Construction method and application of tetracycline induction condition termination replication plasmid

Abstract

The invention discloses a construction method and application of a tetracycline induction condition termination replica plasmid. The plasmid comprises the following components: a repB replica gene, a TetR tetracycline transcriptional regulatory factor, a Pet promoter and a replicon gene element. According to the construction method, a chloramphenicol resistance gene cat, a replicating gene pUCmini and a gene segment PNDKR are seamlessly cloned and assembled to obtain plasmids, the method is suitable for multi-round, rapid and multi-site biological genetic engineering, and the next round of operation process is rapidly entered without multi-round culture and screening of plasmid loss strains; the method can be applied to rapid plasmid loss in the lactococcus lactis, and has important significance on future industrial operation of the lactococcus lactis.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and specifically relates to a method for constructing a tetracycline-induced conditional replication termination plasmid and its application. Technical Background

[0002] In the genetic engineering operation of the target strain, plasmids are often used as expression vectors to integrate and knock out the cell genome. However, since plasmids often carry resistance genes and other protein expression sequences, they interfere with the normal screening process of bacteria and the normal metabolism of cells. Therefore, it is necessary to try to eliminate the plasmid after completing the genetic operation to obtain a plasmid-free derivative strain for the next step. Usually, multi-copy plasmids, that is, relaxed replication plasmids, can be lost spontaneously from bacteria, but the probability of natural loss is very low, and the difference between plasmid-lost strains and non-lost strains is not much, making screening difficult. Currently reported physical and chemical methods for eliminating plasmids, such as electroporation, increasing temperature culture, and adding SDS detergent during culture, have mutagenic properties and can damage the bacterial genome; in molecular biology methods, plasmids can be eliminated by the principle of plasmid incompatibility or by using the CRISPR-Cas system, but the cost is high, the design is difficult, and the efficiency for multi-copy plasmids is low.

[0003] Lactococcus lactis is a non-spore, Gram-positive bacterium with a clear genetic background and a simple metabolic pathway. Because of its good biosafety, it is suitable for expressing and secreting foreign proteins and can be used as a live vector to make vaccines. It has important application prospects in the fields of food industry, biopharmaceuticals and vaccine research. In the genetic engineering of Lactococcus lactis, the temperature-sensitive gene integration vector pG + host, etc. to perform gene manipulation, and change the culture temperature to lose plasmids and screen plasmid-lost strains. In the currently reported genetic engineering of Lactococcus lactis, the temperature-sensitive plasmid pNZts-PnisA-I-sceI constructed by Hong Xia et al. in the patent "Lactococcus lactis with traceless gene integration and construction method" [Patent No.: CN2024110410932] had a loss rate of 12.2% in a single antibiotic-free culture after completing the integration of gene fragments, and the temperature-sensitive integrated plasmid constructed by Qiao Jianjun et al. in the patent "A method for efficient iterative integration of multiple genes in Lactococcus lactis" [Patent No.: CN202310140909.6] had a loss rate of 14%, and the Lactococcus lactis base editor plasmid constructed by Tian et al. [ACS Synthetic Biology 2022 11(11)] had a loss rate of 28% to 37%, which was a low loss efficiency, limiting rapid multi-round gene manipulation. Therefore, the development of a method for rapid elimination of plasmids is of great significance to the future industrial operation of Lactococcus lactis. Summary of the invention

[0004] In order to overcome the disadvantages of low plasmid loss rate of Lactococcus lactis in the prior art, which limits rapid multi-round gene manipulation, the main purpose of the present invention is to provide a method for constructing a tetracycline-induced conditional termination replication plasmid, which solves the problem of requiring multiple rounds of cultivation to screen for plasmid loss strains in the prior art.

[0005] A plasmid that terminates replication under tetracycline induction conditions comprises the following components: a repB replication gene, a TetR tetracycline transcriptional regulatory factor, a Ptet promoter, and a replicon gene element, and is named PNDKR-Empty-TetR-repB1. Its nucleotide sequence is shown in SEQ ID NO.1.

[0006] A method for constructing a tetracycline-induced conditional termination replication plasmid comprises the following steps: seamlessly cloning and assembling a chloramphenicol resistance gene cat, a replication gene pUCmini and a gene fragment PNDKR to obtain a plasmid PNDKR-Empty-TetR-repB1.

[0007] The nucleotide sequence of the artificially synthesized gene fragment PNDKR is shown in SEQ ID NO.2, comprising the repB replication gene, the TetR tetracycline transcriptional regulator gene, the Ptet promoter, and the replication gene element, and is obtained by PCR amplification using primers NdrepB-F and NdrepB20.

[0008] The chloramphenicol resistance gene cat is obtained by PCR amplification using plasmid pNZ5319 as a template and primers NdCm-F and NDcm-R.

[0009] The replication gene pUCmini is obtained by PCR amplification using plasmid pUC57 as a template and primers BSucmini-F and BSucmini-R.

[0010] Application of tetracycline-inducible conditional termination of replication plasmid construction method to rapid plasmid loss in Lactococcus lactis.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The invention provides a method for constructing a tetracycline-induced conditional termination replication plasmid, which has a high plasmid loss rate and a conditional elimination rate of 67.5%. The method is suitable for multi-round, rapid, and multi-site biological genetic engineering, and does not require multiple rounds of cultivation to screen plasmid-lost strains, and can quickly enter the next round of operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Schematic diagram of the construction of PNDKR-Empty-TetR-repB1 plasmid;

[0014] Figure 2 :The results of colony PCR electrophoresis, the DNA marker in the figure is DL5000, and the target band is 1712bp;

[0015] Figure 3 :Conversion experiment of Lactococcus lactis without / with anhydrotetracycline, A is the transformation result of GM17 medium without anhydrotetracycline, B is the transformation result of anhydrotetracycline added to the final concentration of 1 μg / mL;

[0016] Figure 4 : Results of a point experiment of plasmid loss after adding anhydrotetracycline. A is a culture medium without resistance, and B is a culture medium with chloramphenicol resistance (CM 10 μg / mL). Specific implementation plan

[0017] The present invention is further described below in conjunction with specific examples, which are merely exemplary and do not limit the scope of protection of the present invention. The Lactococcus lactis NZ9000 and Escherichia coli DH5α involved in each example can be purchased.

[0018] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0019] Example 1 Construction of tetracycline-induced termination plasmid

[0020] The artificial fragment of PNDKR (SEQ ID NO.2) was synthesized by the company, and its structure is as follows Figure 1 As shown, it contains the repB replication gene (SEQ ID NO.3), TetR tetracycline transcription regulator gene (SEQ ID NO.4), Ptet promoter (SEQ ID NO.5), and replicon (SEQ ID NO.6) gene elements, and uses primers NdrepB-F (SEQ ID NO.13) and NdrepB20 (SEQID NO.14) to perform PCR amplification to obtain the gene fragment PNDKR artificial synthetic fragment;

[0021] Using plasmid pNZ5319 as a template, primers NdCm-F (SEQ ID NO.9) and NDcm-R (SEQ ID NO.10) were used for PCR amplification to obtain the chloramphenicol resistance gene cat;

[0022] Using plasmid pUC57 as a template, PCR amplification was performed using primers BSucmini-F (SEQ ID NO.11) and BSucmini-R (SEQ ID NO.12) to obtain the replication gene pUCmini;

[0023] The gene cat, pUCmini, and PNDKR artificial synthetic fragments were seamlessly cloned and assembled using the New Cyme Seamless Cloning Kit, and the molar ratio of fragment addition was 1:1:1.

[0024] Transform the assembled product into commercial E.coli DH5α competent medium: Add 10 μL of the above assembly reaction solution to 50 μL of the pre-thawed commercial E.coli DH5α competent medium, place on ice for 30 minutes, heat shock in a 42°C water bath for 60 seconds, and then quickly transfer to an ice bath for 2 minutes. Add 500 μL of LB culture medium and culture in a shaking incubator at 220 r / min and 37°C for 45 minutes. Centrifuge and discard 400 μL of supernatant, mix the remaining bacterial solution and spread it on an LB solid plate containing chloramphenicol (final concentration of 25 μg / mL), and culture overnight at 37°C. Colony PCR verification was performed using primers CxAut-F (SEQ ID NO.7) and primer CxAutP-R (SEQ ID NO.8). The length of the correct clone band was 1712 bp ( Figure 2 ), the clone with the correct band was cultured with LB containing chloramphenicol resistance, the plasmid was extracted using Tiangen's plasmid extraction kit, and sent to the company for sequencing. The correct clone was named PNDKR-Empty-TetR-repB1 (SEQ ID NO.1).

[0025] Example 2 Conditional termination of replication expression plasmid transformation into target strain (taking L. lactic NZ9000 as an example)

[0026] 1. Preparation of Lactococcus lactis Electrotransformation Competent Cells

[0027] (1) Take the frozen L.lactic NZ9000 glycerol bacteria in the laboratory, make three-zone lines on the GM17 agar medium plate, and then place it in a 30℃ incubator for static culture. The next day, inoculate the grown single colony into GM17 broth, subculture and activate it for three generations, inoculate it into 50mL GM17 broth, and culture it at 30℃ for about 3-4h until the logarithmic growth phase (OD600≈0.5). Add ampicillin at a final concentration of 20μg / mL and continue to culture at 30℃ for 1h to weaken the cell wall. Use two 50mL centrifuge tubes to collect the bacterial precipitate.

[0028] (2) Resuspend the bacterial pellet from step (1) with 20 mL of 10% glycerol, centrifuge at 8000 rpm for 3 min, repeat this step 3 times, resuspend the bacterial pellet with 1 mL of 10% glycerol, dispense 50 μL into 1.5 mL EP tubes, and quickly store in a -80°C refrigerator for later use.

[0029] 2. Operation steps of electroporation of Lactococcus lactis

[0030] Take L.lactic NZ9000 electroporation competent cells stored at -80℃ and thaw on ice. Pipette 500ng plasmid xx and add it to 50μL L.lactic NZ9000 electroporation competent cells, mix it by gentle pipetting and transfer it to a pre-cooled 2mm electroporation cup. Set the voltage of the electroporator to 2.5kv, press the "pulse" button to start the electric shock, then quickly add 1mL of GM17 broth to revive, mix it and transfer it to a 1.5mL sterile EP tube, and incubate it at 30℃ for 2h. After centrifugation at 8000rpm for 2min, discard 900μL of supernatant, and spread the control group on a GM17 plate containing chloramphenicol (5μg / mL), and the experimental group on a GM17 plate containing chloramphenicol (5μg / mL) and dehydrated tetracycline ATCC (1μg / mL). Culture at 30℃.

[0031] 3. After chloramphenicol screening, the control group transformed and grew normally, and was stably inherited in the resistance culture; the experimental group added ATCC, which interfered with the replication of the plasmid vector and could not replicate normally, and no colonies grew on the chloramphenicol resistance plate ( Figure 3 ), indicating that the plasmid can be transformed into Lactococcus lactis and tetracycline has the function of interfering with plasmid replication.

[0032] Example 3 Conditional termination of replication Expression plasmid replication stability and conditional elimination:

[0033] 1. Conditional termination of replication and stability of expression plasmid replication: The transformants obtained in step 3 of Example 2 were cultured in GM17 medium without resistance, and were propagated for 10 generations using GM17 medium without resistance. They were then transferred to GM17 medium containing chloramphenicol (5 μg / mL) for culture. It was found that the transformants could grow normally in the medium containing resistance, indicating that the chloramphenicol resistance phenotype was stably inherited.

[0034] 2. Conditional termination of replication and conditional elimination of expression plasmid: The transformants obtained in step 3 of Example 2 were cultured in an antibiotic-free GM17 medium supplemented with ATCC (1 μg / mL) at 30°C, and then three-zoned on an antibiotic-free GM17 solid medium plate containing ATCC (1 μg / mL). Single colonies were randomly picked and inoculated simultaneously on ordinary antibiotic-free / chloramphenicol-resistant GM17 solid medium plates. A total of 77 single colonies were picked, of which 52 had plasmid loss, and the conditional elimination rate was as high as 67.5%.

Claims

1. A plasmid that terminates replication using tetracycline-induced conditions, characterized in that The invention comprises the following components: a repB replication gene, a TetR tetracycline transcriptional regulatory factor, a Ptet promoter, and a replication gene element, and is named PNDKR-Empty-TetR-repB1, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. A method for constructing a tetracycline-induced conditional termination replication plasmid, characterized in that: The steps include: 1) Artificially synthesize the PNDKR main fragment, PCR amplify the cat chloramphenicol resistance gene and pUCmini replicon; 2) Assemble the three gene fragments in step 1 by seamless cloning technology and chemically transform them into DH5α Escherichia coli competent cells; 3) Use primers to screen the correct clones, extract the plasmid and sequence it to obtain the tetracycline conditionally induced replication termination plasmid PNDKR-Empty-TetR-repB1.

3. The method for constructing a tetracycline-induced conditional termination replication plasmid according to claim 2, characterized in that: The nucleotide sequence of the artificially synthesized gene fragment PNDKR is shown in SEQ ID NO.2, comprising the repB replication gene, the TetR tetracycline transcriptional regulator gene, the Ptet promoter, and the replication gene element, and is obtained by PCR amplification using primers NdrepB-F and NdrepB20.

4. The method for constructing a tetracycline-induced conditional termination replication plasmid according to claim 2, characterized in that: The chloramphenicol resistance gene cat is obtained by PCR amplification using plasmid pNZ5319 as a template and primers NdCm-F and NDcm-R.

5. The method for constructing a tetracycline-induced conditional termination replication plasmid according to claim 2, characterized in that: The replication gene pUCmini is obtained by PCR amplification using plasmid pUC57 as a template and primers BSucmini-F and BSucmini-R.

6. Use of the tetracycline-inducible conditional termination replication plasmid of claim 1 for rapid loss in Lactococcus lactis.

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