Plasmid with high DNA conversion efficiency and construction method

Abstract

The invention discloses a plasmid with high DNA conversion efficiency and a construction method, and the construction method comprises the following steps: (1) carrying out whole genome three-generation methylation sequencing on lactococcus lactis subsp. Lactis YF11, and analyzing the base modification condition of DNA to obtain a restriction modification system recognition site; (2) analyzing the recognition sites of the restricted modification system on the plasmid pLEB124, and destroying the recognition sites of the restricted modification system on the plasmid pLEB124 by designing primers to avoid the restricted modification system of lactococcus lactis; the method comprises the following steps: by taking a plasmid pLEB124 as a template, respectively carrying out PCR (Polymerase Chain Reaction) to obtain fragments MUT1, MUT2 and MUT3; carrying out seamless cloning assembly on the fragments, transferring the fragments into escherichia coli competent cells, and culturing and selecting positive clones; according to the present invention, the plasmid construction method is simple, the practicability is strong, and the DNA conversion efficiency is significantly improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a plasmid with high DNA conversion efficiency and a construction method. Background Art

[0002] Lactococcus lactis is a non-spore, Gram-positive bacterium with a clear genetic background, a single metabolic pathway, a short growth cycle, and mature genetic manipulation technology. It is widely found in dairy and plant products, is non-pathogenic to humans and animals, and is a recognized safe food-grade microorganism. In recent years, it has become the most ideal cell factory for producing food-grade metabolites. However, little information is known about the genome of industrial non-model Lactococcus lactis, genetic tools are scarce, and long-term mutagenesis makes it difficult to transform exogenous DNA. However, many non-model strains naturally have excellent characteristics required for ideal cell factories, such as tolerance to extreme environments, high yield of target products, stable performance, and strong robustness. With the advancement of synthetic biology technology, many non-model strains will develop into new chassis cells.

[0003] The restriction-modification (RM) system is a common immune system in bacteria and archaea, which can be used to resist attacks from external viruses and bacteriophages and protect host cells from infection by foreign DNA. The bacterial RM system is mainly composed of restriction endonucleases (REases) and DNA methyltransferases (MTases). MTases can recognize bacterial DNA and methylate specific motifs to protect their genomes. REases are highly sensitive to the methylation state of DNA target sequences and degrade unmethylated or inappropriately methylated foreign DNA. Through this mechanism, bacteria effectively distinguish between "self" and "non-self" DNA and limit the invasion of "non-self" DNA.

[0004] At present, the most commonly used genetic transformation method for Lactococcus lactis is electrotransformation. Many studies have optimized the transformation conditions to improve the efficiency of plasmid transformation, including adding glycine to weaken the cell wall, growth OD600, plasmid transfer amount, electric field strength, recovery time, etc. The restriction modification system is the main barrier for exogenous DNA to enter bacteria and achieve stable inheritance. Some studies have weakened the defense system of the strain by knocking out the restriction endonuclease of the strain itself, thereby improving the transformation efficiency of the exogenous vector. It is reported that among all bacteria that have completed genome sequencing, 95% of the strains contain RM systems, and 33% of the strains contain more than four RM systems. The presence of multiple RM systems makes the genetic manipulation of bacteria more difficult. As far as industrial strains are concerned, it is very difficult to perform multi-gene knockout due to the lack of genetic transformation operation tools. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a plasmid with high DNA conversion efficiency.

[0006] The second object of the present invention is to provide a method for constructing a plasmid with high DNA transformation efficiency.

[0007] The technical solution of the present invention is summarized as follows:

[0008] A method for constructing a plasmid with high DNA transformation efficiency comprises the following steps:

[0009] (1) Performing whole-genome third-generation methylation sequencing on Lactococcus lactis subsp. lactis YF11, with a deposit number of CGMCCNO.12429, to analyze the base modification of DNA and obtain the recognition sites of the restriction modification system;

[0010] (2) analyzing the restriction modification system recognition sites on the plasmid pLEB124, and destroying the restriction modification system recognition sites on the plasmid pLEB124 by designing primers to circumvent the restriction modification system of Lactococcus lactis subsp. lactis YF11;

[0011] The steps of circumventing the restriction modification system of Lactococcus lactis subsp. lactis YF11 are:

[0012] Using plasmid pLEB124 as a template, primers NoRM-F and NoRM-R were used to perform PCR amplification to obtain fragment MUT1, using plasmid pLEB124 as a template, primers XL124-R and NoRM-F1 were used to perform PCR amplification to obtain fragment MUT2, using plasmid pLEB124 as a template, primers NoRM-R1 and XL124-F were used to perform PCR amplification to obtain fragment MUT3;

[0013] The nucleotide sequence of NoRM-F is shown in SEQ ID NO.3;

[0014] The nucleotide sequence of NoRM-R is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of XL124-R is shown in SEQ ID NO.5;

[0016] The nucleotide sequence of NoRM-F1 is shown in SEQ ID NO.6;

[0017] The nucleotide sequence of NoRM-R1 is shown in SEQ ID NO.7;

[0018] The nucleotide sequence of XL124-F is shown in SEQ ID NO.1;

[0019] The fragments MUT1, MUT2 and MUT3 were seamlessly cloned and assembled, and then transferred into Escherichia coli TG1 competent cells, and then spread on LB plates containing 180 μg / mL erythromycin resistance and cultured to select positive clones; plasmid extraction was performed using a plasmid mini-extraction kit to obtain a plasmid with high DNA conversion efficiency, the plasmid with high DNA conversion efficiency is abbreviated as pLEB124-MUT, and the nucleotide sequence of the plasmid pLEB124-MUT is shown in SEQ ID NO.2.

[0020] The plasmid with high DNA transformation efficiency constructed by the above construction method.

[0021] Advantages of the invention

[0022] The plasmid construction method of the invention is simple, has strong feasibility, and significantly improves DNA conversion efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram for the construction of plasmid pLEB124-MUT.

[0024] Figure 2 Comparison of electroporation efficiency of pLEB124 and pLEB124-MUT: Figure 2 A is the complete coating of pLEB124 after incubation. Figure 2 B is pLEB124-MUT full coating, Figure 2 C is pLEB124-MUT1 / 10. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific embodiments. These embodiments are merely exemplary and do not limit the protection scope of the present invention.

[0026] The Lactococcus lactis subsp. lactis YF11 used in the present invention has a deposit number of CGMCC No. 12429 and is deposited in the General Microbiology Center of China Culture Collection Administration, the address of which is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, and the deposit date is May 10, 2016.

[0027] Lactococcus lactis subsp. lactis YF11 has a deposit number of CGMCC No. 12429, and is disclosed in Patent No. 201811439305.7, a recombinant Lactococcus lactis that produces high levels of nisin and a construction method.

[0028] Example 1 A method for constructing a plasmid with high DNA conversion efficiency comprises the following steps:

[0029] (1) Commissioning BGI to perform whole-genome third-generation methylation sequencing on Lactococcus lactis subsp. lactis YF11 (CGMCC NO.12429, hereinafter referred to as YF11), analyze the base modification of DNA, and obtain the restriction modification system recognition site (see Table 1); the restriction modification is referred to as RM;

[0030] Table 1

[0031]

[0032]

[0033] (2) Analysis of the restriction modification system recognition sites on plasmid pLEB124 (commercial product) with a total of 4 sites (see Figure 1 ), by designing primers to destroy the restriction modification system recognition site on plasmid pLEB124 (commercial product), circumventing the restriction modification system of YF11;

[0034] The steps to circumvent the restriction modification system of Lactococcus lactis subsp. lactis YF11 are:

[0035] Using plasmid pLEB124 as a template, primers NoRM-F (SEQ ID NO.3) and NoRM-R (SEQ ID NO.4) were used to perform PCR amplification to obtain fragment MUT1, using plasmid pLEB124 as a template, primers XL124-R (SEQ ID NO.5) and NoRM-F1 (SEQ ID NO.6) were used to perform PCR amplification to obtain fragment MUT2, using plasmid pLEB124 as a template, primers NoRM-R1 (SEQ ID NO.7) and XL124-F (SEQ ID NO.1) were used to perform PCR amplification to obtain fragment MUT3;

[0036] The fragments MUT1, MUT2 and MUT3 were seamlessly cloned and assembled using the New Cyme seamless cloning kit and then transferred into Escherichia coli TG1 competent cells. After being spread on an LB plate containing 180 μg / mL erythromycin resistance, the positive clones were selected by culturing at 37°C overnight. The plasmid was extracted using Tiangen's plasmid extraction kit to obtain a plasmid with high DNA conversion efficiency. The plasmid with high DNA conversion efficiency is abbreviated as pLEB124-MUT, and the nucleotide sequence of the plasmid pLEB124-MUT is shown in SEQ ID NO.2.

[0037] (3) Preparation of YF11 electroporation competent cells

[0038] 1) Take the YF11 glycerol bacteria stored in the laboratory, make three-zone lines on the GM17 agar medium plate, and then place it in a 30℃ incubator for static culture.

[0039] 2) Inoculate the grown single colony into GM17 broth medium, activate it for three generations, inoculate it into 50 mL SMGG medium (M17 broth + 0.5 M sucrose + 0.5% glucose + 1% glycine) at a 2% inoculum, and culture it at 30°C for about 5-6 hours until the logarithmic growth phase (OD600≈0.5).

[0040] 3) Use two 50 mL centrifuge tubes to collect the bacterial solution and balance it. Centrifuge at 8000 rpm for 3 min in a precooled 4°C high-speed centrifuge to collect the bacterial cells and discard the supernatant.

[0041] 4) Add 20 mL of washing solution (water + 10% glycerol + 0.5 M sucrose) to the precipitate and resuspend it. Centrifuge at 8000 rpm for 3 min to collect the cells, discard the supernatant, and repeat this step 3 times.

[0042] 5) Finally, add 1 mL of washing solution and mix by pipetting slowly. Dispense the resuspended bacterial solution into 1.5 mL EP tubes at 50 μL per tube to obtain YF11 electroporation competent cells. Quickly store them in a -80°C refrigerator for later use.

[0043] (4) Plasmid transfer into YF11

[0044] Take the YF11 electroporation competent cells obtained in step (3) stored at -80°C and thaw on ice. Take plasmids pLEB124 and pLEB124-MUT, respectively, and pipette 500ng of plasmids into 50μL YF11 electroporation competent cells. Gently pipette and mix well, and then transfer to a pre-cooled electroporation cup. Set the electroporator voltage to 13kV / cm in advance, and press the "pulse" button to start the electric shock. Then add 1mL of electroporation recovery solution SMGMC (M17 broth + 0.5M sucrose + 0.5% glucose + 1mM MgCl2 + 0.1mM CaCl2), mix well, and transfer to a 1.5mL sterile EP tube, place it in a 30°C incubator for 2h, centrifuge at 8000rpm for 2min, and keep about 100μL of supernatant for resuspending the bacterial precipitate. The precipitate was mixed evenly and spread on a GM17 agar medium plate containing erythromycin resistance (5 μg / mL). After overnight culture in a 30°C incubator, the colonies were counted.

[0045] (5) pLEB124-MUT destroyed the restriction modification recognition site of pLEB124, and the final total length was 3563 bp, which was 1042 bp shorter than the original pLEB124. Figure 2 Comparison of electroporation efficiency of pLEB124 and pLEB124-MUT: Figure 2 A is the complete coating of pLEB124 after incubation. Figure 2 B is the full coating of pLEB124-MUT after incubation. Figure 2 C is 1 / 10 of pLEB124-MUT. Figure 2 It can be seen that the transformation efficiency of pLEB124 is about 70 cfu / μg DNA, while the transformation efficiency of pLEB124-MUT is as high as 1.4×10 4 cfu / μgDNA, which is 200 times higher than the original pLEB124.

Claims

1. A method for constructing a plasmid with high DNA conversion efficiency, characterized in that: The steps include: (1) Performing whole-genome third-generation methylation sequencing on Lactococcus lactis subsp. lactis YF11, with a deposit number of CGMCCNO.12429, to analyze the base modification of DNA and obtain the recognition sites of the restriction modification system; (2) Analyze the restriction modification system recognition sites on the plasmid pLEB124, and design primers to destroy the restriction modification system recognition sites on the plasmid pLEB124 to circumvent the restriction modification system of Lactococcus lactis subsp. lactis YF11; The steps of circumventing the restriction modification system of Lactococcus lactis subsp. lactis YF11 are: Using plasmid pLEB124 as a template, primers NoRM-F and NoRM-R were used to perform PCR amplification to obtain fragment MUT1, using plasmid pLEB124 as a template, primers XL124-R and NoRM-F1 were used to perform PCR amplification to obtain fragment MUT2, using plasmid pLEB124 as a template, primers NoRM-R1 and XL124-F were used to perform PCR amplification to obtain fragment MUT3; The nucleotide sequence of NoRM-F is shown in SEQ ID NO.3; The nucleotide sequence of NoRM-R is shown in SEQ ID NO.4; The nucleotide sequence of XL124-R is shown in SEQ ID NO.5; The nucleotide sequence of NoRM-F1 is shown in SEQ ID NO.6; The nucleotide sequence of NoRM-R1 is shown in SEQ ID NO.7; The nucleotide sequence of XL124-F is shown in SEQ ID NO.1; The fragments MUT1, MUT2 and MUT3 were seamlessly cloned and assembled, and then transferred into Escherichia coli TG1 competent cells, and then spread on LB plates containing erythromycin resistance and cultured to select positive clones; plasmid extraction was performed using a plasmid mini-extraction kit to obtain a plasmid with high DNA conversion efficiency. The plasmid with high DNA conversion efficiency is abbreviated as pLEB124-MUT, and the nucleotide sequence of the plasmid pLEB124-MUT is shown in SEQ ID NO.

2.

2. A plasmid with high DNA transformation efficiency constructed by the construction method of claim 1.

Images