A pBeloBCA11-MCS(+) recombinant plasmid and its construction method and application

Abstract

The present invention provides a pBeloBCA11-MCS(+) recombinant plasmid, a construction method, and an application thereof. The recombinant plasmid can expand the endonuclease recognition sites of the low-copy plasmid vector pBeloBCA11 at the molecular level, improve the recombination between the pBeloBCA11 plasmid and the target fragment, promote the construction of large-fragment genomic libraries, and facilitate applications in gene cluster-related research.

Description

Technical Field

[0001] The present invention relates to a recombinant plasmid, in particular to a pBeloBCA11-MCS(+) recombinant plasmid and a construction method thereof, and applications in research related to the construction of large-fragment genomic libraries and gene clusters. Background Art

[0002] Many important traits of organisms involve complex physiological and biochemical reactions, involving the participation and regulation of multiple genes (Yin J, Hoffmann M, etc.. Direct cloning and heterologous expression of the salinomycinbiosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomycescoelicolor A3(2). Sci Rep, 2015, 5: 15081.) or gene clusters (Wang G, Zhao Z, etc.. CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. Nat Microbiol, 2019, 4(12): 2498-2510.). Cloning and utilizing these important functional sequences to study the structure, function and evolution of these sequences has become one of the hot topics in the field of genetics. At the same time, physical mapping of complex genomes (Zouali H, Chamaillard M, etc.. Genetic refinement and physical mapping of a chromosome 16q candidate region for inflammatory bowel disease. Eur J Hum Genet, 2001, 9(10): 731-742.) and map-based cloning of genes (Suster ML, AbeG, etc.. Transposon-mediated BAC transgenesis in zebrafish. Nat Protoc, 2011, 6(12): 1998-2021.) also involve the study of large fragments. Therefore, improving the vector's capacity and recombination efficiency has always been one of the important development directions of cloning vectors.

[0003] The pBACs series of bacterial chromosomal cloning vectors, constructed based on the Escherichia coli fertility factor F plasmid (F plasmid), contain elements such as oriS (a unidirectional replicon), repE (controls F plasmid replication), and parA and parB (controls copy number). The pBeloBAC11 plasmid, one of the pBACs, exhibits low chimera frequency, a low copy number (1-2 copies), limited intracellular plasmid rearrangements, exclusion of exogenous F plasmids, high transformation efficiency, and a large cloning capacity (50-300 kb).

[0004] Blue-white screening is a recombinant screening method that utilizes α-complementation. The pBeloBAC11 plasmid contains the sP6 and T7 promoters linked to the regulatory sequence of the B-galactosidase gene (lacZ) and a 146-amino acid coding sequence. A 31-bp multiple cloning site (MCS) is inserted into this coding sequence. The inserted MCS sequence does not affect the reading frame or post-translational function of the B-galactosidase gene (lacZ) regulatory sequence. The DH10 electroporation-competent strain (genotype: F-mcrAΔ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15ΔlacX74 recA1 endA1 araD139Δ(ara,leu)7697 galUgalKλ-rpsL nupG) encodes a partial lacZ C-terminal sequence and possesses the ability to electroporate exogenous plasmids. When DH10B cells are electroporated with the pBeloBAC11 plasmid and cell viability is restored, the lacZ regulatory sequences from the pBeloBAC11 plasmid and the C-terminal portion of the lacZ from DH10B are expressed simultaneously and co-localized within the cell, forming an enzymatically active lacZ. This complementary mechanism between the host cell lacking these regulatory sequences and the plasmid carrying the complete regulatory sequences is known as α-complementation. In the presence of the inducer IPTG, lacZ in the DH10B strain cleaves the colorless compound 5-bromo-4-chloro-3-indole-β-D-galactoside (X-gal) into galactose and the dark blue substance 5-bromo-4-indigo. 5-bromo-4-indigo gives the entire colony a blue color.

[0005] When the exogenous nucleic acid sequence is inserted into the MCS restriction site of the pBeloBAC11 plasmid to form a recombinant plasmid, it will almost inevitably interfere with the expression of the B-galactosidase gene regulatory sequence in the plasmid (not translated or translated to produce an amino acid fragment without α-complementary ability), making it impossible for DH10B carrying the recombinant plasmid to form enzymatically active lacZ to cut X-gal, resulting in the DH10B colonies carrying the recombinant plasmid appearing white.

[0006] The present invention provides a method for constructing a pBeloBCA11-MCS(+) recombinant plasmid and the pBeloBCA11-MCS(+) recombinant plasmid obtained by the construction method. The recombinant plasmid can expand the endonuclease recognition site of the low-copy plasmid vector pBeloBCA11 at the molecular level, improve the recombination between the pBeloBCA11 plasmid and the target fragment, promote the construction of large-fragment genomic libraries, and promote applications in gene cluster-related research. Summary of the Invention

[0007] The present invention provides a pBeloBCA11-MCS(+) recombinant plasmid, its construction method, and applications. The recombinant plasmid can expand the endonuclease recognition sites of the low-copy plasmid vector pBeloBCA11 at the molecular level, improving the recombination between the pBeloBCA11 plasmid and the target fragment, promoting the construction of large-fragment genomic libraries, and facilitating applications in gene cluster research.

[0008] The technical solution adopted by the present invention to solve the technical problem is:

[0009] The present invention first protects a method for constructing a pBeloBCA11-MCS(+) recombinant plasmid. The pBeloBCA11 plasmid containing a chloramphenicol screening tag is used as a vector. An artificially synthesized sequence for homodizyme cleavage is inserted between the restriction endonuclease sites BamHI and HindIII of pBeloBCA11. The sequence contains multiple restriction endonuclease recognition sites including PmeI and PacI, thereby constructing a pBeloBCA11-MCS(+) recombinant plasmid containing PmeI, PacI and a chloramphenicol screening tag.

[0010] Specifically, the method includes the following steps:

[0011] 1) Artificially synthesizing a nucleotide sequence containing multiple restriction endonuclease recognition sites including PmeI and PacI;

[0012] 2) Double enzyme digestion of pBeloBCA11 plasmid, identification of enzyme digestion efficiency by agarose gel electrophoresis, and recovery;

[0013] 3) Double enzyme digestion of the artificially synthesized sequence in step 1), identification of the digestion efficiency by agarose gel electrophoresis, and recovery;

[0014] 4) Ligating the purified and recovered pBeloBCA11 vector fragment and the purified and recovered artificially synthesized sequence;

[0015] 5) The recombinant plasmid was transformed into competent bacteria, blue spot clones were selected for expansion and sequencing, and the pBeloBCA11-MCS(+) recombinant plasmid was extracted.

[0016] As a preferred technical solution of the present application, the artificially synthesized sequence is shown as SEQ ID NO: 1.

[0017] As a preferred technical solution of the present application, in step 2), the reaction system for double enzyme digestion of pBeloBCA11 plasmid is as follows: pBeloBCA11 1ug, BamHI 1.0uL (10U), HindIII 1.0uL (10U), 10X FastDigest Green buffer 5.0uL, 0.2% BSA 5.0uL, and ultrapure water to 50uL.

[0018] As a preferred technical solution of the present application, in step 3), the double enzyme digestion of the artificially synthesized sequence in step 1) is carried out in the following reaction system: 1 ug of artificially synthesized sequence, 1.0 uL (10 U) of BamHI, 1.0 uL (10 U) of HindIII, 5.0 uL (10 U) of 10X FastDigestGreen buffer, and 5.0 uL of 0.2% BSA.

[0019] As the preferred technical solution of the present application, the ligation system in step 4) is as follows: 10X T4 Ligase buffer 2uL, pBeloBCA11 1uL, artificially synthesized target fragment 1uL, T4 Lidase 0.5ul (15U), and ultrapure water to 20uL.

[0020] As a preferred technical solution of the present application, the sequencing primers in step 5) are selected from the following first pair of primers or second pair of primers:

[0021] The first pair of primers:

[0022] Forward primer: 5′-TGTTGGCGGGTGTCGGGGCTGGCTT-3;

[0023] Reverse primer: 5′-TGCGATGAGTGGCAGGGCGGGGCGT-3;

[0024] The second pair of primers:

[0025] Forward primer: 5′-GCACAGATGCGTAAGGAGAA-3′;

[0026] Reverse primer: 5′-GATAATAAGCGGATGAATGG-3.

[0027] In a second aspect, the present invention also protects the pBeloBCA11-MCS(+) recombinant plasmid constructed by the construction method of the pBeloBCA11-MCS(+) recombinant plasmid described above.

[0028] The recombinant plasmid of the present invention can be dissolved in water or TE buffer to form a solution, or can be prepared into dry powder by freeze drying.

[0029] The present invention also protects the use of the aforementioned pBeloBCA11-MCS(+) recombinant plasmid in constructing large-fragment genomic libraries and gene cluster-related research.

[0030] Beneficial effects

[0031] The present invention provides a method for constructing a pBeloBCA11-MCS(+) recombinant plasmid and the pBeloBCA11-MCS(+) recombinant plasmid obtained by the construction method. The recombinant plasmid can expand the endonuclease recognition site of the low-copy plasmid vector pBeloBCA11 at the molecular level, improve the recombination of the pBeloBCA11 plasmid and the target fragment, promote the construction of large-fragment genomic libraries, and promote applications in gene cluster-related research.

[0032] The recombinant plasmid clones were cloned while maintaining the original plasmid clones (C (Cho) =20μg-40 / ml culture medium), it has better chloramphenicol resistance (BCA11-2 E) and higher physiological activity (better growth rate, BCA11-2 B). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a preliminary screening of the recombinant plasmid pBeloBCA-MCS(+). Well 1, marker; Wells 2 (A), 6 (E), and 8 (G): preliminary identification of the target band amplified from the recombinant plasmid pBeloBCA-MCS(+); Wells 3 (B), 5 (D), and 9 (H): preliminary identification of the target band amplified from the original plasmid pBeloBCA.

[0034] Figure 2 The sequence of the MCS region of the recombinant plasmid pBeloBCA11-MCS(+) is compared with the MCS region of the original plasmid pBeloBCA11;

[0035] Figure 3 and Figure 4 These are the restriction endonuclease recognition sites in the MCS region of the pBeloBCA11 plasmid and the pBeloBCA11-MCS(+) recombinant plasmid, respectively;

[0036] Figure 5 The trait characteristics of pBeloBCA11 plasmid and pBeloBCA11-MCS(+) recombinant plasmid 15 hours after electroporation;

[0037] Figure 6 The trait characteristics of pBeloBCA11 plasmid and pBeloBCA11-MCS(+) recombinant plasmid 21 hours after electroporation;

[0038] Figure 7 The trait characteristics of pBeloBCA11 plasmid and pBeloBCA11-MCS(+) recombinant plasmid 24 hours after electroporation;

[0039] Figure 8 The trait characteristics of pBeloBCA11 plasmid and pBeloBCA11-MCS(+) recombinant plasmid 36 hours after electroporation;

[0040] Figure 9 This is the electrophoresis diagram of the PCR amplification of the exogenous fragment to be inserted (133bp-psbA); wells 4 (D) and 5 (E) were preliminarily identified as the target bands of PCR amplification and were subjected to end-smoothing treatment (system B); well F is a marker;

[0041] Figure 10 The recombinant plasmid pBeloBCA11-MCS(+) was digested with PmeI and then ligated with the exogenous fragment (133bp-psbA). After electroporation, positive clones were identified (white spot screening).

[0042] Figure 11 for Figure 10 White spot strains (positive clones of exogenous fragments) and Figure 8 The middle blue spot strain (positive clone strain of the recombinant plasmid) was sequenced after PCR using the second pair of sequencing primers, and the sequence comparison results were obtained. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to the examples. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0044] Example 1 Construction of pBeloBCA11-MCS(+) recombinant plasmid

[0045] The method for constructing the pBeloBCA11-MCS(+) recombinant plasmid comprises the following steps:

[0046] 1) Design a double-stranded nucleotide sequence containing two restriction endonuclease recognition sites, PmeI and PacI

[0047] 104 bp, the nucleotide sequence is shown in SEQ ID NO: 1;

[0048] 2) Double enzyme digestion of pBeloBCA11 plasmid, the specific reaction system is as follows:

[0049] pBeloBCA11 1ug, BamHI 1.0uL (10U), HindIII 1.0uL (10U), 10X FastDigestGreen buffer 5.0uL, 0.2% BSA 5.0uL, and ultrapure water to 50uL. Mix all components and incubate at 37°C in a water bath for 30min. Analyze the digestion efficiency by 1% agarose gel electrophoresis (120V / 45min) and purify and recover the target fragment. Determine the target fragment concentration (ng / uL) and adjust to 100ng / uL.

[0050] 3) Double digestion of 104 bp artificially synthesized sequence with BamHI and HindIII. The specific reaction system is as follows:

[0051] 104bp synthetic sequence (1 μg), BamHI 1.0 μL (10 U), HindIII 1.0 μL (10 U), 10X Fast Digest Green buffer 5.0 μL, 0.2% BSA 5.0 μL, and ultrapure water to 50 μL. Mix all components and incubate at 37°C in a water bath for 30 min. Analyze the digestion efficiency by 5% agarose gel electrophoresis (90 V / 25 min) and purify the fragment. Determine the target fragment concentration (ng / μL) and adjust the concentration to 100 ng / μL.

[0052] 4) After the electrophoresis-purified target band is recovered, the plasmid vector pBeloBCA11 and the artificially synthesized target fragment ligation system are constructed as follows:

[0053] 10X T4 Ligase buffer 2uL, pBeloBCA11 1uL, artificially synthesized target fragment 1uL, T4 Lidase 0.5ul (15U), make up to 20uL with ultrapure water, mix all components and incubate in a 25℃ water bath for 1h;

[0054] 5) The recombinant plasmid was transformed into DH10B competent bacteria, and blue spot clones were screened for PCR amplification of the bacterial solution and preliminary electrophoresis detection. The results were as follows Figure 1 The recombinant plasmid MCS region was sequenced and identified. If the identification result showed that the MCS region contained an artificially synthesized fragment, the pBeloBCA11-MCS(+) recombinant plasmid was extracted.

[0055] 6) DNA sequencing analysis was performed on the pBeloBCA11-MCS(+) recombinant plasmid extracted in step 5). The bidirectional sequencing mode was selected and the universal primers of the pBeloBCA11 plasmid were used to detect the bases across the MCS region of the plasmid. The detection results were compared with the original plasmid (pBeloBCA11) sequence. The results were as follows: Figure 2 shown.

[0056] In step 5), the specific method of sequencing identification can be:

[0057] Use the first pair of primers to perform colony PCR amplification, and perform agarose gel electrophoresis on the amplified product. If the length of the obtained target fragment (782 bp) is between 750 bp and 1000 bp of the DL2000 marker, it is preliminarily identified as a recombinant plasmid. The first pair of primers are:

[0058] Forward primer: 5,-TGTTGGCGGGTGTCGGGGCTGGCTT-3;

[0059] Reverse primer: 5,-TGCGATGAGTGGCAGGGCGGGGCGT-3;

[0060] Use the second pair of primers to perform colony PCR amplification, and perform agarose gel electrophoresis on the amplified product. If the length of the obtained target fragment (614 bp) is between 500 bp and 750 bp of the 100 bp DNA ladder, it is preliminarily identified as a recombinant plasmid. The second pair of primers are:

[0061] Forward primer: 5,-GCACAGATGCGTAAGGAGAA-3;

[0062] Reverse primer: 5'-GATAATAAGCGGATGAATGG-3.

[0063] The PCR amplification system was constructed according to the reference instructions of the selected enzymes. The annealing temperatures of the first and second pairs of primers were 50°C and 48°C, respectively.

[0064] In step 6), the DNA sequencing analysis is to send the pBeloBCA11-MCS(+) recombinant plasmid extracted in step 5) to China Qingke Biotechnology Co., Ltd. for DNA sequencing analysis: the results of the selected detection are compared with the base sequence of the MCS region of pBeloBCA11 as follows:

[0065] The results showed that the 9-base insertion of "TAATTAAGT" occurred at position 361, forming the PacI restriction endonuclease recognition site "TTAATTAA";

[0066] The results showed that the 9-base insertion of "ACTGTTTAA" occurred at site 369, forming the PmeI restriction endonuclease recognition site "GTTTAAAC";

[0067] The MCS region of the pBeloBCA11 plasmid and the pBeloBCA11-MCS(+) recombinant plasmid were tested by restriction enzymes, and the results were as follows: Figure 3 and Figure 4 As shown:

[0068] The results showed that in addition to all the restriction endonuclease recognition sites in the MCS region of the pBeloBCA11 plasmid, two restriction endonuclease recognition sites, PmeI and PacI, were added to the MCS region of the pBeloBCA11-MCS(+) recombinant plasmid, completing the transformation of the pBeloBCA11-MCS(+) recombinant plasmid.

[0069] Example 2 Transformation of pBeloBCA11-MCS(+) plasmid

[0070] The pBeloBCA11-MCS(+) recombinant plasmid was transformed into DH10 competent cells. The specific steps are as follows:

[0071] 1) The pBeloBCA11-MCS(+) plasmid containing the chloramphenicol resistance selection gene was transformed into DH10 competent cells by electroporation. The specific transformation steps are as follows:

[0072] (1) Clean a 0.1 mm electrophoresis cuvette three times with 75% alcohol, rinse again with anhydrous ethanol, invert for 5-7 minutes, and then upright for 5-7 minutes. After the inner tank of the electrophoresis cuvette is dry, place it on ice for precooling.

[0073] (2) Thaw 100 μL of DH10 electroporation competent cells and 15 μL of pBeloBCA11-MCS(+) plasmid on ice;

[0074] (3) Pipette 10 μL of pBeloBCA11-MCS(+) plasmid and gently mix it into 100 μL of DH10 electroporation competent medium. Keep this step on ice.

[0075] (4) Transfer the system in step (3) to an electroporation cup, prevent bubbles from forming and wipe the outer wall of the electroporation cup dry during electroporation to prevent arcing during electroporation, and cover the cup with a lid; the electroporation steps are as follows:

[0076] (5) Set the electroporator parameters: C = 25uF, PC = 200Ω, V = 2.5kV (Bio-Rad GenePulser electroporator), quickly place the electroporation cup into the electroporation tank, and quickly insert it into ice after electroporation is completed;

[0077] (6) After 2 min, remove the electroporation cup from the ice and add 700 μL of sterile SOC medium (room temperature) without chloramphenicol. Mix by gently pipetting the bottom of the electroporation cup several times. Transfer the culture to a 2 mL sterile centrifuge tube and incubate at 37°C, 200 rpm / 1 h.

[0078] (7) In a clean bench, draw 100 μL of the bacterial solution from step (6) and evenly spread it on LB solid medium preheated to 37°C containing chloramphenicol, X-Gal, and IPTG;

[0079] (8) Place the culture dish upside down in a 37°C incubator for 24-36 hours until the plaques turn blue.

[0080] The experiment found that the pBeloBCA11-MCS(+) recombinant plasmid still maintained the blue-white screening characteristics after the artificially synthesized exogenous fragment was inserted into the MCS region. The artificially synthesized exogenous nucleic acid fragment containing BamHI, PacI, XbaI, SalI, PmeI, PstI, BspMI, SphI, and HindIII restriction endonuclease recognition sites was inserted into the MCS region of pBeloBCA11, without changing the blue-white screening characteristics of the pBeloBCA11 plasmid.

[0081] Performance Testing

[0082] To compare the characteristic properties of the recombinant plasmid (pBeloBCA11-MCS(+)) and / or the original plasmid (pBeloBCA11) in host competent cells (DH10B), the following experiment was designed:

[0083] Relying on LB (IPTG + X-Gal) solid medium, the resistance performance of recombinant plasmid (pBeloBCA11-MCS (+)) clone and original plasmid (pBeloBCA11) clone to chloramphenicol (Cho) hormone was constructed respectively. The working concentration screening gradient was constructed as follows: A, C (Cho) =20 μg / ml; B, C (Cho) =30 μg / ml; C, C (Cho) =40 μg / ml; D, C (Cho) =50 μg / ml; E, C (Cho) =60 μg / ml; electroporation, incubation at 37°C in the dark.

[0084] Here are the results:

[0085] 1. Such as Figure 5 As shown, 15 h after electroporation, (Cho) =30 μg / ml culture medium, the original plasmid clone did not show obvious plaques (plate number: BCA11 B), while the recombinant plasmid clone showed obvious plaques (plate number: BCA11-2 B), indicating that the recombinant plasmid clone showed a better growth rate in the 30 μg / ml chloramphenicol selection medium.

[0086] 2. Such as Figure 6 As shown, 21 h after electroporation, (Cho) =20 μg / ml and C (Cho)=30μg / ml culture medium, both the original plasmid clones (plate numbers: BCA11 A, BCA11 B) and the recombinant plasmid clones (plate numbers: BCA11-2 A, BCA11-2 B). This indicates that the recombinant plasmid clones retained the resistance of the original plasmid clones at a chloramphenicol working concentration of 20-30μg / ml, which was consistent with experimental expectations.

[0087] 3. Such as Figure 7 As shown, after 24 h of electroporation, the cells were grown at C (Cho) =20μg / ml culture medium, the original plasmid clone (plate number: BCA11 A) and the recombinant plasmid clone (plate number: BCA11-2 A) both showed blue spots. (Cho) =30μg / ml culture medium showed blue spots, while the original plasmid clone (plate culture number: BCA11 B) did not show blue spot characteristics. This shows that ① the recombinant plasmid clone in C (Cho) =20μg / ml culture medium maintained the blue spot characteristics of the original plasmid clone, which was in line with experimental expectations. ② The recombinant plasmid clone was cloned in C (Cho) =30μg / ml culture medium showed higher physiological recovery activity than the original plasmid clone.

[0088] At the same time, the recombinant plasmid was cloned in C (Cho) =40 μg / ml, C (Cho) =50μg / ml, C (Cho) = 60 μg / ml culture medium, compared with the original plasmid clones, all showed significant plaque growth (recombinant plasmid clones plate number: BCA11-2 C, BCA11-2 D, BCA11-2 E; original plasmid clones plate number: BCA11-C, BCA11-D, BCA11-E). This shows that the recombinant plasmid clones have significantly improved chloramphenicol tolerance compared to the original plasmid clones.

[0089] 4. Such as Figure 8 As shown, after 36 h of electroporation, the cells were grown at C (Cho) =20 μg / ml, C (Cho) =30 μg / ml, C (Cho) =40μg / ml culture medium, the original plasmid clones (plate number: BCA11A, BCA11 B, BCA11 C) and the recombinant plasmid clones (plate number: BCA11-2 A, BCA11-2 B, BCA11-2 C) all showed blue spots. The recombinant plasmid clone (plate number: BCA11-2 D) (Cho)=50μg / ml culture medium showed blue spots, while the original plasmid clone (plate culture number: BCA11-D) showed weak blue spot characteristics. This shows that ① the recombinant plasmid clone in C (Cho) = 20-50μg / ml culture medium continuously showed blue spot characteristics; ② recombinant plasmid cloned in C (Cho) =60μg / ml culture medium is more active than the original plasmid clone.

[0090] From the above experimental results, we can conclude that the recombinant plasmid clone maintains the original plasmid clone (C (Cho) =20μg-40 / ml culture medium), it has better chloramphenicol resistance (BCA11-2 E) and higher physiological recovery activity (better growth rate, BCA11-2 B).

[0091] Example 3 Application of recombinant plasmid pBeloBCA11-MCS(+)

[0092] 1) The recombinant enzyme cleavage site of the recombinant plasmid pBeloBCA11-MCS(+): pmeI was internally cut and dephosphorylated (the construction system is as follows: System A), and the gel was cut after electrophoresis for recovery.

[0093] 2) PCR amplified a 133 bp specific DNA sequence in psbA (psbA: soybean chloroplast gene photosystem II protein D1), the results are as follows Figure 9 The primer pair is:

[0094] Forward primer: 5,-GCGAAAGCCTATG-3;

[0095] Reverse primer: 5,-GCAATAAAAGCGATAA-3;

[0096] The PCR amplification system was constructed according to the instructions for the selected enzymes. The primer pair annealing temperature was 50°C. The PCR amplification products extracted by CTAB were smoothed using a blunt-end enzyme (system B). After electrophoresis, the gel was cut and recovered for later use.

[0097] 3) Ligate the 5'-end dephosphorylated pmeI-cleaved recombinant plasmid (Step 1) with the end-blunted PCR product (Step 2) (ligation system as follows: System C). Ligation conditions: 16°C / 8h, 65°C / 10min;

[0098] The recombinant plasmid in step 3) was electroporated into electrocompetent DH10B cells, and 700 μL of antibiotic-free SOC medium was added for recovery (37°C / 1 h), and then spread onto LB (IPTG+X-Gal) solid medium containing chloramphenicol (20 μg / ml) and incubated at 37°C / 24 h.

[0099] 4) After preliminary PCR detection of white spots, sequencing verification was performed. The PCR detection primer pair was the second universal primer pair for the pBeloBCA11 plasmid:

[0100] Forward primer: 5,-GCACAGATGCGTAAGGAGAA;

[0101] Reverse primer: 5,-GATAATAAGCGGATGAATGG-3;

[0102] Among them, system A:

[0103] Recombinant plasmid pBeloBCA11-MCS(+) 1 μg, PmeI 1.0 μL (10 U), dephosphatase 1.5 μL (15 U), 10X Fast Digest Green buffer 5.0 μL, 0.2% BSA 5.0 μL, and ultrapure water to 50 μL. Mix all components and incubate at 37°C in a water bath for 30 min. Analyze enzymatic digestion efficiency by 1% agarose gel electrophoresis (90 V / 25 min) and purify and recover the target fragment. Determine the target fragment concentration (ng / μL) and adjust the concentration to 50 ng / μL.

[0104] System B:

[0105] 1 μg of PCR amplification product, 2.0 μL (20 U) of Endase, 5.0 μL of 10X Endase buffer, 5.0 μL of 0.2% BSA, and ultrapure water were added to a volume of 50 μL. Mix all components thoroughly and incubate in a 20°C water bath for 1 hour. Purify by 5% agarose gel electrophoresis (90 V / 25 min) and recover the target fragment. Determine the target fragment concentration (ng / μL) and adjust to 50 ng / μL.

[0106] System C:

[0107] 5'-end dephosphorylated PmeI endonuclease recombinant plasmid (step 1) 2.0uL (100ng), end-smoothed PCR product (step 2) 4.0uL (200ng), 10X T4 buffer 1.0uL, PEG400 1uL, T4 ligase 0.1uL (34U), ultrapure water to 10uL, mix all components, 16℃ / 8h, 65℃ / 10min.

[0108] The results are as follows Figure 10 and Figure 11As shown, the recombinant plasmid pBeloBCA11-MCS(+) connected with the exogenous DNA fragment maintained the blue-white spot screening characteristics of the exogenous fragment insertion, that is, white spot screening positive clones; sequencing results showed that the exogenous DNA fragment (133bp-psbA) was inserted into the new restriction site PmeI constructed in the MCS region of the recombinant plasmid pBeloBCA11-MCS(+). At the same time, the length of the exogenous insert fragment (133bp-psbA) (133bp) and the length of the artificially synthesized fragment (SEQ ID NO: 1) (104bp) were relatively equal. The white spot and blue spot characteristics exhibited by each of them can be compared to indicate the sequence specificity of the artificially synthesized fragment (SEQ ID NO: 1) in the recombinant plasmid pBeloBCA11-MCS(+).

[0109] The above results show that the recombinant plasmid of the present invention maintains the blue-white spot screening property and expands the scope of use of the plasmid.

[0110] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A method for constructing a pBeloBCA11-MCS(+) recombinant plasmid, characterized in that: Using the pBeloBCA11 plasmid containing a chloramphenicol selection tag as a vector, a double-enzyme-cuttable artificially synthesized sequence containing PmeI and PacI restriction endonuclease recognition sites was inserted between the restriction endonuclease sites BamHI and HindIII of pBeloBCA11, thereby constructing a pBeloBCA11-MCS(+) recombinant plasmid containing PmeI, PacI and a chloramphenicol selection tag; Wherein, the artificially synthesized sequence is shown as SEQ ID NO:

1.

2. The method for constructing the pBeloBCA11-MCS(+) recombinant plasmid according to claim 1, wherein: The method comprises the following steps: 1) Artificially synthesize a nucleotide sequence containing multiple restriction endonuclease recognition sites, including PmeI and PacI; 2) Double-digest the pBeloBCA11 plasmid, determine the digestion efficiency by agarose gel electrophoresis, and recover the fragments; 3) Double enzyme digestion of the artificially synthesized sequence in step 1) is performed, and the digestion efficiency is verified by agarose gel electrophoresis and recovered; 4) Ligating the pBeloBCA11 vector fragment purified and recovered in step 2) and the artificially synthesized sequence purified and recovered in step 3); 5) Transform the recombinant plasmid into competent bacteria, select blue-spot clones for expansion and sequencing, and extract the pBeloBCA11-MCS(+) recombinant plasmid.

3. The method for constructing the pBeloBCA11-MCS(+) recombinant plasmid according to claim 2, wherein: In step 2), the reaction system for double-enzyme digestion of the pBeloBCA11 plasmid is as follows: pBeloBCA11 1ug, BamHI 1.0uL 10U, HindIII 1.0uL 10U, 10X FastDigest Green buffer 5.0uL, 0.2% BSA 5.0uL, and make up to 50uL with ultrapure water.

4. The method for constructing the pBeloBCA11-MCS(+) recombinant plasmid according to claim 2, wherein: In step 3), the double enzyme digestion of the artificially synthesized sequence in step 1) is carried out in the following reaction system: 1 μg of artificially synthesized sequence, 10 U of BamHI 1.0 μL, 10 U of HindIII 1.0 μL, 5.0 μL of 10× FastDigest Green buffer, and 5.0 μL of 0.2% BSA.

5. The method for constructing the pBeloBCA11-MCS(+) recombinant plasmid according to claim 2, wherein: The ligation system in step 4) is as follows: 10 X T4 Ligase buffer 2uL, pBeloBCA11 1uL, artificially synthesized target fragment 1uL, T4 Ligase 0.5ul 15U, and ultrapure water to 20uL.

6. The method for constructing the pBeloBCA11-MCS(+) recombinant plasmid according to claim 2, wherein: In step 5), the sequencing primers are selected from the following first pair of primers or second pair of primers: The first pair of primers: Forward primer: 5 ， -TGTTGGCGGGTGTCGGGGCTGGCTT-3 ， ; Reverse primer: 5 ， -TGCGATGAGTGGCAGGGCGGGGCGT-3 ， ; The second pair of primers: Forward primer: 5 ， -GCACAGATGCGTAAGGAGAA-3 ， ; Reverse primer: 5 ， -GATAATAAGCGGATGAATGG-3 ， .

7. The pBeloBCA11-MCS(+) recombinant plasmid constructed by the construction method of the pBeloBCA11-MCS(+) recombinant plasmid according to any one of claims 1 to 6.

8. The recombinant plasmid according to claim 7, characterized in that The recombinant plasmid can be dissolved in water or TE buffer to form a solution, or can be prepared into a dry powder using freeze-drying method.

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