecule whose nucleotides are from positions 1 to 5945 of SEQ ID No: 1; the pSC101 replicon is a molecule whose nucleotides are from positions 5946 to 7592 of SEQ ID No: 1; the arabinose-inducible promoter nucleotides are from positions 10332 to 10616 of SEQ ID No: 1; the rhamnose-inducible promoter nucleotides are from positions 1927 to 2045 of SEQ ID No: 2; the araC gene is a molecule whose nucleotides are from positions 9427 to 10305 of SEQ ID No: 1; the rhaRS gene is a molecule whose nucleotides are from positions 22 to 1781 of SEQ ID No: 2; and the ampicillin gene is a molecule whose nucleotides are from positions 8422 to 9388 of SEQ ID No: 1.
[0011] Furthermore, the nucleotide sequence of the auxiliary plasmid in step 1) is SEQ ID No: 1 or a DNA molecule obtained by replacing positions 9427 to 10616 in SEQ ID No: 1 with SEQ ID No: 2.
[0012] In a specific embodiment, the auxiliary plasmid may be pSC101-aa or pSC101-rap, the nucleotide sequence of the SC101-aa is SEQ ID No: 1, and the nucleotide sequence of the pSC101-ra plasmid is the sequence obtained by replacing positions 9427 to 10616 in SEQ ID No: 1 with SEQ ID No: 2.
[0013] In the above method, in step 2), the M13 phage replicon and packaging signal are molecules whose nucleotides are from positions 1 to 432 of SEQ ID No: 3; the p15A replicon is a molecule whose nucleotides are from positions 515 to 1060 of SEQ ID No: 3; the chloramphenicol resistance gene is a molecule whose nucleotides are from positions 1289 to 1948 of SEQ ID No: 3; and the kanamycin resistance gene is a molecule whose nucleotides are from positions 1423 to 2217 of SEQ ID No: 4.
[0014] Furthermore, in step 2), the nucleotide sequence of the donor plasmid is a molecule of SEQ ID No: 3 or SEQ ID No: 4.
[0015] In a specific embodiment, the donor plasmid may be pDonor-pc plasmid (sequence shown in SEQ ID No: 3).
[0016] In a specific embodiment, the donor plasmid may be pDonor-pk plasmid (sequence shown in SEQ ID No: 4).
[0017] The aforementioned helper plasmid and / or donor plasmid also fall within the scope of protection claimed by the present invention.
[0018] The present invention also provides a method for improving the delivery efficiency of target DNA, the method comprising:
[0019] 1) transforming the aforementioned helper plasmid into a host cell, and using an inducer to induce the aforementioned helper plasmid to express the phage-related protein;
[0020] 2) Simultaneously, the donor plasmid described above is transformed into a host cell, the donor plasmid described above is packaged into M13 phage to obtain packaged M13 phage, and the packaged M13 phage is used to infect a host cell carrying an F plasmid or a plasmid derived therefrom; the donor plasmid contains the target DNA.
[0021] In the above method, the inducer is L-arabinose or L-rhamnose.
[0022] The present invention also provides the use of the above-mentioned method in improving the efficiency of target DNA delivery.
[0023] The present invention also provides the use of the aforementioned helper plasmid and / or donor plasmid in improving phage infection and transduction.
[0024] The present invention provides a method for preparing and delivering M13 phage based on a helper plasmid. First, the packaging signal sequence of the M13 phage and its downstream replication initiation sequence are cloned into a plasmid to form a donor plasmid pDonor, which allows the insertion of a DNA sequence of interest. Except for the packaging signal sequence and replication-related sequences, other sequences of the M13 phage genome are cloned into a plasmid carrying the pSC101 or RK2 replicon, and an inducible promoter is inserted upstream of gII. Afterwards, the helper plasmid and the donor plasmid are transformed into Escherichia coli together, and a single clone is inoculated into a liquid culture medium. After the transfer, an inducer is added to induce the M13 helper plasmid to express phage-related proteins, and the donor plasmid is packaged into the M13 phage intracellularly and released. Finally, the prepared phage is infecting a host cell carrying an F plasmid or a plasmid derived therefrom to achieve efficient delivery of the donor plasmid.
[0025] Compared with the existing conventional M13KO7 helper phage technology, the synthesis of phages in the present invention depends on the helper plasmid. The content of phages containing helper plasmids in the prepared phages is much lower than that of the M13KO7 helper phage. It is expected that during the library construction or editing process in which the M13 phage repeatedly infects the host, the influence of residual phages containing helper plasmids (or helper phages) on the library construction or editing efficiency can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the plasmid map of pSC101-aa.
[0027] Figure 2 This is the plasmid map of pDonor-pc.
[0028] Figure 3 Comparison of phage titers prepared from different helper plasmids and M13KO7 helper phage.
[0029] Figure 4 Comparison of the residual amounts of auxiliary components in different preparation methods. DETAILED DESCRIPTION
[0030] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.
[0031] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0032] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.
[0033] The pSC101-AmpR EV plasmid in the following examples was purchased from Addgene with ID #208184.
[0034] The pTD103aiiA (Cm) plasmid in the following examples was purchased from Addgene with ID #48886.
[0035] The T7-LacO-QF_p15A plasmid in the following examples was purchased from Addgene, ID # 171651. The public can obtain this biological material from the applicant and it is only used to repeat the experiments of the present invention and cannot be used for other purposes.
[0036] The BW25113 strain used in the following examples is described in Lessard, IA et al. (1998) Homologs of the vancomycin-resistant D-Ala-D-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli, and Synechocystis: attributes of catalytic efficiency, stereoselectivity, and regulation with implications for function. Chem. Biol. The public can obtain this biological material from the applicant for use solely in replicating the experiments described herein and not for any other purpose.
[0037] The MG1655 strain used in the following examples is described in Blattner et al. (1997) The complete genome sequence of Escherichia coli K-12. Science. The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and not for any other purpose.
[0038] The Trans1-Blue strain was purchased from Beijing Quanshijin Biological Company (catalog number: CD401-02).
[0039] Trans1-T1 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd. (catalog number: CD501-02).
[0040] M13KO7 helper phage was purchased from New England Biolabs (Cat. No. N0315S).
[0041] M13mp18 phage was purchased from New England Biolabs (Cat. No. N4040S).
[0042] The strains used in the present invention are shown in Table 1, the primers used are shown in Table 2, and the plasmids involved are shown in Table 3.
[0043] Table 1. Strains used in the present invention
[0044]
[0045] Table 2. Primer information used in the present invention
[0046]
[0047]
[0048] Table 3. Plasmids used and constructed in the present invention
[0049] plasmids Plasmid information pSC101-AmpR EV ampR,pSC101 pTD103aiiA(Cm) chlR,p15A T7-LacO-QF_p15A kanR,p15A pSC101-aa ampR, pSC101, Para, araC, M13 phage gI-gXI pSC101-ra ampR, pSC101, Prha, rhaRS, M13 phage gI-gXI pDonor-pk kanR, p15A, M13 ori, M13 packaging signal pDonor-pc chlR, p15A, M13 ori, M13 packaging signal
[0050] Example 1, construction of auxiliary plasmid
[0051] The specific steps for constructing the helper plasmid are as follows:
[0052] The first step is to obtain the M13 phage gI-gXI sequence fragment A.
[0053] Using the M13KO7 phage genome as a template, PCR amplification was performed with primers 389 and 390 (see Table 2 for the specific sequence) to obtain a PCR product of approximately 5.9 kb, which is the M13 phage gI-gXI sequence fragment A (positions 1 to 5945 of SEQ ID No: 1).
[0054] The PCR amplification system consisted of 10 μL of New England Biolabs Phusion 5× buffer, 1 μL of dNTPs (2.5 mM each), 0.1 μL of template, 2 μL of each primer (10 μM), 0.5 μL of Phusion High-Fidelity DNA Polymerase (2.5 U / μL), and 34.9 μL of distilled water, in a total volume of 50 μL. Amplification conditions were: initial denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, and extension at 72°C for 2.5 minutes (30 cycles); and extension at 72°C for 5 minutes (1 cycle). Unless otherwise specified, the amplification system and conditions used below are the same as those used here.
[0055] In the second step, the pSC101-AmpR EV plasmid backbone fragment B was obtained.
[0056] Using the pSC101-AmpR EV plasmid as a template, PCR amplification with primers 391 and 392 (see Table 2 for the specific sequence) yielded a 3.2 kb PCR product, the pSC101 plasmid backbone fragment B. This fragment contains the ampR gene and the pSC101 replicon, and its sequence is shown in SEQ ID No: 1, positions 5946 to 7592.
[0057] The third step is to obtain fragments C and D of Para and its accessory genes and Prha promoter and its accessory genes.
[0058] Using the genome of strain MG1655 as a template, primers 393 and 394 were used for amplification to obtain a PCR product of approximately 1.3 kb, which is fragment C containing Para and araC. The sequence is shown in positions 9427 to 10616 of SEQ ID No: 1.
[0059] Using the genome of strain MG1655 as a template, primers 395 and 396 were used to amplify the fragment, resulting in a 2 kb PCR product, fragment D containing Prha and rhaRS, with the sequence shown in SEQ ID No: 2. For more information, see Table 4.
[0060] Table 4. Fragments used to construct plasmids in the present invention
[0061] snippet Components included Length (kb) Use primers A M13 phage gI-gXI 5.9 389,390 B ampR,pSC101 3.5 391,392 C Para,araC 1.3 393，394 D Prha,rhaRS 2 395，396 E M13 ori, M13 packaging signal 0.4 400，401 F chlR,p15A 1.6 402，403 G kanR,p15A 1.9 404,405
[0062] In the fourth step, Gibson assembly was performed to obtain the final plasmid.
[0063] Using Gibson technology (Hieff Universal II One Step Cloning Kit, Yisheng Biotechnology (Shanghai) Co., Ltd.) was used to assemble the above-mentioned fragments A, B and C; and fragments A, B and D. Chemical transformation was carried out into competent cells TransT1 (Beijing Quanshijin Biotechnology Co., Ltd.), and the resulting clones were verified by PCR using primers 397 and 398 (specific sequences are shown in Table 2). The band size of the positive clone was about 1.6 kb. Plasmid DNA was extracted from the positive clones and sent for sequencing analysis, and the correct pSC101-aa (nucleotide sequence is SEQ ID No: 1) and pSC101-ra (the difference in nucleotide sequence from the pSC101-aa plasmid is only that positions 9427 to 10616 of SEQ ID No: 1 are replaced by SEQ ID No: 2), of which the pSC101-aa plasmid map is shown in Figure 1 .
[0064] Example 2: Construction of donor plasmid
[0065] The first step was to obtain fragment E (SEQ ID No: 8), containing the M13 phage replication initiation sequence and packaging signal sequence. Using the M13mp18 phage genome as a template, PCR amplification was performed using primers 400 and 401 (see Table 2 for specific sequences). A 0.4 kb PCR product was obtained, which is the M13 phage replication initiation sequence and packaging signal sequence fragment E.
[0066] The second step was to obtain the pTD103aiiA(Cm) plasmid backbone fragment F and the T7-LacO-QF_p15A plasmid backbone fragment G (see Table 4). Using the pTD103aiiA(Cm) plasmid as a template, PCR amplification was performed using primers 402 and 403 (see Table 2 for their specific sequences). A 1.6 kb PCR product, pTD103aiiA(Cm) plasmid backbone fragment F, was obtained. This fragment contains the chlR gene and the p15A replicon.
[0067] Using the T7-LacO-QF_p15A plasmid as a template, PCR amplification with primers 404 and 405 (see Table 2 for specific sequences) yielded a 1.9 kb PCR product, the T7-LacO-QF_p15A plasmid backbone fragment G. This fragment contains the kanR gene and the p15A replicon.
[0068] The third step is to obtain the final plasmid by Gibson assembly. The above fragments E and F; and fragments E and G were assembled using the Universal II One Step Cloning Kit, Yisheng Biotechnology (Shanghai) Co., Ltd.
[0069] The assembly products of fragment EF and fragment EG were chemically transformed into competent cells TransT1 (Beijing Quanshijin Biotechnology Co., Ltd.). The clones obtained by assembling fragment EF were verified by PCR using primers 400 and 406 (specific sequences are shown in Table 2). The band size of the positive clone was about 0.6 kb. Plasmid DNA was extracted from the positive clone and sent for sequencing analysis, and the correct pDonor-pc (nucleotide sequence is SEQ ID No: 3) was obtained. The plasmid map is shown in Figure 2 The clones obtained by fragment GI assembly were verified by PCR using primers 401 and 406 (for specific sequences, see Table 2). The band size of the positive clone was approximately 1.9 kb. Plasmid DNA was extracted from the positive clone and sent for sequencing analysis, resulting in the correct pDonor-pk plasmid (nucleotide sequence: SEQ ID No: 4).
[0070] Example 3: Transformation of helper plasmid and donor plasmid into Escherichia coli to prepare M13 phage
[0071] 1. Preparation of electroporation competent cells
[0072] Inoculate BW25113 strain into liquid LB medium and culture at 37°C, 250 rpm overnight to obtain seed solution. Inoculate the seed solution into 50 mL fresh liquid LB medium at a ratio of 1%, and culture at 37°C, 250 rpm to obtain OD 600 The Trans1-Blue cells were collected at 6000 rpm, 4°C, for 5 minutes. The cells were washed three times with pre-cooled 10% glycerol, then resuspended in 1 mL of 10% glycerol and aliquoted at 100 μL / tube for later use.
[0073] 2. Electroporation auxiliary plasmid and donor plasmid
[0074] Mix 300 ng of the helper plasmid pSC101-aa with 300 ng of the pDonor-pk plasmid and add it to the electroporation-competent medium of the BW25113 strain. Gently mix on ice and transfer to a cuvette. Electroporate at 1.8 kV, 25 μF, and 200 Ω. Immediately after electroporation, add 37°C preheated LB medium, mix thoroughly, transfer to a 1.5 mL centrifuge tube, and recover at 37°C, 200 rpm for two hours. After recovery, collect the cells by centrifugation and plate them on LB plates containing ampicillin (100 μg / mL, the same final concentration as described below) and kanamycin (50 μg / mL, the same final concentration as described below). Incubate overnight at 37°C to obtain the BW25113 strain harboring the pSC101-aa and pDonor-pk plasmids.
[0075] Similarly, 300 ng of the helper plasmid pSC101-ra was mixed with 300 ng of the pDonor-pk plasmid, and the BW25113 strain containing the pSC101-ra and pDonor-pk plasmids was obtained according to the above method.
[0076] 300 ng of the helper plasmid pSC101-aa was mixed with 300 ng of the pDonor-pc plasmid and electroporated into competent cells of the BW25113 strain as described above, generating a BW25113 strain harboring both the pSC101-aa and pDonor-pc plasmids. Similarly, 300 ng of the helper plasmid pSC101-ra was mixed with 300 ng of the pDonor-pc plasmid and generated a BW25113 strain harboring both the pSC101-ra and pDonor-pc plasmids as described above.
[0077] 3. Induce phage production
[0078] The BW25113 strain containing the pSC101-aa and pDonor-pk plasmids and the BW25113 strain containing the pSC101-ra and pDonor-pk plasmids generated in the second step were inoculated into 2 mL of liquid LB medium containing ampicillin and kanamycin, respectively. After overnight culture at 37°C, the culture was transferred to 20 mL of liquid LB medium containing ampicillin and kanamycin at a ratio of 1%, and the corresponding inducer (pSC101-aa plasmid corresponding to a final concentration of 2 g / L L-arabinose, pSC101-ra plasmid corresponding to 1 g / L L-rhamnose) was added. After culture at 37°C and 200 rpm for 12 h, the culture was centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was filtered using a 0.22 μm filter membrane to obtain a phage suspension packaging the pDonor-pk plasmid.
[0079] Following the same steps as above, single clones of the BW25113 strain containing the pSC101-aa and pDonor-pc plasmids and the BW25113 strain containing the pSC101-ra and pDonor-pc plasmids were inoculated into 2 mL of liquid LB medium containing ampicillin and chloramphenicol (34 μg / mL, the same final concentrations below). After overnight culture at 37°C, the clones were transferred to 20 mL of liquid LB medium containing ampicillin and chloramphenicol (34 μg / mL) at a ratio of 1%. The corresponding inducers (the inducer for the pSC101-aa plasmid was a final concentration of 2 g / L L-arabinose, and the inducer for the pSC101-ra plasmid was 1 g / L L-rhamnose) were added. After culture at 37°C and 200 rpm for 12 h, the clones were centrifuged at 8000 rpm at 4°C for 10 min, and the supernatant was filtered using a 0.22 μm filter to obtain a phage suspension packaging the pDonor-pc plasmid.
[0080] Example 4. Comparison of phage titers prepared from different types of plasmid combinations and helper phage M13KO7
[0081] 1. Phage preparation method based on auxiliary M13KO7
[0082] The donor plasmids pDonor-pk and pDonor-pc were transformed into the Trans1-Blue strain respectively. Fresh colonies were inoculated into 50 ml of LB liquid medium containing 70 μg / ml kanamycin and grown at 37°C and 250 rpm until slightly turbid (<10 Klett, OD 600 <0.05). Add 50 μL of M13KO7 helper phage (final concentration of 1×10 8Pfu / ml) and shake continuously for 60-90 minutes. Add kanamycin to a final concentration of 70 μg / ml and grow at 37°C, 250 rpm for 14-18 hours. Spin at 4000 × g for 10 minutes. Filter the supernatant through a 0.22 μm filter into a fresh tube to obtain the phage suspension.
[0083] 2. Determination of phage titer (i.e., DNA delivery ability)
[0084] In the first step, the Trans1-Blue strain was inoculated into LB liquid medium containing tetracycline and cultured overnight at 37°C and 200 rpm. After that, 1% of the strain was transferred to 1 mL of LB liquid medium without antibiotics and cultured at 37°C and 200 rpm until the OD 600 About 0.1-0.2. The second step is to dilute the phage suspension. Take 1 μL of phage suspension to 1mL LB liquid culture medium and mix well. Then take 1 μL of the diluted suspension to 1mL LB liquid culture medium and mix well. Take 10 μL of the final diluted suspension to the above 1mL bacterial solution and culture at 37°C and 200rpm for 45 minutes. Finally, take 50 μL of bacterial solution and spread it on the corresponding resistance plate (pDonor-pc corresponds to chloramphenicol, pDonor-pk corresponds to kanamycin). The titer of the phage is obtained by multiplying the number of clones by the dilution multiple. The calculation formula is titer (CFU / mL) = 2×10 9 × Number of clones. According to the above method, a series of phages were prepared and tested to compare the titer difference between the method disclosed in the present invention and the traditional method based on helper phage M13KO7.
[0085] The results are as follows Figure 3 As shown in the figure: The comparison of phage titers of pDonor-pc and pDoonor-pk plasmids showed that the phage titers prepared by the present invention were slightly higher than those of M13KO7 version, both of which were above 10 12 The titer of phage prepared by the method of the present invention fluctuated between 18.1±0.9 and 25.3±0.7 CFU / mL, and the titer of phage containing the donor plasmid prepared using the helper phage M13KO7 method fluctuated between 16.2±1.6 and 16.6±1.2 CFU / mL.
[0086] 3. Comparison of residual auxiliary elements between the phage prepared by the present invention and the phage prepared by M13KO7
[0087] When calculating the residual amount of M13KO7 helper phage in the phage suspension, the method is the same as step 2, and finally the plate is plated on a kanamycin-resistant plate. When calculating the residual amount of phage packaging helper plasmid in the phage suspension, the method is the same as step 2, and finally the plate is plated on an ampicillin-resistant plate. The residual percentage of the helper plasmid or helper phage is calculated by dividing the residual titer by the phage titer corresponding to the donor plasmid. Since pDonor-pk and the helper phage M13KO7 have the same kanamycin resistance, the residual amount of this group of helper phages was not tested. For specific results, see Figure 4 .
[0088] Depend on Figure 4 It can be seen that the content of M13KO7 helper phage in the phage suspension packaging pDonor-pc plasmid is about 1.6×10 10 CFU / mL, accounting for 99.0×10 -4 The titer of phage prepared based on the auxiliary plasmid pSC101-aa of the present invention can reach 2.5×10 12 CFU / mL, the phage packaged with the helper plasmid had only 2.3×10 8 CFU / mL, accounting for 0.9×10 -4 Therefore, compared with the helper phage M13KO7 method, the method of the present invention can not only produce high-concentration phages, but also reduce the residual rate of the helper plasmid by 110 times, and should have broad application prospects.
[0089] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.
Claims
1. A method for preparing M13 phage based on a helper plasmid, characterized in that: The method comprises the following steps: 1) constructing a helper plasmid, wherein the helper plasmid contains a partial sequence A in the M13 phage genome, a pSC101 replicon, an arabinose-inducible promoter or a rhamnose-inducible promoter, a gene encoding an arabinose-inducible promoter regulatory protein araC or a gene encoding a rhamnose-inducible promoter activator protein rhaRS, and an ampicillin resistance gene; the partial sequence A in the M13 phage genome does not contain the remaining sequences after the replicon and packaging signal of the M13 phage genome; 2) constructing a donor plasmid containing an M13 phage replicon and packaging signal, a p15A replicon, a kanamycin resistance gene or a chloramphenicol resistance gene, and a target donor sequence; 3) Transforming the helper plasmid described in step 1) and the donor plasmid described in step 2) into a recipient strain to induce the production of the phage.
2. The method according to claim 1, characterized in that Step 1) The partial sequence A in the M13 phage genome is a molecule whose nucleotides are the 1st to 5945th positions of SEQ ID No: 1; the pSC101 replicon is a molecule whose nucleotides are the 5946th to 7592th positions of SEQ ID No: 1; the arabinose-inducible promoter nucleotides are the 10332th to 10616th positions of SEQ ID No: 1; the rhamnose-inducible promoter nucleotides are the 1927th to 2045th positions of SEQ ID No: 2; the araC gene is a molecule whose nucleotides are the 9427th to 10305th positions of SEQ ID No: 1; the rhaRS gene is a molecule whose nucleotides are the 22nd to 1781st positions of SEQ ID No: 2; and the ampicillin gene is a molecule whose nucleotides are the 8422th to 9388th positions of SEQ ID No:
1.
3. The method according to claim 1 or 2, characterized in that The nucleotide sequence of the helper plasmid in step 1) is SEQ ID No: 1, or a DNA molecule obtained by replacing positions 9427 to 10616 in SEQ ID No: 1 with SEQ ID No:
2.
4. The method according to any one of claims 1 to 3, characterized in that: Step 2) The M13 phage replicon and packaging signal are molecules with nucleotides from positions 1 to 432 of SEQ ID No: 3; the p15A replicon is a molecule with nucleotides from positions 515 to 1060 of SEQ ID No: 3; the chloramphenicol resistance gene is a molecule with nucleotides from positions 1289 to 1948 of SEQ ID No: 3; and the kanamycin resistance gene is a molecule with nucleotides from positions 1423 to 2217 of SEQ ID No:
4.
5. The method according to claim 4, characterized in that Step 2) The nucleotide sequence of the donor plasmid is a molecule of SEQ ID No: 3 or SEQ ID No:
4.
6. The helper plasmid or / and donor plasmid described in any one of the methods of claims 1-5.
7. A method for improving the efficiency of target DNA delivery, characterized in that: The method comprises: 1) transforming the helper plasmid according to claim 6 into a host cell, and using an inducer to induce the helper plasmid according to claim 6 to express the phage-related protein; 2) Simultaneously, transforming the donor plasmid of claim 6 into a host cell, packaging the donor plasmid of claim 6 into an M13 phage to obtain packaged M13 phage, and infecting a host cell carrying an F plasmid or a plasmid derived therefrom with the packaged M13 phage; the donor plasmid contains the target DNA.
8. The method according to any one of claims 7, characterized in that The inducer is L-arabinose or L-rhamnose.
9. Use of the method according to any one of claims 1 to 5 in improving the efficiency of target DNA delivery.
10. Use of the plasmid according to claim 6 in improving phage infection and transduction.