CN117535265A - Recombinant 9 DEG N DNA polymerase and application thereof in DNA information storage - Google Patents
Recombinant 9 DEG N DNA polymerase and application thereof in DNA information storage Download PDFInfo
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- CN117535265A CN117535265A CN202311522803.9A CN202311522803A CN117535265A CN 117535265 A CN117535265 A CN 117535265A CN 202311522803 A CN202311522803 A CN 202311522803A CN 117535265 A CN117535265 A CN 117535265A
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- dna polymerase
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- information storage
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Classifications
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- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12Y—ENZYMES
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- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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Abstract
A recombinant 9 DEG N DNA polymerase and an application thereof in DNA information storage belong to the technical fields of biochemistry and molecular biology. The invention prepares high-purity recombinant polymerase through chemical synthesis of recombinant polymerase genes optimized by escherichia coli preferential codons, then constructing the recombinant polymerase genes on a prokaryotic expression vector, carrying out protein expression strain transformation and resistance screening, and combining nickel column affinity chromatography and cation exchange chromatography for two-step purification; and then, the oligonucleotide containing stored information is used as a template, an amplification system is optimized, and the enzymatic properties of the recombinant polymerase and the application effect of the recombinant polymerase in DNA information storage are systematically evaluated. The result shows that the recombinant polymerase can realize complete decoding of stored information, effectively reduce the number of errors, especially base substitution type errors, reduce the occurrence probability of errors between purine and pyrimidine, improve the precision and efficiency of information storage encoding and decoding, reduce the cost of information storage, and have wide application prospects in DNA information storage.
Description
Technical Field
The invention belongs to the technical fields of biochemistry and molecular biology, and particularly relates to a recombinant 9 DEG N DNA polymerase and application thereof in DNA information storage.
Background
With the explosive growth of mass data in the information age, international data companies predict that 2025-year global internet data volume will increase to 163ZB each year, and existing storage media using silicon resources as materials cannot support the future exponentially growing demands of data; DNA is a powerful alternative to existing data carriers as a storage medium for natural genetic information. The DNA information storage technology has the advantages of good long-term storage stability, high information storage density, low energy consumption, capability of bearing extreme environments and the like, and has great potential and subversion advantages compared with the traditional storage medium. However, the current DNA information storage technology is still in the initial stage of research, and the problems of unbalanced sequence, base loss, base mutation, high cost and the like exist in DNA synthesis, amplification and sequencing, so that the future application of the technology is restricted.
In order to achieve more efficient DNA information storage, there is a need to develop a class of high performance tool enzymes that overcomes the above limitations, reduces errors in information storage, improves accuracy of information storage data, and reduces cost of information storage. The current information storage coding workflow mainly depends on in vitro synthesis of single-stranded DNA, but the high throughput sequencing technology for information decoding requires DNA polymerase to copy the single-stranded DNA into double-stranded DNA to read data, and has higher requirements on the concentration and purity of the DNA to be detected.
DNA polymerase is a tool enzyme widely applied to DNA replication, plays a vital role in nucleic acid synthesis and intracellular genetic information transmission, can realize exponential amplification of the quantity of target DNA through Polymerase Chain Reaction (PCR), and rapidly improves the concentration of the target DNA, and has become a research hotspot in the field of molecular biology and plays a vital role in the field of modern bioengineering.
9 DEG N DNA polymerase is a thermostable enzyme isolated from marine thermophilic archaea, the nucleotide sequence of the coding region of the enzyme gene is 2325 bases, 775 amino acids are coded, the molecular weight is about 89kDa, and the enzyme has 5'-3' polymerase activity and 3'-5' exonuclease activity, so that the incorporated wrong bases can be removed in the DNA polymerization process, and the enzyme has higher fidelity. In DNA information storage, DNA polymerases with high fidelity have a number of advantages: (1) The error occurrence rate in the synthesis process can be reduced, and the phenomenon that stored information cannot be decoded due to non-specific amplification of a stored information sequence and accumulation of the number of error bases is avoided; (2) Under the same data volume, the DNA polymerase with higher fidelity can improve the accuracy of data reading and reduce the cost of information storage; (3) The error rate in the information storage flow can be reduced, so that a large amount of physical redundancy is avoided from being required for storing data to avoid information loss in the storage process, the pressure of an encoding algorithm is reduced, and the density of information storage is further improved. Therefore, the recombinant 9 DEG N DNA polymerase provided by the invention can be used as a key enzyme molecule of a PCR and sequencing technology, can participate in the writing and reading process of DNA information storage data, is hopeful to become a DNA information storage tool enzyme, builds a good platform for encoding and decoding of DNA information storage, and promotes the application of the DNA polymerase in the fields of information storage, encryption and the like.
Disclosure of Invention
The invention aims to provide a recombinant 9 DEG N DNA polymerase and an application thereof in DNA information storage. The invention constructs the recombinant 9 DEG N DNA polymerase gene with optimized codons on a prokaryotic expression vector through chemically synthesizing the recombinant 9 DEG N DNA polymerase gene with optimized codons of escherichia coli, carries out protein expression strain transformation and resistance screening, and combines nickel column affinity chromatography and cation exchange chromatography to obtain high-purity recombinant 9 DEG N DNA polymerase through two-step purification; and then, taking the oligonucleotide containing stored information as a template, and after optimizing an amplification system, evaluating the enzymatic properties of the recombinant 9 DEG N DNA polymerase and the application effect of the recombinant 9 DEG N DNA polymerase in DNA information storage by the system.
The recombinant 9 DEG N DNA polymerase is prepared by the method comprising the following steps:
(1) Construction of recombinant 9 DEG N DNA polymerase engineering bacteria
Carrying out engineering bacterium codon optimization treatment on a gene sequence for encoding 9 DEG N DNA polymerase, wherein the optimized nucleotide sequence is shown as SEQ ID No.1 and is used as a gene for encoding recombinant 9 DEG N DNA polymerase;
adding a 6 XHis tag at the 5' end of a DNA polymerase gene for coding and recombining 9 DEG N, and connecting the DNA polymerase gene for coding and recombining 9 DEG N added with the 6 XHis tag to an expression vector pET-30a through NdeI and HindIII enzyme cutting sites to construct a recombinant expression vector pET-30a/9 DEG N;
transferring the recombinant expression vector pET-30a/9 DEG N into competent cell escherichia coli Trans5 alpha, then coating the competent cell escherichia coli Trans5 alpha onto a kanamycin resistance plate for screening, culturing overnight at 37 ℃, and then picking up a monoclonal for enzyme verification; extracting a recombinant expression vector pET-30a/9 DEG N with correct verification results in the escherichia coli Trans5α through a plasmid kit, transferring the recombinant expression vector pET-30a/9 DEG N into a protein expression competent cell escherichia coli BL21 (DE 3), and constructing a recombinant 9 DEG N DNA polymerase gene expression engineering bacterium;
(2) Acquisition of recombinant 9℃N DNA polymerase
1, the method comprises the following steps: inoculating recombinant 9 deg. N DNA polymerase gene expression engineering bacteria into LB culture medium with 80-120 (v/v) inoculum size, culturing at 37deg.C to optical density OD of culture 600 Reaching 0.6 to 0.8; adding IPTG with the final concentration of 0.5mM, and inducing for 4-8 hours at 37 ℃; centrifuging the culture at 5000-8000 r/min for 20-30 min to collect thalli, adding 50mM phosphate water solution into the thalli according to the ratio of 1:8-15 (w/v), uniformly mixing, and then carrying out ultrasonic crushing for 20-30 min; heating the obtained bacterial disruption solution in water bath at 80-90 ℃ for 20-30 min, cooling to room temperature, centrifuging at 7000-8000 r/min for 15-20 min, and collecting supernatant to obtain crude enzyme solution;
incubating the crude enzyme solution and the nickel column material at the temperature of 4 ℃ for 1-3 hours, eluting the hybrid protein by 10mM imidazole, and eluting the target protein by 75mM imidazole; centrifuging the eluted target protein sample for 15-25 min at 5000-7000 r/min by a 15kDa ultrafiltration tube, and repeatedly centrifuging for 2-4 times to remove imidazole during eluting the target protein; and then selecting a cation exchange chromatographic column, linearly eluting with a buffer B solution containing 1M sodium chloride, collecting an eluted sample, centrifuging the eluted sample for 15-25 min at 5000-7000 r/min through a 15kDa ultrafiltration tube, repeatedly centrifuging for 2-4 times, thereby obtaining the recombinant 9 DEG N DNA polymerase, and storing the recombinant 9 DEG N DNA polymerase in a storage buffer solution.
Preparation System of LB Medium (w/v): 1 to 2 percent of yeast powder, 1 to 2 percent of peptone, 0.5 to 2 percent of sodium chloride, and the pH value is 7.0, and the reagent is fully dissolved in deionized water to obtain an LB culture medium;
the buffer B solution preparation system comprises: 50-60 mM potassium phosphate buffer (pH 7.4) containing 1-1.5M sodium chloride and 5-10% glycerol (v/v), and the above-mentioned reagents were sufficiently dissolved in deionized water to obtain buffer B solution.
The preparation system of the preservation buffer solution is as follows: 5-15 mM of tris (hydroxymethyl) aminomethane hydrochloride (pH 7.4), 50-120 mM of sodium chloride, 1-1.5 mM of dimercaptosulfitol, 0.1-0.5 mM of ethylenediamine tetraacetic acid, 0.1-0.5% of 4- (1, 3-tetramethylbutyl) phenyl-polyethylene glycol and 20-50% of glycerol (v/v), and the above reagents are fully dissolved in deionized water to obtain a preservation buffer solution.
The method for purifying the recombinant 9 DEG N DNA polymerase can obtain high-purity tool enzyme molecules, and the optimization of PCR reaction conditions can greatly improve the catalysis efficiency of the recombinant 9 DEG N DNA polymerase, reduce the generation of nonspecific bands, improve the quality of amplified target products and facilitate downstream purification and subsequent sequencing analysis. The activity of the recombinant 9 DEG N DNA polymerase can be determined by in vitro fluorescence analysis, the good thermal stability is a necessary condition for PCR cyclic reaction, and the recombinant 9 DEG N DNA polymerase can resist the high temperature of the cyclic reaction condition and maintain higher catalytic efficiency in the reaction process, thereby being beneficial to the efficient amplification of target DNA. The fidelity of the recombinant 9 DEG N DNA polymerase endows the recombinant 9 DEG N DNA polymerase with good correction capability, and the incorporated error base can be removed in time to repair mismatched fragments, so that the accuracy of synthesizing target DNA is improved.
The recombinant 9 DEG N DNA polymerase can improve the fidelity of DNA information storage. And (3) taking an oligonucleotide pool obtained by storing digital information through Reed-Solomon (Reed-Solomon) erasure code coding as a template, optimizing a PCR reaction amplification system, purifying the obtained sample by a column type DNA purification kit to obtain a high-purity DNA amplification product, and performing high-throughput sequencing analysis. The recombinant 9 DEG N DNA polymerase can amplify all the stored information sequences in the oligonucleotide pool to realize lossless decoding of stored digital information, completely recover the stored digital information content, effectively reduce the number of errors generated in the DNA replication process, especially the type of errors mainly depending on the enzyme catalysis performance, reduce the occurrence probability of errors between purine and pyrimidine, improve the precision of information storage coding and decoding, and has wide application prospect as a tool enzyme applicable to DNA information storage.
The nucleotide sequence of the coding recombinant 9 DEG N DNA polymerase gene is shown as SEQ ID No. 1. The nucleotide sequence is obtained by optimizing the codon of a corresponding engineering bacterium by a 9 DEG N protein sequence derived from marine thermophilic archaea (Thermococcus sp.9 DEG N-7), and the coded recombinant 9 DEG N DNA polymerase has good purity, yield and catalytic activity after purification, can reduce the cost of DNA information storage, and improves the quality and efficiency of information storage data decoding.
The solutions of the present invention are deionized water solutions unless specifically stated. The PCR reaction system buffer solution of the invention is 10× ThermoPol Reaction Buffer, and can provide salt ions and metal ions for the polymerase catalytic reaction, and is purchased from NEB company in the United states. The dNTP mixed solution can provide a substrate for DNA synthesis reaction of polymerase, wherein the concentration of the four dNTPs is 4 mu M, and the dNTP mixed solution is purchased from NEB company in the United states. The beta-galactosidase (beta-gal) of the present invention can degrade the substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside (X-gal) to produce a blue product based on the expression control mechanism of the lacZ lactose operon, which when mutated produces white colonies when the polymerase replicates the lacZ gene, for use in blue-white screening to calculate polymerase fidelity, all reagents used in the assays were purchased from Sigma, USA. The oligonucleotide pool related by the invention is a mixture composed of oligonucleotides (DNA) containing various different sequences and obtained by coding a Reed-Solomon erasure code for a digital file, and the mixture is used for storing data, and is synthesized on a 12K chip by an electrochemical method and purchased from Nanjing Jinsry company. The PicoGreen dsDNA quantification kit of the invention is purchased from Thermo Fisher company of America. The Taq DNA polymerase of the present invention is used for amplification of oligonucleotide pools and is purchased from Beijing Bao Ri doctor materials technology Co. The Q5 DNA polymerase of the present invention, used for amplification of oligonucleotide pools, was purchased from NEB Inc. of America. The DNA sample carrying buffer solution and the protein sample carrying buffer solution are used for preparing electrophoresis samples by stopping the reaction and are purchased from Beijing Bao Ri doctor materials technology Co. Ssm13mp18 DNA according to the present invention was purchased from NEB Inc. of America as a DNA template for PCR reaction. The escherichia coli Trans5α and escherichia coli BL21 (DE 3) related by the invention are taken as competent host bacteria for carrying and transforming plasmids and are purchased from Beijing full gold biotechnology Co. The primers related to the invention are all purchased from Nanjing Jinsri company.
Drawings
FIG. 1 is a plasmid map of the recombinant expression vector pET-30a/9℃N obtained in example 1.
FIG. 2 is a photograph showing the gel electrophoresis of a denatured polyacrylamide gel digested with the recombinant expression vector obtained in example 1; lanes from left to right represent: m. nucleic acid molecular weight standard; 1. untreated plasmid; plasmid obtained after single cleavage of ndei; a plasmid obtained after single cleavage of HindIII; the plasmid obtained after double digestion of NdeI and HindIII.
FIG. 3 is a denaturing polyacrylamide gel electrophoresis of the IPTG induction expression time optimization assay obtained in example 2; m: protein molecular weight standard.
FIG. 4 is a denaturing polyacrylamide gel electrophoresis analysis of DNA polymerase purified by the method of example 3; m: protein molecular weight standard.
FIG. 5 shows agarose gel electrophoresis analysis of amplification results obtained by the method of example 4 at different DNA polymerase activities; lanes from left to right represent 1. Nucleic acid molecular weight standard, respectively; 2.0.08U;3.0.40U;4.0.80U;5.1.60U.
FIG. 6 is an enzyme activity assay curve of DNA polymerase measured by the in vitro fluorescent probe method obtained by the method of example 5; a: time course-fluorescence curves for Taq DNA polymerase with different activities; the abscissa is the synthetic reaction time of the DNA polymerase, and the ordinate is the fluorescence value measured by the fluorescence enzyme label instrument; b: the abscissa of the fluorescence quantitative standard curve of the activity of Taq DNA polymerase is the initial slope of the fluorescence curve under different activities calculated by the results of the A graph, and the ordinate is the corresponding activity unit.
FIG. 7 is an agarose gel electrophoresis chart of the evaluation of the thermal stability of the DNA polymerase obtained by the method of example 6; a: incubating at 95 ℃ for different times; b: incubate at 100℃for different times. 1. A nucleic acid molecular weight standard; 2.0 min; 3. 5min; 4. for 10min; 5. 20min; 6. 30min; 7. for 40min; 8. 60min; 9. for 90min; 10. 120min.
Fig. 8 is a schematic diagram of DNA information storage contents and a storage flow according to example 8 and example 9.
FIG. 9 is a bar graph of the error types and numbers of three DNA polymerase amplifications analyzed by high throughput sequencing obtained by the method of example 8. The abscissa is the DNA polymerase group, including commercial Taq DNA polymerase, commercial Q5 DNA polymerase, and recombinant 9℃N DNA polymerase purified in example 3; the ordinate is the base type and the number percentage obtained after high-throughput sequencing comparison.
FIG. 10 is a bar graph showing the analysis of the base substitution of three DNA polymerase amplifications obtained by the method of example 9. The X-axis is the desired base type, the Y-axis is the substitution base type, and the Z-axis is the probability of substitution for each base.
Detailed Description
The following examples are set forth to provide a further understanding of the present invention to those skilled in the art. The examples given are not to be construed as limiting the scope of the invention, and thus, insubstantial modifications and variations made by a person skilled in the art in light of the above disclosure should also be considered as being within the scope of the invention.
Example 1:
the construction of the recombinant 9 DEG N DNA polymerase engineering bacteria comprises the following specific processes: firstly, carrying out engineering bacterium codon optimization treatment on a gene sequence for encoding 9 DEG N DNA polymerase by an on-line codon optimization tool (https:// www.genscript.com.cn/gene-code-optimization. Html), wherein the optimized nucleotide sequence is shown as SEQ ID No.1 and is used as a gene for encoding recombinant 9 DEG N DNA polymerase. The nucleotide sequence shown in SEQ ID No.1 is constructed on an expression vector pET-30a (a general E.coli expression vector) through NdeI and HindIII enzyme cutting sites to obtain a recombinant expression vector pET-30a/9 DEG N, so that the recombinant 9 DEG N DNA polymerase can be efficiently expressed in E.coli.
The plasmid map of the recombinant expression vector pET-30a/9 DEG N is shown in figure 1, the start codon is ATG, and the stop codon is TAA. Firstly, adding a 6 XHis tag at the 5' end of a coding recombinant 9 DEG N DNA polymerase gene for subsequent purification, entrusting Nanjin Style company to synthesize the gene according to the nucleotide sequence, and connecting the coding recombinant 9 DEG N DNA polymerase gene added with the 6 XHis tag to an expression vector pET-30a through NdeI and HindIII enzyme cutting sites to construct a recombinant expression vector pET-30a/9 DEG N. Transferring the recombinant expression vector into competent cell escherichia coli Trans5α, then coating onto a kanamycin resistance plate for screening, culturing overnight at 37 ℃, selecting a monoclonal, and carrying out enzyme assay verification, wherein the results are shown in a graph in fig. 2, and group 2 and group 3 are respectively obtained through single enzyme digestion treatment of NdeI and HindIII, are single strips, and have the strip size lower than that of a recombinant expression vector pET-30a/9 DEG N group of group 1, so that the sequences of two enzyme digestion sites of NdeI and HindIII are correctly constructed, and the corresponding enzyme digestion sites on the recombinant expression vector can be identified and the recombinant expression vector is cut; group 4 is the result of double digestion with NdeI and HindIII, and the two bands are the enzyme-digested expression vector pET-30a and the recombinant 9N DNA polymerase gene with the 6 XHis tag, respectively, again demonstrating that both digestion site sequences of NdeI and HindIII are constructed correctly. Meanwhile, the recombinant expression vector pET-30a/9 DEG N entrusted vincristocet company carries out first generation sequencing analysis on sequences between the digestion sites NdeI and HindIII, and the sequencing result is completely consistent with the sequence in SEQ ID No.1, so that successful construction of the recombinant expression vector pET-30a/9 DEG N is proved. The recombinant expression vector pET-30a/9 DEG N in the escherichia coli Trans5α is extracted by a plasmid kit and is transferred into a protein expression competent cell escherichia coli BL21 (DE 3) to construct recombinant 9 DEG N DNA polymerase gene expression engineering bacteria.
Example 2:
the optimization method of the expression conditions of the recombinant 9 DEG N DNA polymerase gene expression engineering bacteria is as follows: preparing system (w/v) of LB culture medium of recombinant 9-degree NDNA polymerase gene expression engineering bacteria: 1% yeast powder, 1.6% peptone, 0.5% sodium chloride, pH 7.0, and dissolving the above reagents in deionized water to obtain LB medium. The recombinant 9°n DNA polymerase gene expression engineering bacteria constructed in example 1 were inoculated with 1:100 ratio (v/v) inoculating and recovering in the LB medium, culturing at 37deg.C until the optical density OD of the culture is reached 600 Reaching 0.65. Then isopropyl- β -D-thiogalactoside (IPTG) was added at a final concentration of 0.5mM and induced at 37℃for 0h, 1h, 2h, 4h, 6h, 8h, respectively. Centrifuging the obtained bacterial culture solution at 5000r/min for 20min to collect thalli, adding commercial protein sample loading buffer solution, heating for 10min in a constant temperature metal bath instrument at 100 ℃, cooling the sample, loading the sample, performing modified polyacrylamide gel electrophoresis, staining with coomassie brilliant blue staining solution, decolorizing with decolorizing solution, and observing the bands. As a result, as shown in FIG. 3, the protein expression amount increased with the increase of the induction time, and the equilibrium was reached at 6 hours, which is the optimal time for the induction of the protein expression.
Example 3:
in this example, the recombinant 9 ° NDNA polymerase of the present invention is obtained by extracting and purifying from recombinant 9 ° N DNA polymerase gene expression engineering bacteria by two steps of protein purification through nickel column affinity chromatography and ion exchange chromatography, and the specific steps are as follows: 1, the method comprises the following steps: 100 (v/v) inoculum size recombinant 9℃N DNA polymerase gene expression engineering bacteria were inoculated into LB medium of example 2 and cultured at 37℃to the optical density OD of the culture 600 Up to 0.65;adding IPTG with the final concentration of 0.5mM, and inducing at 37 ℃ for 6h; centrifuging the culture at 5000r/min for 20min to collect thallus, adding 50mM phosphate water solution (pH of 8.0) into thallus according to 1:10 (w/v), mixing uniformly, and ultrasonic crushing for 20min; heating the obtained bacterial disruption solution in water bath at 85deg.C for 30min, cooling to room temperature, centrifuging at 8000r/min for 15min, and collecting supernatant to obtain crude enzyme solution; carrying out protein purification by nickel column affinity chromatography, incubating crude enzyme solution and nickel column material at 4 ℃ for 1h, eluting the hybrid protein by 10mM imidazole, and eluting the target protein by 75mM imidazole; centrifuging the eluted target protein sample at 6000r/min for 20min through a 15kDa ultrafiltration tube, and repeating the centrifugation for three times to remove imidazole during eluting the target protein; then carrying out ion exchange chromatography, selecting a cation exchange chromatography column, linearly eluting with buffer B solution containing 1M sodium chloride, collecting an eluted sample, centrifuging the eluted sample for 20min at 6000r/min through a 15kDa ultrafiltration tube, repeatedly centrifuging for three times, and storing the obtained recombinant 9 DEG N DNA polymerase in a storage buffer solution.
Wherein the buffer B solution preparation system is as follows: 50mM potassium phosphate buffer (pH 7.4) containing 1M sodium chloride and 5% glycerol (v/v) was dissolved in deionized water to obtain buffer B solution. The preparation system of the preservation buffer was 10mM tris hydrochloride (pH 7.4), which contained 100mM sodium chloride, 1mM dithiothreitol, 0.1mM ethylenediamine tetraacetic acid, 0.1%4- (1, 3-tetramethylbutyl) phenyl-polyethylene glycol, 25% glycerol (v/v), and the above-mentioned reagents were sufficiently dissolved in deionized water to obtain a preservation buffer. The purified recombinant 9 DEG N DNA polymerase is stored in the storage buffer system, so that the catalytic activity and stability of the DNA polymerase can be maintained for a long time.
Respectively taking a protein sample which is not added with IPTG to induce bacterial precipitation, bacterial precipitation when 0.5mM IPTG is added to induce for 6 hours, protein sample which is eluted by 75mM imidazole in nickel column affinity chromatography and protein sample which is subjected to ion exchange chromatography, adding a commercial protein sample loading buffer solution, heating for 10 minutes in a metal bath instrument at 100 ℃, cooling the sample, loading the sample, carrying out denaturing polyacrylamide gel electrophoresis, staining by using coomassie brilliant blue staining solution, and observing the bands after decolorizing by using a decolorizing solution. As a result, as shown in FIG. 4, after the continuous two-step purification of proteins by Ni column affinity chromatography and ion exchange chromatography, there was only a single band in the sample of the ion exchange chromatography group, and the size of the band was consistent with that of the 9℃N DNA polymerase of the target protein, indicating that the recombinant 9℃N DNA polymerase was successfully prepared in high purity.
Example 4:
the enzyme concentration optimization method of the recombinant 9 DEG N DNA polymerase amplification system is as follows: firstly preparing a PCR amplification reaction buffer system comprising 1 mu M of one-way primer F1 (5'-GACACTCGTATGCAGTAGCC-3'), 300ng of template M1 (5'-ACAACCATTTATGTAGCATTTATGAAATTTTT AAATCAATTTACTATTGGCTACTGCATACGAGTGTC-3'), 2.5mM dNTP and 10X ThermoPol Reaction Buffer (NEB company), adding the recombinant 9 DEG N DNA polymerase obtained in the example 3 into the amplification reaction buffer system according to concentration gradients of 0.08U, 0.40U, 0.80U and 1.60U, supplementing the reaction system to 100 mu L by deionized water, evenly distributing the system into special PCR tubes, and carrying out an amplification reaction under a preset PCR circulation program for each tube by 25 mu L. The PCR cycling program was: 95 ℃ for 5min; cycling for 25 times at 95℃for 30sec,55℃for 30sec, and 72℃for 30 sec; and at 72℃for 10min. And adding a commercial DNA sample loading buffer solution to terminate the reaction after the amplification reaction is finished, blowing and mixing uniformly, loading samples into 2% agarose gel for electrophoresis under the conditions of 120V and 25min, and then observing and analyzing in a gel imager. As shown in FIG. 5, the obtained DNA polymerase has optimal activity at an enzyme concentration of 0.8U in the PCR amplification reaction, and the DNA polymerase gene encoding recombinant 9 DEG N can be amplified well at the concentration, the band is single and no pollution of nonspecific bands is caused.
Example 5:
the enzyme activity of the recombinant 9°n DNA polymerase was determined as follows: a subsequent study was conducted according to the optimum DNA polymerase concentration obtained by optimizing in Experimental example 4, by first preparing a pre-reaction mixture containing 1. Mu.M of one-way primer S1 (5'-CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCC-3'), 300ng ssm13mp18 DNA (available from NEB Co.), 10X ThermoPol Reaction Buffer (available from NEB Co.), adding deionized water to the reaction system to 20. Mu.L, pre-annealing at 95℃and then cooling on ice, adding the recombinant 9℃N DNA polymerase obtained in example 3 and 2.5mM substrate dNTP, reacting for different times at 72℃and sampling to obtain a sample of 1:200 (v/v) in a TE buffer diluted PicoGreen working solution (available from Thermo Fisher Co.) was added, wherein the TE buffer was formulated as 10mM Tris hydrochloride (pH 8.0) containing 1mM EDTA. The above reagent was sufficiently dissolved in deionized water to obtain a TE buffer. The fluorescence signal generated when PicoGreen fluorescent dye is combined with the synthesized DNA is detected by a fluorescence spectrophotometer, so that the synthesized DNA is quantified, and the enzyme catalytic activity is calculated by comparing the initial slopes of the DNA polymerase with known enzyme activity and the DNA polymerase to be detected. As shown in FIG. 6, the initial slope calculation shows that the enzyme activity of the recombinant 9 DEG N DNA polymerase is 8U/. Mu.L, which is higher than the enzyme activity of the commercial Taq DNA polymerase by 5U/. Mu.L, and the recombinant 9 DEG N DNA polymerase has good DNA amplification activity and important potential in the aspects of synthesis and sequencing in DNA information storage.
Example 6:
the method for evaluating the thermal stability of the recombinant 9°n DNA polymerase is as follows: based on the optimized optimal PCR reaction system, adding the recombinant 9 DEG N DNA polymerase into deionized water diluted 1X ThermoPol Reaction Buffer, respectively carrying out thermal incubation at 95 ℃ and 100 ℃, sampling at 0, 5, 10, 20, 30, 40, 60, 90 and 120min, further carrying out incubation on the samples at 72 ℃ for 2min to balance the temperature of each sample, completing the reaction system for carrying out PCR reaction, and evaluating the thermal stability of the recombinant 9 DEG N DNA polymerase, wherein the PCR reaction condition is as described in example 4. As shown in FIG. 7, the activity of the recombinant 9℃DNA polymerase was hardly reduced after 120min incubation at 95℃and the recombinant 9℃DNA polymerase still had good catalytic activity after 120min incubation at 100℃indicating that the recombinant 9℃DNA polymerase had good thermal stability, which is a necessary condition for the application of the DNA polymerase to PCR reactions.
Example 7:
the method for fidelity analysis of the recombinant 9°n DNA polymerase was as follows: based on the expression regulatory mechanism of the lacZ lactose operon, a blue-white screening method was selected to evaluate the fidelity of recombinant 9℃N DNA polymerase. The lacZ gene on pUC19 plasmid is excised by selecting Hind III and Nde I restriction endonucleases, plasmid skeleton and excised gene are recovered by gel recovery kit, excised gene lacZ is amplified by recombination 9 DEG N DNA polymerase, amplified product is recovered by gel and is re-connected to plasmid skeleton, transferred into E.coli Trans5 alpha competent cells, coated on LB plate added with 0.5M IPTG and 20mg/mL X-gal, inverted overnight at 37 deg.C, and the colony growth condition is observed and counted. The blue colony is a normal colony, and the white colony is a colony containing mutant gene plasmids. Formulation System of LB plates (w/v): 1% yeast powder, 1.5% peptone, 1% sodium chloride, 1% agar, and the above reagents were sufficiently dissolved in deionized water at pH 7.5 to obtain LB plates.
The mismatch rate of DNA polymerase amplification was calculated by the formula er=mf/(bp×d). Wherein ER is the mismatch rate, mf is the mutation frequency, bp is the lacZ gene length, d is the template multiplying power, and the specific calculation mode is shown in the lower part of the table 1. The results are shown in Table 1, where the mismatch rate of the recombinant 9℃N DNA polymerase is 1.91X 10 compared to the commercial Taq polymerase -4 The fidelity is about 5.8 times of that of Taq polymerase, so that the recombinant 9 DEG N DNA polymerase can more accurately realize DNA amplification and reduce the occurrence of errors.
TABLE 1 evaluation of DNA polymerase fidelity by blue-white screening
Colony clone numbers were the sum of independent assays for each group.
a mutation frequency = number of mutant colony clones/number of total colony clones.
b template multiplying power calculation formula 2 d = (amount of PCR product)/(amount of starting target), quantitative calculation was performed by Image J software.
The calculation formula of the c mismatch rate is ER=mf/(bp×d), wherein ER is the mismatch rate, mf is the mutation frequency, bp is the lacZ target gene size (265 bp), and d is the template multiplying power.
Example 8:
the information storage encoding process is as follows: the information storage flow chart is shown in fig. 8. The Chinese and English names of Jilin university in the text format and the pictures of Jilin university in the jpg format are encoded through Reed-Solomon erasure codes, and are converted into nucleotide sequences (wherein the Reed-Solomon erasure codes are open source codes, the codes and the specific using method can be obtained through link downloading uploaded on Github by a developer, and the downloading website is https:// Github.com/reinhardh/dna_rs_coding). Obtaining an oligonucleotide pool containing coding DNA sequence information, amplifying the oligonucleotide pool by using the recombinant 9 DEG N DNA polymerase, and then carrying out high-throughput sequencing analysis on an amplified sample; and then clustering analysis processing is carried out on the data obtained by sequencing by taking the homologous arm sequence as a clustering characteristic, and the nucleotide sequence is converted back into a binary sequence through a decoding algorithm in the Reed-Solomon erasure code, so that the stored data information is recovered.
The coding scheme for converting the binary sequence into the DNA sequence adopts a method of converting two bits into one base, and the conversion preset corresponding relation is as follows: 00-A, 01-C, 10-G, 11-T. For example, the Chinese character Ji is 11100101 10010000 10001001 binary, and the nucleotide sequence TGCC GCAA GAGC is obtained through conversion. Wherein A is adenine, T is thymine, G is guanine, and C is cytosine. Coding the file containing the Chinese and English names of Jilin university and the logo picture of Jilin university according to the steps provided in the Github link, wherein parameters are set to be-n=70, -k=47 (n, k parameters are selected to be related to the file size, and are specifically selected to be suggested by a developer in a standard reference link), and the rest parameters are default parameters, so that an oligonucleotide pool containing the file can be obtained, wherein the oligonucleotide pool is a nucleotide sequence with the total length of 126480 (the capacity of the oligonucleotide pool obtained by coding is related to the size of the file to be coded).
For the coding DNA sequence in the oligonucleotide pool, homologous arm sequences are added at both ends for company synthesis and subsequent PCR amplification in the following manner: 5 '-ACACGACGCTCTACCGATCT-DNA coding sequence-AGTTCGGAAGAGCACACGTCT-3'. The above obtained sequence was subjected to chemical synthesis by Nanjing Jinsri company and used for the subsequent experiments.
Example 9:
the recombinant 9°n DNA polymerase amplification oligonucleotide pool and decoding process were as follows: the PCR amplification system was prepared as shown in Table 2, and the oligonucleotide pool was subjected to replication amplification by recombinant 9℃N DNA polymerase, and the PCR cycling procedure was: 95 ℃ for 5min; cycling for 25 times at 95℃for 30sec,55℃for 30sec, and 72℃for 30 sec; and at 72℃for 10min.
Table 2: the PCR amplification system is as follows:
| 10×ThermoPol Reaction Buffer | 10μL |
| dNTP mixture 4. Mu.M | 5μL |
| Primer P1 100. Mu.M (5'-ACACGACGCTCTACC GATCT-3') | 2μL |
| Primer P2 100. Mu.M (5'-AGACGTGTGCTCTTCCGAACT-3') | 2μL |
| Oligonucleotide pool (10 ng/. Mu.L) | 10μL |
| Recombinant 9°n DNA polymerase | 0.8 U |
| Deionized water | Up to 100 mu L |
Amplification product of recombinant 9 DEG N DNA polymerase obtained by PCR (polymerase chain reaction) circular replication by using DNA (deoxyribonucleic acid) purification kitPurifying to remove salt ion and protein impurities until the optical density OD 260 /OD 280 The ratio is 1.8-2.0, and the concentration is more than or equal to 25 ng/. Mu.L, and high-throughput sequencing analysis is performed.
The sequencing result is subjected to cluster analysis processing by taking the homology arm sequence as a clustering characteristic, and the processed data is decoded by a decoding algorithm in a Reed-Solomon erasure code (the decoding step refers to a use instruction provided by a code developer, as described in example 8), so that the 'Jilin university Chinese and English names and Jilin university logo pictures' stored in DNA can be recovered. In addition, SNP rolling analysis is performed on the sequencing data, the DNA sequence obtained by sequencing is compared with the nucleotide sequence in the oligonucleotide pool, and the type and the number of errors generated are analyzed. As shown in FIG. 9, the correct base ratio after amplification by recombinant 9℃N DNA polymerase treatment was 99.00% and the base ratio for error was 1.00%. The proportions of the three types of errors commonly found are 0.19%, 0.41% and 0.40%, respectively, of insertions, deletions and base substitutions. The ratio of three types of errors, insertion, deletion and base substitution, which are common after commercial Taq DNA polymerase treatment amplification, is 0.17%, 0.35% and 0.57%, respectively. The ratio of the three types of errors, insertions, deletions and base substitutions, which are common after commercial Q5 DNA polymerase treatment amplification, was 0.15%, 0.37% and 0.44%, respectively. Both insertion and deletion errors can be further reduced by improvements and perfection of the error correction algorithm, but existing algorithms cannot effectively reduce the type of base substitution. Compared with commercialized Taq DNA polymerase and Q5 DNA polymerase, the recombinant 9 DEG NDNA polymerase can effectively reduce the base substitution, which is mainly dependent on the error type of DNA polymerase fidelity, and proves that compared with the commercialized DNA polymerase, the recombinant 9 DEG N DNA polymerase has the advantages in the aspect of amplifying an oligonucleotide pool, can reduce the error occurrence in the replication process, and is crucial for further improving the robustness of the information storage technology.
The nucleotide sequence in the oligonucleotide pool is taken as a desired sequence, the DNA sequence obtained by sequencing is taken as an actual sequence, and the two sequences are compared one by one. Taking the X axis as a desired base type, taking the Y axis as a replacement base type, taking the Z axis as the probability of each base replacement, and making a three-dimensional bar graph to further analyze the base replacement errors. As shown in FIG. 10, the recombinant 9℃N DNA polymerase can reduce the number of errors of various types of bases, and particularly can reduce the occurrence probability of errors between purine and pyrimidine, thereby improving the accuracy of information storage.
The above description is only of the preferred embodiments of the present invention and is not limited to the present invention, but various modifications and changes will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A recombinant 9°n DNA polymerase characterized by: is prepared by the method comprising the following steps,
(1) Construction of recombinant 9 DEG N DNA polymerase engineering bacteria
Carrying out engineering bacterium codon optimization treatment on a gene sequence for encoding 9 DEG N DNA polymerase, wherein the optimized nucleotide sequence is shown as SEQ ID No.1 and is used as a gene for encoding recombinant 9 DEG N DNA polymerase;
adding a 6 XHis tag at the 5' end of a DNA polymerase gene for coding and recombining 9 DEG N, and connecting the DNA polymerase gene for coding and recombining 9 DEG N added with the 6 XHis tag to an expression vector pET-30a through NdeI and HindIII enzyme cutting sites to construct a recombinant expression vector pET-30a/9 DEG N;
transferring the recombinant expression vector pET-30a/9 DEG N into competent cell escherichia coli Trans5 alpha, then coating the competent cell escherichia coli Trans5 alpha onto a kanamycin resistance plate for screening, culturing overnight at 37 ℃, and then picking up a monoclonal for enzyme verification; extracting a recombinant expression vector pET-30a/9 DEG N with correct verification results in the escherichia coli Trans5α through a plasmid kit, transferring the recombinant expression vector pET-30a/9 DEG N into a protein expression competent cell escherichia coli BL21 (DE 3), and constructing a recombinant 9 DEG N DNA polymerase gene expression engineering bacterium;
(2) Acquisition of recombinant 9℃N DNA polymerase
1, the method comprises the following steps: 80-120 (v/v) inoculum size recombinant 9 DEG N DNA polymerase gene expression engineering bacteria are inoculated in LB cultureCulturing in culture medium at 37deg.C to optical density OD of culture 600 Reaching 0.6 to 0.8; adding IPTG with the final concentration of 0.5mM, and inducing for 4-8 hours at 37 ℃; centrifuging the culture at 5000-8000 r/min for 20-30 min to collect thalli, adding 50mM phosphate water solution into the thalli according to the ratio of 1:8-15 (w/v), uniformly mixing, and then carrying out ultrasonic crushing for 20-30 min; heating the obtained bacterial disruption solution in water bath at 80-90 ℃ for 20-30 min, cooling to room temperature, centrifuging at 7000-8000 r/min for 15-20 min, and collecting supernatant to obtain crude enzyme solution;
incubating the crude enzyme solution and the nickel column material at the temperature of 4 ℃ for 1-3 hours, eluting the hybrid protein by 10mM imidazole, and eluting the target protein by 75mM imidazole; centrifuging the eluted target protein sample for 15-25 min at 5000-7000 r/min by a 15kDa ultrafiltration tube, and repeatedly centrifuging for 2-4 times to remove imidazole during eluting the target protein; and then selecting a cation exchange chromatographic column, linearly eluting with a buffer B solution containing 1M sodium chloride, collecting an eluted sample, centrifuging the eluted sample for 15-25 min at 5000-7000 r/min through a 15kDa ultrafiltration tube, repeatedly centrifuging for 2-4 times, thereby obtaining the recombinant 9 DEG N DNA polymerase, and storing the recombinant 9 DEG N DNA polymerase in a storage buffer solution.
2. A recombinant 9°n DNA polymerase according to claim 1, wherein: the gene sequence encoding 9°n DNA polymerase was derived from the 9°n protein sequence of marine thermophilic archaea (thermo coccus sp.9°n-7).
3. A recombinant 9°n DNA polymerase according to claim 1, wherein: the preparation system (w/v) of the LB culture medium comprises 1-2% of yeast powder, 1-2% of peptone, 0.5-2% of sodium chloride and pH of 7.0, and the reagent is fully dissolved in deionized water to obtain the LB culture medium; the buffer B solution is prepared by dissolving the above reagent in deionized water fully, wherein the buffer B solution is 50-60 mM potassium phosphate buffer with pH of 7.4, contains 1-1.5M sodium chloride and 5-10% glycerol (v/v); the preparation system of the preservation buffer solution is 5-15 mM of tris hydrochloride with the pH of 7.4, contains 50-120 mM of sodium chloride, 1-1.5 mM of dithiothreitol, 0.1-0.5 mM of ethylenediamine tetraacetic acid, 0.1-0.5% of 4- (1, 3-tetramethylbutyl) phenyl-polyethylene glycol and 20-50% of glycerol (v/v), and the reagent is fully dissolved in deionized water to obtain the preservation buffer solution.
4. Use of a recombinant 9°n DNA polymerase according to any one of claims 1 to 3 in DNA information storage.
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