PLS DNA polymerase constructed by DNA polymerase-DNA binding protein and application of PLS DNA polymerase in DNA information storage

Abstract

The invention discloses a PLS DNA polymerase constructed by DNA polymerase-DNA binding protein and application of the PLS DNA polymerase in DNA information storage, and belongs to the technical field of biochemistry and molecular biology. The fusion protein PLS DNA polymerase is constructed by connecting Sso7dDNA binding protein to the C end of 9-degree N DNA polymerase through flexible oligopeptide. The method comprises the following steps: chemically synthesizing a codon-optimized PLS DNA polymerase gene, and purifying to prepare the high-purity PLS DNA polymerase; the system evaluates the enzymatic property of the PLS DNA polymerase and the application effect of addressing in in-vitro DNA information storage and assembling DNA movable type blocks in in-vivo DNA information storage. The result shows that after the DNA binding protein is fused, the continuous synthesis capacity, fidelity, PCR efficiency and other performance of PLS DNA polymerase are remarkably improved, a specific file can be recovered from a mixed file pool, and random addressing is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry and molecular biology, and specifically relates to a PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein and its application in DNA information storage. Background Art

[0002] In recent years, the shift of industrial network demand to the cloud and the surge in smart devices connected to the Internet have driven the rapid growth of global digital data output. With the explosive growth of data, traditional storage devices, such as magnetic, optical, and solid-state devices, are approaching their physical limits and are unable to meet the requirements of digital storage. If ZB-level data is to be stored, a large amount of physical space is required. In addition, the limited preservation time of silicon-based data storage is also a huge challenge for existing storage devices. Therefore, alternative storage media are urgently needed to fill this growing gap. The huge demand for information storage in the field of big data has promoted the cross-integration of biotechnology and information technology, and derived a DNA-based information storage paradigm. As a natural carrier of genetic information, DNA has the characteristics of environmental friendliness, long-term preservation stability, and information density scalability, and is considered to be a potential alternative to solve the problems of traditional information storage methods.

[0003] In the process of DNA-based data storage, binary 0 / 1 data is first converted into a quaternary DNA sequence, and then the substrate containing chemically active protective groups is activated step by step through the phosphoramidite chemical synthesis method to achieve controllable synthesis of DNA. For data reading, a specific DNA sequence is selected and sequenced, and the measured DNA sequence information is converted back to the original data, thereby restoring the stored digital file. As a type of tool enzyme widely used in DNA replication in molecular biology, DNA polymerase plays an important role in the entire DNA data storage process: (1) Through the design of primers, DNA polymerase can amplify specific DNA sequences from a large number of DNA sequences, thereby achieving addressing and retrieval of specific files in mixed files; (2) As an important protein in nanopore sequencing, DNA polymerase controls the movement speed of the DNA chain through the nanopore, thereby improving the accuracy of the current change signal generated when different bases pass through the nanopore, which helps to improve the accuracy of nanopore sequencing; (3) DNA polymerase is used to extend the hybridized overlapping DNA fragments to achieve the assembly of long DNA fragments, overcoming the length limitation of chemical de novo synthesis of DNA.

[0004] Sso7d DNA-binding protein is a small molecule DNA-binding protein from extremely thermophilic archaea. It has the property of non-specific binding to double-stranded deoxyribonucleic acid (dsDNA) and plays a key role in genome stability in thermophilic archaea. Summary of the invention

[0005] The purpose of the present invention is to provide a PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein and its application in DNA information storage.

[0006] Through the protein engineering method, the present invention fuses the 9°N DNA polymerase isolated from marine thermophilic archaea with the Sso7d DNA binding protein to construct a 9°N-Sso7d fusion protein polymerase (named PLS DNA polymerase). The PLS DNA polymerase can be fixed on dsDNA like a sliding clamp to form a more stable fusion protein-primer-template complex, thereby assisting the DNA polymerase to quickly identify and extend DNA, and helping to enhance the continuous synthesis ability of the 9°N DNA polymerase. Therefore, the PLS DNA polymerase described in the present invention, as a key DNA polymerase molecule in the DNA information storage process, can participate in the DNA information storage data writing and addressing process, build a good platform for DNA information storage encoding and decoding, and promote the application of DNA polymerase in the fields of in vivo information storage and in vitro information storage.

[0007] The PLS DNA polymerase is composed of 9°N DNA polymerase, a flexible linker peptide and a Sso7d DNA binding protein. The N-terminus of the flexible linker peptide is connected to the C-terminus of the 9°N DNA polymerase domain, and the C-terminus of the flexible linker peptide is connected to the N-terminus of the Sso7d DNA binding protein domain; the flexible linker peptide is used to prevent the proteins on both sides from misfolding and causing impaired activity, and its amino acid sequence is shown in SEQ ID No. 2.

[0008] The PLS DNA polymerase gene was optimized for Escherichia coli preferred codons, and the codon-optimized PLS DNA polymerase gene was constructed into a prokaryotic expression vector. The plasmid containing the fusion protein gene was synthesized by chemical method, and then the plasmid transformation and expression strain construction were carried out. High-purity PLS DNA polymerase was prepared by two-step purification combined with nickel column affinity chromatography and cation exchange chromatography. Using DNA containing storage information as a template and after optimizing the amplification system, the addressing ability of the PLS DNA polymerase in DNA information storage and its effect in assembling short-fragment DNA were systematically evaluated.

[0009] The preparation method of the PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein of the present invention comprises the following steps:

[0010] (1) Construction of PLS ​​DNA polymerase engineered bacteria

[0011] The gene sequence encoding the PLS DNA polymerase is subjected to engineering bacteria codon optimization treatment, and the optimized nucleotide sequence is shown in SEQ ID No. 1, which is used as the gene encoding the PLS DNA polymerase;

[0012] A 6×His tag was added to the 5' end of the gene encoding PLS DNA polymerase, and the gene encoding PLS DNA polymerase with the 6×His tag was connected to the expression vector pET-30a through the NdeⅠ and HindⅢ restriction sites to construct a recombinant expression vector pET-30a / PLS;

[0013] The recombinant expression vector pET-30a / PLS was transferred into competent cells of Escherichia coli Trans5α, and then coated on kanamycin-resistant plates for screening. After overnight culture at 37°C, single clones were picked for enzyme digestion experiment verification. The recombinant expression vector pET-30a / PLS with the correct verification result in Escherichia coli Trans5α was extracted using a plasmid kit and transferred into protein expression competent cells of Escherichia coli BL21 (DE3), thereby constructing an engineering bacterium expressing the PLS DNA polymerase gene;

[0014] (2) Acquisition of PLS ​​DNA polymerase

[0015] The PLS DNA polymerase gene expression engineered bacteria were inoculated into LB medium at an inoculum of 1:80-120 (v / v) and cultured at 37°C until the optical density of the culture reached OD 600 Reach 0.6-0.8; add IPTG with a final concentration of 0.5mM and induce at 37°C for 4-8h; collect the bacteria by centrifugation at 5000-8000r / min for 20-30min, add 50mM phosphate buffer to the bacteria at a ratio of 1:8-15 (w / v), mix well and then ultrasonically disrupt for 20-30min; heat the obtained bacterial disrupted liquid in a water bath at 80-90°C for 20-30min, cool to room temperature, centrifuge at 7000-8000r / min for 15-20min, collect the supernatant and obtain the crude enzyme liquid;

[0016] The crude enzyme solution and the nickel column material are incubated at 4°C for 1 to 3 hours, and the impurity proteins are eluted with 10mM imidazole and the target protein is eluted with 75mM imidazole; the eluted target protein sample is centrifuged at 5000 to 7000 r / min for 15 to 25 minutes through a 15kDa ultrafiltration tube, and the centrifugation is repeated 2 to 4 times to remove the imidazole when eluting the target protein; then a cation exchange chromatography column is selected, and linear elution is performed with a buffer B solution containing 1M sodium chloride, and the eluted sample is collected, and the eluted sample is centrifuged at 5000 to 7000 r / min for 15 to 25 minutes through a 15kDa ultrafiltration tube, and the centrifugation is repeated 2 to 4 times, thereby obtaining PLS DNA polymerase, and storing it in a storage buffer.

[0017] Preparation system of LB medium (w / v): 1-2% yeast powder, 1-2% peptone, 0.5-2% sodium chloride, pH 7.0, fully dissolve the above reagents in 1000 mL of deionized water to obtain LB medium;

[0018] The buffer B solution preparation system is: 50-60 mM potassium phosphate buffer (pH 7.4), containing 1-1.5 M sodium chloride, 5-10% glycerol (v / v), and the above reagents are fully dissolved in 1000 mL of deionized water to obtain a buffer B solution;

[0019] The preparation system of the preservation buffer is: 5-15mM tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4), 50-120mM sodium chloride, 1-1.5mM dithiothreitol, 0.1-0.5mM ethylenediaminetetraacetic acid, 0.1-0.5% by mass of 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, and 20-50% by mass of glycerol (v / v). The above reagents are fully dissolved in 1000mL of deionized water to obtain a preservation buffer.

[0020] The protein purification method selected by the present invention can obtain high-purity PLS DNA polymerase, and the optimization of PCR reaction conditions can greatly improve the catalytic efficiency of PLS ​​DNA polymerase, reduce the generation of non-specific bands, improve the quality of amplified target products, and facilitate downstream purification and subsequent sequencing analysis. Compared with 9°N DNA polymerase, the thermal stability, fidelity, salt ion tolerance and continuous synthesis ability of PLS ​​DNA polymerase are all improved. Good thermal stability is a necessary condition for PLS DNA polymerase to be applied to PCR cycle reaction, so that PLS DNA polymerase can tolerate the high temperature of the cycle reaction conditions and maintain a high catalytic efficiency during the reaction process, which is conducive to the efficient amplification of target DNA. Fidelity gives PLS DNA polymerase good correction ability, which can timely remove the incorporated erroneous bases to achieve the repair of mismatched fragments, thereby improving the accuracy of synthesizing target DNA. The improvement of salt ion tolerance broadens the resistance of PLS ​​DNA polymerase to complex environments and has a broader application prospect. The improvement of continuous synthesis ability enables PLS DNA polymerase to amplify DNA faster and can continuously and stably amplify longer DNA fragments.

[0021] The PLS DNA polymerase of the present invention can improve the fidelity of DNA information storage. Using the oligonucleotide pool obtained by encoding and storing digital information with Reed-Solomon erasure code as a template, after optimizing the PCR reaction amplification system, the obtained sample is purified by a column DNA purification kit to obtain a high-purity DNA amplification product, and high-throughput sequencing analysis is performed. In addition, the PLS DNA polymerase can amplify a DNA sequence of a specified file encoding from a mixed DNA sample containing two different file encodings through the design of an orthogonal barcode, realize in vitro random addressing of multi-file digital information storage, and completely restore the stored digital information content. At the same time, it can also effectively reduce the number of errors generated during DNA replication, especially base substitution, which is an error type that mainly depends on the catalytic performance of the enzyme, reduce the probability of errors between purines and pyrimidines, and improve the accuracy of DNA information storage encoding and decoding.

[0022] The PLS DNA polymerase described in the present invention can reduce the cost of DNA information storage. Current DNA-based data storage generally involves de novo chemical synthesis of DNA. However, due to the limitations of synthesis technology, it is currently impossible to de novo synthesize DNA fragments exceeding 200nt. Therefore, the original data must be divided into many blocks and synthesized separately to overcome the limitation of the length of de novo synthesis by chemical methods. In contrast, enzyme-based methods allow DNA synthesis to have higher fidelity and longer length. Combining the advantages of high throughput of chemical synthesis and high synthesis length of enzymatic synthesis, short DNA blocks are first chemically synthesized, and then a cyclic assembly reaction based on DNA polymerase is used to achieve one-step splicing of multiple short DNA fragments to form longer DNA fragments, which can reduce the synthesis cost.

[0023] The nucleotide sequence of the gene encoding PLS DNA polymerase of the present invention is shown in SEQ ID No. 1. The encoded PLS DNA polymerase has good purity, yield and catalytic activity after purification, can reduce the cost of DNA information storage, and improve the quality and efficiency of information storage data decoding.

[0024] Unless otherwise specified, the solutions described in the present invention are all deionized aqueous solutions. The PCR reaction system buffer involved in the present invention is 10×ThermoPol Reaction Buffer, which can provide salt ions and metal ions for DNA polymerase catalytic reactions and is purchased from NEB, USA. The dNTP mixed solution involved in the present invention can provide a substrate for DNA polymerase to replicate DNA reactions, wherein the concentrations of the four dNTPs are all 4 μM and are purchased from NEB, USA. The oligonucleotide pool involved in the present invention is a mixture of oligonucleotides (DNA) containing a variety of different sequences obtained by encoding digital files through Reed-Solomon erasure codes, which is used to store data and is synthesized on a 12K chip by electrochemical methods and purchased from Nanjing GenScript. The restriction endonucleases, DNA loading buffers and protein loading buffers involved in the present invention are used to cut DNA and terminate reactions to prepare electrophoresis samples, respectively, and are purchased from Beijing Bao Ri Yi Biological Technology Co., Ltd. The ssm13mp18 DNA and λDNA involved in the present invention, as DNA templates for PCR reactions, are purchased from NEB, USA. The E. coli Trans5α and E. coli BL21 (DE3) involved in the present invention, as competent host bacteria carrying transformation plasmids, were purchased from Beijing Quanshijin Biotechnology Co., Ltd. The primers involved in the present invention were purchased from Nanjing GenScript Co., Ltd. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a denaturing polyacrylamide gel electrophoresis diagram of the recombinant expression vector obtained in Example 1 after enzyme digestion; the lanes from left to right represent: M: nucleic acid molecular weight standard; 1: untreated plasmid; 2: plasmid obtained after NdeⅠ digestion; 3: plasmid obtained after HindⅢ digestion; 4: plasmid obtained after NdeⅠ and HindⅢ digestion.

[0026] Figure 2 This is a denaturing polyacrylamide gel electrophoresis diagram of the optimization analysis of IPTG-induced expression time in Example 2; the lanes from left to right represent: M: protein molecular weight standard; 1: no induction; 2: induced for 1 h; 3: induced for 2 h; 4: induced for 4 h; 5: induced for 6 h; 6: induced for 8 h.

[0027] Figure 3 This is a denaturing polyacrylamide gel electrophoresis analysis of the PLS DNA polymerase purified by the method of Example 3; the lanes from left to right represent: M: protein molecular weight standard; 1: uninduced; 2: after induction; 3: nickel column affinity chromatography; 4: cation exchange column chromatography.

[0028] Figure 4This is an agarose gel electrophoresis analysis of the amplification results obtained under different PLS DNA polymerase activities by the method of Example 4; the lanes from left to right represent: M: nucleic acid molecular weight standard; 1: 0.08U; 2: 0.40U; 3: 0.80U; 4: 1.60U.

[0029] Figure 5 The agarose gel electrophoresis diagrams of the thermal stability evaluation of 9°N DNA polymerase and the DNA polymerase obtained by the method of Example 5; Figure A is 9°N DNA polymerase, and Figure B is PLS DNA polymerase. The lanes from left to right represent: M: nucleic acid molecular weight standard; M: 0min; 1: 5min; 2: 10min; 3: 20min; 4: 30min; 5: 40min; 6: 60min; 7: 90min; 8: 120min.

[0030] Figure 6 Agarose gel electrophoresis diagrams of the salt ion tolerance evaluation analysis of 9°N DNA polymerase and the DNA polymerase obtained by the method of Example 6; Figure A is 9°N DNA polymerase, and Figure B is PLS DNA polymerase. M: nucleic acid molecular weight standard; from left to right lanes represent KCl concentrations, 1: 0mM; 2: 10mM; 3: 20mM; 4: 30mM; 5: 40mM; 6: 50mM; 7: 60mM.

[0031] Figure 7 This is an agarose gel electrophoresis diagram of the PCR efficiency analysis obtained by the method of Example 8 based on 9°N DNA polymerase and PLS DNA polymerase (added with 20mM KCl); Figure A is 72°C 30s, and Figure B is 72°C 60s; M: nucleic acid molecular weight standard; from left to right, the lanes represent the expected lengths of the synthesized DNA, 1: 1kb; 2: 2kb; 3: 4kb; 4: 6kb; 5: 8kb.

[0032] Figure 8 This is an overview diagram of the in vitro DNA information storage addressing and in vivo DNA information storage assembly DNA fragments involved in Examples 9, 10 and 11.

[0033] Fig. 9 This is a bar graph of the error types and quantities of two DNA polymerase amplifications obtained by high-throughput sequencing analysis using the method of Example 10. The horizontal axis is the DNA polymerase group, including 9°N DNA polymerase and PLS DNA polymerase, and the vertical axis is the base type and quantity percentage obtained after high-throughput sequencing comparison.

[0034] Fig.10This is a bar graph analyzing the base substitutions amplified by two DNA polymerases obtained by the method of Example 10. The horizontal axis is the expected base type, the vertical axis is the replacement base type, and the value is the probability of each base being replaced.

[0035] Fig.11 The agarose gel electrophoresis diagram of assembling multiple DNA fragments at different PLS DNA polymerase concentrations obtained by the method of Example 11. The lanes from left to right represent the concentrations of PLS ​​DNA polymerase, respectively; M: Marker; 1: 1.0 pM; 2: 2.0 pM; 3: 3.0 pM; 4: 4.0 pM; 5: 5.0 pM; 6: 6.0 pM; 7: 7.0 pM; 8: 8.0 pM.

[0036] Fig.12 The bar graph is a frequency chart of three error types on different type blocks after alignment of the sequencing data obtained by the method of Example 11. DETAILED DESCRIPTION

[0037] The following examples are provided to further illustrate the present invention, so that those skilled in the art can more fully understand the present invention. However, the examples provided cannot be understood as limiting the scope of protection of the present invention, and thus non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention content should also fall within the scope of protection of the present invention.

[0038] Embodiment 1:

[0039] The specific process of constructing the PLS DNA polymerase engineering bacteria is as follows: First, the gene sequence encoding the PLS DNA polymerase is optimized by the online codon optimization tool (https: / / www.genscript.com.cn / gensmart-free-gene-codon-optimization.html), and the optimized nucleotide sequence is shown in SEQ ID No. 1. The nucleotide sequence shown in SEQ ID No. 1 is constructed on the expression vector pET-30a (a universal Escherichia coli expression vector) through the NdeⅠ and HindⅢ restriction sites to obtain the recombinant expression vector pET-30a / PLS, so that the PLS DNA polymerase can be efficiently expressed in Escherichia coli.

[0040] The start codon of the recombinant expression vector pET-30a / PLS is ATG, and the stop codon is TAA. First, a 6×His tag is added to the 5' end of the gene encoding PLS DNA polymerase for subsequent purification. The gene encoding PLS DNA polymerase with the added 6×His tag is connected to the expression vector pET-30a through the NdeⅠ and HindⅢ restriction sites to construct the recombinant expression vector pET-30a / PLS. Nanjing GenScript was commissioned to synthesize the recombinant expression vector according to the above nucleotide sequence. The recombinant expression vector was transferred into the competent cell Escherichia coli Trans5α, and then spread on a kanamycin-resistant plate for screening. After overnight culture at 37°C, a single clone was picked and an enzyme cutting experiment was performed to verify the results. Figure 1 As shown, Group 2 and Group 3 are the results obtained by NdeⅠ or HindⅢ restriction digestion, both of which are single bands, and the band size is lower than that of the recombinant expression vector pET-30a / PLS group in Group 1, proving that the two restriction site sequences of NdeⅠ and HindⅢ on the recombinant expression vector are correctly constructed, and can respectively identify the corresponding restriction sites on the recombinant expression vector and cut the recombinant expression vector; Group 4 is the result of adding NdeⅠ and HindⅢ restriction digestion at the same time, and the two bands are the expression vector pET-30a cut by two restriction endonucleases and the PLS DNA polymerase gene with a 6×His tag, which again proves that the two restriction site sequences of NdeⅠ and HindⅢ on the recombinant expression vector are correctly constructed. At the same time, Changchun Kumei Company was commissioned to sequence and analyze the sequence between the restriction sites NdeⅠ and HindⅢ in the recombinant expression vector pET-30a / PLS, and the sequencing results are completely consistent with the sequence described in SEQ ID No.1, proving the successful construction of the recombinant expression vector pET-30a / PLS. The recombinant expression vector pET-30a / PLS in Escherichia coli Trans5α was extracted using a plasmid kit and transferred into protein expression competent cells Escherichia coli BL21 (DE3) to construct PLS DNA polymerase gene expression engineering bacteria.

[0041] Embodiment 2:

[0042] The method for optimizing the expression conditions of the PLS DNA polymerase gene expression engineering bacteria is as follows: the PLS DNA polymerase gene expression engineering bacteria constructed in Example 1 are inoculated into LB medium at a ratio of 1:100 (v / v), and cultured at 37°C until the optical density OD of the culture reaches 600Reach 0.65. Then, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5mM, and induce culture at 37℃ for 0h, 1h, 2h, 4h, 6h, and 8h respectively. The obtained bacterial culture solution was centrifuged at 5000r / min for 20min to collect the bacteria, add commercial protein loading buffer, and heat in a constant temperature metal bath at 100℃ for 10min. After the sample is cooled, load it and perform denaturing polyacrylamide gel electrophoresis, then stain it with Coomassie Brilliant Blue staining solution, and observe the bands after decolorization with decolorizing solution. The results are as follows Figure 2 As shown in the figure, with the increase of induction time, the protein expression amount increases accordingly, and tends to be balanced at 6h, which is the optimal time for protein induction expression.

[0043] The preparation system of LB medium (w / v): 1.5% yeast powder, 1.5% peptone, 1% sodium chloride, pH 7.0, the above reagents were fully dissolved in 1000 mL of deionized water to obtain LB medium.

[0044] Embodiment 3:

[0045] In this example, the PLS DNA polymerase of the present invention was extracted and purified from the PLS DNA polymerase gene expression engineering bacteria by a two-step protein purification method of nickel column affinity chromatography and ion exchange chromatography. The specific steps are as follows: the PLS DNA polymerase gene expression engineering bacteria were inoculated into the LB medium of Example 2 at an inoculum of 1:100 (v / v), and cultured at 37°C until the optical density OD of the culture reached 600 Reach 0.65; add IPTG with a final concentration of 0.5mM and induce at 37℃ for 6h; collect the bacteria by centrifugation at 5000r / min for 20min, add 50mM phosphate buffer (pH 8.0) to the bacteria at a ratio of 1:10 (w / v), mix well and then ultrasonically disrupt for 20min; heat the obtained bacterial disrupted liquid in a water bath at 85℃ for 30min, cool to room temperature, centrifuge at 8000r / min for 15min, collect the supernatant and obtain the crude enzyme liquid; purify the protein by nickel column affinity chromatography, incubate the crude enzyme liquid with the nickel column material at 4℃ for 1h, elute the impurities with 10mM imidazole, and elute the target protein with 75mM imidazole; centrifuge the eluted target protein sample at 6000r / min for 20min through a 15kDa ultrafiltration tube, repeat the centrifugation three times to remove the imidazole when eluting the target protein; then perform cation exchange chromatography, use a buffer containing 1M sodium chloride Solution B was linearly eluted, and the eluted samples were collected and centrifuged at 6000r / min for 20min through a 15kDa ultrafiltration tube. The centrifugation was repeated 3 times, and the obtained PLS DNA polymerase was stored in a storage buffer.

[0046] The buffer B solution preparation system is: 50mM potassium phosphate buffer (pH 7.4), containing 1M sodium chloride, 5% glycerol (v / v), the above reagents are fully dissolved in 1000mL deionized water to obtain buffer B solution. The preparation system of the preservation buffer is 10mM tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4), containing 100mM sodium chloride, 1mM dithiothreitol, 0.1mM ethylenediaminetetraacetic acid, 0.1% 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol by mass fraction, 25% glycerol (v / v), the above reagents are fully dissolved in 1000mL deionized water to obtain the preservation buffer. The purified PLS DNA polymerase is stored in the preservation buffer, which can maintain the catalytic activity and stability of the DNA polymerase for a long time.

[0047] Take the bacterial precipitate without IPTG induction, the bacterial precipitate after 6 hours of induction with 0.5mM IPTG, the protein sample eluted with 75mM imidazole in nickel column affinity chromatography, and the protein sample collected by ion exchange chromatography, add commercial protein loading buffer, heat in a 100℃ metal bath for 10min, load the sample after cooling, perform denaturing polyacrylamide gel electrophoresis, and then stain with Coomassie Brilliant Blue staining solution, and observe the bands after decolorization with decolorizing solution. Figure 3 As shown, after two consecutive steps of protein purification by nickel column affinity chromatography and ion exchange chromatography, there was only a single band in the samples of the ion exchange chromatography group, and the size of the band was consistent with the size of the target protein PLS DNA polymerase, indicating that high-purity PLS DNA polymerase was successfully prepared.

[0048] Embodiment 4:

[0049] The method for optimizing the enzyme concentration of the PLS DNA polymerase amplification system is as follows: first prepare a PCR amplification reaction buffer system, including 1 μM unidirectional primer F1 (5'-GACACTCGTATGCAGTAGCC-3'), 300 ng template M1 (5'-ACAACCATTTATGTAGCATTTATGAAATTTTT AAATCAATTTACTATTGGCTACTGCATACGAGTGTC-3'), 2.5 mM dNTP, and 10×ThermoPol Reaction Buffer (NEB Company), and add the PLS DNA polymerase obtained in Example 3 to the amplification reaction buffer system according to a concentration gradient of 0.08 U, 0.40 U, ​​0.80 U, and 1.60 U, and add deionized water to the reaction system to 100 μL, and evenly distribute the system into PCR-specific tubes, 25 μL in each tube, and perform amplification reaction under a preset PCR cycle program. The PCR cycle program is: 95℃ 5min; 95℃ 30sec, 55℃ 30sec, 72℃ 30sec, 25 cycles; 72℃ 10min. After the amplification reaction is completed, add commercial DNA loading buffer to terminate the reaction, pipette to mix, load the sample on a 2% agarose gel for electrophoresis, the electrophoresis conditions are 120V, 25min, and then observe and analyze on a gel imager. The results are as follows Figure 4 As shown, when the activity of PLS ​​DNA polymerase is 0.8U, the PCR amplification reaction can obtain good template M1 gene amplification, with a single band and no contamination by nonspecific bands.

[0050] Embodiment 5:

[0051] The thermal stability evaluation method of PLS ​​DNA polymerase is as follows: Based on the optimized optimal PCR reaction system, 9°N DNA polymerase and PLS DNA polymerase are added to 1×ThermoPol Reaction Buffer diluted with deionized water, respectively, and samples are taken after incubation at 95°C for 0, 5, 10, 20, 30, 40, 60, 90 and 120 minutes, and then the above samples are further incubated at 72°C for 2 minutes to balance the temperature of each sample. After completing the reaction system as described in Example 4, PCR reaction is performed to evaluate the thermal stability of PLS ​​DNA polymerase. Figure 5 As shown, after incubation at 95°C for 120 min, the activity of PLS ​​DNA polymerase was significantly higher than that of 9°N DNA polymerase, indicating that the addition of DNA binding protein makes the fusion protein PLS DNA polymerase have better thermal stability and effectively improves the thermal tolerance of DNA polymerase when used in PCR reactions.

[0052] Embodiment 6:

[0053] The salt ion tolerance evaluation method of PLS ​​DNA polymerase is as follows: Based on the optimized optimal PCR reaction system, a PCR amplification reaction buffer system is first prepared, including 1 μM primer F1 (5'-TCATGCATTGCCTGCTCTGCC-3'), 1 μM primer R1 (5'-GCAATGGCGATGACGCATCCTCACG-3'), 300 ngλDNA template, 2.5 mM dNTP, 10×ThermoPolReaction Buffer, 0.8 U of PLS ​​DNA polymerase or 9°N DNA polymerase, and then KCl 0mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM is added to each tube of PCR system, and PCR reaction is performed with reference to the complementary reaction system described in Example 4 to evaluate the salt ion tolerance of PLS ​​DNA polymerase. The results are shown in FIG. Figure 6 As shown in the figure, with the increase of KCl salt ion concentration, the enzymatic activity of 9°N DNA polymerase was inhibited and the amplification band became darker, but PLS DNA polymerase still had good amplification ability, indicating that the salt ion tolerance of PLS ​​DNA polymerase was higher than that of 9°N DNA polymerase, which means that the addition of DNA binding protein significantly improved the salt tolerance of the fusion protein PLS DNA polymerase, and it is expected to be suitable for a wider range of application scenarios.

[0054] Embodiment 7:

[0055] The processivity evaluation method of PLS ​​DNA polymerase is as follows: Pre-anneal the fluorescent labeled primer M13-F (5'-FAM-GTTTTCCCAGTCACGACGTTGTAAAACGACGGCC-3') and ssm13mp18DNA, and then add 300μM dNTPs and DNA polymerase in sequence. Add different concentrations of DNA polymerase to different reaction tubes (DNA polymerase: template after primer annealing = 1:100-1:2000) to explore the conditions for different DNA polymerases to reach the processivity state (usually enzymes with high processivity require a higher ratio of DNA polymerase: template after primer annealing to reach the processivity state). The amplification reaction was carried out at 37°C in the dark. At the end of the reaction, 50mM EDTA was added to each reaction tube to terminate the reaction. The reaction products were commissioned to Sangon Biotech (Shanghai) Co., Ltd. for AFLP sequencing detection to obtain the length data of amplification products of different lengths and the corresponding fluorescence signal intensity of amplification products of each length. The average product amplification length of DNA polymerase was calculated according to the following formula:

[0056] log(nI / nT)=(n-1)logPI+log(1-PI)

[0057] The length of each signal peak above the background is recorded as nI, and the sum of the signal intensities of all products is nT. Plot log(nI / nT) against n-1, where n is the number of bases polymerized at the end of the primer. According to the above formula: the average primer amplification length is 1 / (1-PI), and PI represents the possibility that the polymerization does not stop at I. The results are statistically obtained in Table 1. The average primer amplification length of PLS ​​DNA polymerase is higher than that of 9°N DNA polymerase, that is, the fusion of DNA binding protein enhances the continuous synthesis ability of PLS ​​DNA polymerase.

[0058] Table 1: Evaluation of DNA polymerase processivity

[0059] Micro-Persistence Parameter (PI) Average primer extension length (nt) [1 / (1±PI)] 9°N 0.927±0.01 13.7±2.1 PLS 0.962±0.003 26.8±1.7

[0060] Embodiment 8:

[0061] The PCR efficiency evaluation method of PLS ​​DNA polymerase is as follows: DNA fragments of different lengths are amplified from the DNA template at the same time, and the reaction system is referred to Example 6. In Example 6, we found that the addition of the DNA binding protein domain improved the salt ion tolerance of PLS ​​DNA polymerase, and compared with 9°N polymerase, the salt ion concentration at which PLS DNA polymerase exerts the optimal catalytic efficiency is 20mM. Therefore, we performed PCR efficiency analysis under the condition of increasing salt ions to evaluate the amplification ability of PLS ​​DNA polymerase. The results are shown in Figure 7 As shown in the figure, in the absence of salt ion concentration, 9°N DNA polymerase can amplify a product length of 4kb, while PLS DNA polymerase can amplify a product length of 8kb under 20mM KCl concentration conditions, and its catalytic efficiency increases with the increase of extension time. The above results show that the addition of DNA binding protein can improve the catalytic efficiency of PLS ​​DNA polymerase in PCR applications, extending the same length of target DNA in a shorter time or amplifying longer DNA fragments in the same extension time.

[0062] Embodiment 9:

[0063] The information storage encoding process is as follows: The addressing application part of in vitro DNA information storage is as follows Figure 8 As shown. The "Jilin University logo image in jpg format" and the "Jilin University School of Life Sciences logo image in jpg format" are encoded by Reed-Solomon erasure code, and converted into nucleotide sequences (where Reed-Solomon erasure code is open source code, and its code and specific usage method can be downloaded from the link uploaded by the developer on Github, the download address is https: / / github.com / reinhardh / dna_rs_coding), and an oligonucleotide pool containing coding DNA sequence information is obtained.

[0064] An oligonucleotide pool is a general customized product that can be used in DNA information storage research. By synchronously synthesizing thousands of oligonucleotide sequences on a microarray chip at one time, and then cutting the synthesized oligonucleotides and dissolving them in a single small tube, an oligonucleotide pool is obtained. After the DNA polymerase amplifies the oligonucleotide pool, high-throughput sequencing analysis is performed on the amplified sample. Taking the homologous arm sequence as the clustering feature, the sequenced data is subjected to clustering analysis. The nucleotide sequence is converted back to a binary sequence through the decoding algorithm in the Reed-Solomon erasure code to restore the stored data information.

[0065] The encoding scheme for converting the above binary sequence into a DNA sequence uses the method of converting two bits into one base. The preset corresponding relationship for the conversion is: 00→A, 01→C, 10→G, 11→T. For example, the binary representation of the Chinese character "吉" is 11100101 10010000 1001001, and the converted nucleotide sequence is TGCC GCAAGAGC. The "Jilin University school emblem picture" and the "Jilin University College of Life Sciences school emblem picture" are respectively encoded to obtain two oligonucleotide pools containing the above files (the capacity of the encoded oligonucleotide pool is related to the size of the file to be encoded). They are physically mixed at the same concentration to obtain a mixed oligonucleotide pool containing both files. For the DNA sequences encoded for the two files, barcodes are designed at both ends respectively. The barcode sequences of the two files have orthogonality and file uniqueness to avoid sequence collisions between different files affecting subsequent addressing. The orthogonal barcode serves as the binding site for the subsequent PCR amplification primer and is used to address and amplify a specific file. The oligonucleotide pool added with the orthogonal barcode and the amplification primer sequence are both commissioned to Nanjing GenScript Corporation for chemical synthesis for subsequent experiments. The orthogonal barcode sequence and the primer sequences for amplifying the two files respectively are as follows.

[0066] Table 2: Orthogonal barcode sequence and amplification primer sequence

[0067]

[0068] Example 10:

[0069] The PLS DNA polymerase amplifies the mixed oligonucleotide pool to randomly address and decode the school emblem picture. The process is as follows: Prepare the PCR amplification system as shown in Table 3. The oligonucleotide pool is replicated and amplified by the PLS DNA polymerase. The PCR cycling program is: 95°C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, cycle 25 times; 72°C for 10 min.

[0070] Table 3: PCR amplification system

[0071] 10×ThermoPol Reaction Buffer 10μL dNTP mixture 4 μM 2μL School badge F 100μM 2μL Institute logo R 100μM 2μL Oligonucleotide pool (10 ng / μL) 5μL PLS DNA polymerase 0.8U Deionized water Make up to 100 μL

[0072] The amplified product obtained by PCR cycle replication was purified by DNA purification kit to remove impurities such as salt ions and proteins until the optical density OD 260 / OD 280 The ratio was 1.8 to 2.0, and the concentration was ≥25 ng / μL for high-throughput sequencing analysis.

[0073] The barcode sequence is used as the clustering feature to perform cluster analysis on the sequencing results. The processed data is decoded by the decoding algorithm in the Reed-Solomon erasure code (the decoding steps refer to the instruction manual provided by the code developer, as described in Example 9), and the "Jilin University logo image" stored in the DNA can be restored. In addition, SNP calling analysis is performed on the sequencing data, and the DNA sequence obtained by sequencing is compared with the coding DNA sequence to analyze the type and number of errors generated. The comparison results are shown in Figure 1. Fig. 9 As shown, the proportion of correct bases after amplification by 9°N DNA polymerase was 98.63%, and the proportion of incorrect bases was 1.37%. Among them, the proportions of the three common error types, insertion, deletion, and base substitution, were 0.70%, 0.51%, and 0.22%, respectively. The proportion of correct bases after amplification by PLS DNA polymerase was 98.79%, and the proportions of the three common error types, insertion, deletion, and base substitution, were 0.60%, 0.44%, and 0.15%, respectively. The two errors of insertion and deletion can be further reduced by improving and perfecting the error correction algorithm, but the error type of base substitution is mainly introduced by the PCR process in DNA information storage. Therefore, DNA polymerases with high fidelity are the key strategy to reduce base substitution errors in DNA information storage. Compared with 9°N DNA polymerase, PLS DNA polymerase can effectively reduce base substitution, an error type that mainly depends on the fidelity of DNA polymerase, which proves the advantage of PLS ​​DNA polymerase in amplifying oligonucleotide pools and can reduce the occurrence of errors during replication, which is crucial to further improve the robustness of information storage technology.

[0074] The coding DNA sequence is the expected sequence, and the sequenced DNA sequence is the actual sequence. The two sequences are compared one by one to further analyze the base substitution errors. The horizontal axis is the expected base type, the vertical axis is the replacement base type, and the value is the probability of each base substitution. The results are as follows Fig.10 As shown, compared with 9°N DNA polymerase, PLS DNA polymerase can better reduce the number of errors of various types of bases, especially the probability of errors between purine and pyrimidine, thereby improving the accuracy of information storage.

[0075] Embodiment 11:

[0076] Application of assembled DNA fragments in in vivo DNA information storage Figure 8 As shown. PLS DNA polymerase assembles DNA type blocks to obtain DNA type units, and then continues to assemble DNA type units to obtain longer DNA fragments. The ancient poem "Looking at the Waterfall at Mount Lu" is selected as the file to be stored for type storage. Each type unit represents a complete word / character, including index type blocks, check type blocks and content type blocks. The whole poem has 44 Chinese characters, punctuation marks and line breaks, and the final encoding obtains 44 type units. The index type block is used to indicate the position of the type in the entire file; the check type block is used for error correction, which can tolerate a small number of errors and ensure the normal decoding of the type; the content type block is the DNA sequence obtained by encoding the type. Based on the principle of DNA polymerase cyclic assembly reaction, the concentration gradient of PLS ​​DNA polymerase was optimized, and the three movable type blocks of "index movable type, verification movable type and content movable type" were used as primers and templates, and the concentrations of PLS ​​DNA polymerase added were 1.0pM, 2.0pM, 3.0pM, 4.0pM, 5.0pM, 6.0pM, 7.0pM, and 8.0pM, respectively, and PCR reaction was carried out according to the reaction conditions of Example 10. The results are shown in FIG. Fig.11 As shown, after optimization according to the above enzyme concentration gradient, 8.0pM PLS DNA polymerase concentration can realize one-step assembly of index movable type, verification movable type and address movable type, obtain complete movable type units, and preserve them on plasmids and store them in bacteria through homologous recombination, realizing in vivo movable type array DNA information storage. The assembled fragments were sequenced, and the results are shown in Table 4. After comparison of the sequencing results, the proportion of correct bases was as high as 99.9079%, and the wrong bases accounted for only about 0.092%, of which insertion errors were about 0.0039%, deletion errors were 0.0764%, and substitution errors were about 0.0117%. The position where the error occurred corresponds to the position of the movable type block. The results are shown in Table 4. Fig.12 , mostly occurs in the index type and proof type positions, and has little impact on the content type.

[0077] Table 4: Sequencing results of active type assembly

[0078]

[0079] This embodiment can connect 44 movable type blocks and assemble a short DNA fragment of 50 bp into a long DNA fragment of 7 kb. Since the length of chemically synthesized DNA fragments is less than 200 bp, and the cost increases significantly with the increase of length, the technology adopted by this patent uses short DNA fragments as raw materials and uses PLS DNA polymerase for enzymatic assembly, without the need for chemical de novo synthesis of long DNA fragments, thus effectively reducing the cost of DNA information storage.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may be subject to various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein, characterized in that: The PLSDNA polymerase consists of a 9°N DNA polymerase, a flexible connecting short peptide and a Sso7d DNA binding protein, wherein the N-terminus of the flexible connecting short peptide is connected to the C-terminus of the 9°N DNA polymerase domain, and the C-terminus of the flexible connecting short peptide is connected to the N-terminus of the Sso7d DNA binding protein domain; the nucleotide sequence encoding the PLSDNA polymerase gene is shown in SEQ ID No.

1.

2. The PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein as claimed in claim 1, characterized in that: The amino acid sequence of the flexible linker peptide is shown in SEQ ID No.

2.

3. A method for preparing a PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein according to claim 1 or 2, comprising the following steps: (1) Construction of PLS ​​DNA polymerase engineered bacteria The gene sequence encoding PLSDNA polymerase is subjected to engineering bacteria codon optimization treatment, and the optimized nucleotide sequence is shown in SEQ ID No.1, which is used as the gene encoding PLSDNA polymerase; A 6×His tag was added to the 5' end of the gene encoding PLS DNA polymerase, and the gene encoding PLS DNA polymerase with the 6×His tag was connected to the expression vector pET-30a through the NdeⅠ and HindⅢ restriction sites to construct the recombinant expression vector pET-30a / PLS; The recombinant expression vector pET-30a / PLS was transferred into competent cells of Escherichia coli Trans5α, and then coated onto kanamycin-resistant plates for screening. After overnight culture at 37°C, single clones were picked for enzyme digestion experiment verification. The recombinant expression vector pET-30a / PLS with the correct verification result in Escherichia coli Trans5α was extracted using a plasmid kit and transferred into protein expression competent cells of Escherichia coli BL21 (DE3), thereby constructing an engineering bacterium expressing the PLSDNA polymerase gene. (2) Acquisition of PLS ​​DNA polymerase The PLS DNA polymerase gene expression engineering bacteria were inoculated into LB medium at an inoculum of 1:80-120 (v / v) and cultured at 37°C until the optical density of the culture reached OD 600 Reach 0.6-0.8; add IPTG with a final concentration of 0.5mM and induce at 37°C for 4-8h; collect the bacteria by centrifugation at 5000-8000r / min for 20-30min, add 50mM phosphate buffer to the bacteria at a ratio of 1:8-15 (w / v), mix well and then ultrasonically disrupt for 20-30min; heat the obtained bacterial disrupted liquid in a water bath at 80-90°C for 20-30min, cool to room temperature, centrifuge at 7000-8000r / min for 15-20min, collect the supernatant and obtain the crude enzyme liquid; The crude enzyme solution and the nickel column material are incubated at 4°C for 1 to 3 hours, and the impurity proteins are eluted with 10mM imidazole and the target protein is eluted with 75mM imidazole; the eluted target protein sample is centrifuged at 5000 to 7000 r / min for 15 to 25 minutes through a 15kDa ultrafiltration tube, and the centrifugation is repeated 2 to 4 times to remove the imidazole when eluting the target protein; then a cation exchange chromatography column is selected, and linear elution is performed with a buffer B solution containing 1M sodium chloride, and the eluted sample is collected, and the eluted sample is centrifuged at 5000 to 7000 r / min for 15 to 25 minutes through a 15kDa ultrafiltration tube, and the centrifugation is repeated 2 to 4 times, thereby obtaining PLSDNA polymerase, which is stored in a storage buffer.

4. The method for preparing a PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein as claimed in claim 3, characterized in that: The preparation system of LB medium (w / v): 1-2% yeast powder, 1-2% peptone, 0.5-2% sodium chloride, pH 7.0, the above reagents are fully dissolved in 1000 mL deionized water to obtain LB medium; the preparation system of buffer B solution is: 50-60 mM potassium phosphate buffer (pH 7.4), containing 1-1.5 M sodium chloride, 5-10% glycerol (v / v), the above reagents are fully dissolved in 1000 mL deionized water to obtain buffer Solution B; the preparation system of the storage buffer is: 5-15mM tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4), 50-120mM sodium chloride, 1-1.5mM dithiothreitol, 0.1-0.5mM ethylenediaminetetraacetic acid, 0.1-0.5% by mass of 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, and 20-50% by mass of glycerol (v / v). The above reagents are fully dissolved in 1000mL of deionized water to obtain a storage buffer.

5. Application of the PLS DNA polymerase constructed by a DNA polymerase-DNA binding protein as described in claim 1 or 2 in in vitro DNA information storage and in vivo DNA information storage.

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