CN110643560A - Engineering strain for recombinant expression of restriction endonuclease and construction method thereof - Google Patents

Abstract

The invention discloses a kind of engineering strain for recombinant expression restriction endonuclease and its construction method. The engineering strain is Escherichia coli ER2566, and the engineering strain comprises the following conditions: compatible with exogenous methyltransferase; The prokaryotic expression vector pACYC184 of the methyltransferase gene based on the universal sequence; the prokaryotic expression vector pBAD containing the restriction endonuclease gene whose recognition cutting sequence comprises the four-base universal sequence; wherein, the four-base universal sequence includes: TCGA, TGCA , TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC; compared with the prior art, the present invention does not need to screen specific methylation protection strains for each restriction enzyme, greatly Reduces the number of methyltransferase-protected strains required to produce restriction enzymes.

Description

Engineering strain for recombinant expression restriction endonuclease and construction method thereof

technical field

The present invention relates to biochemical and engineering strains, in particular to engineering strains for recombinant expression restriction endonucleases and construction methods thereof.

Background technique

Restriction endonucleases, referred to as restriction enzymes, are a large class of endonucleases that can hydrolyze phosphodiester bonds from the middle of DNA molecules to cut double-stranded DNA. They are one of the most important biomedical tool enzymes and are widely used in DNA recombination, gene localization and cloning, gene structure research, DNA sequence analysis and determination, gene synthesis and other fields of biomedicine. Natural restriction enzymes are mainly derived from prokaryotes, and their corresponding methyltransferases form a "restriction-modification" system. "Restriction-modification" is mainly used to resist the entry of foreign DNA molecules, and its biological significance is to protect the host from Phage infection, in the original host, methyltransferase will methylate specific sequences of the host genome and plasmids contained in its own cells, and the corresponding restriction enzymes cannot recognize and cut these methylated DNAs. When DNA enters the host, because it is not methylated, it will be cleaved into fragments by restriction enzymes, thus losing its function. Most of the early restriction enzymes were isolated from their original strains, with high cost and low yield, and many natural microorganisms were not suitable for industrial cultivation and could not be used for restriction enzyme extraction and isolation. Subsequently, scientists cloned the restriction enzyme genes and transferred them into Recombinant expression into engineered strains to increase its yield. However, due to the ability of restriction enzymes to cut DNA that is not protected by methylation, in engineered strains without corresponding methyltransferase protection, restriction enzymes have strong cytotoxicity, which makes recombinant expression extremely difficult. The most effective recombinant expression strategy of restriction endonucleases at present is mainly to use a specific "restriction-modification" system: firstly, the specific methyltransferase gene that is homologous to a specific restriction enzyme, recognizes and modifies the same DNA sequence Transfer it into a suitable E. coli expression strain to express the methyltransferase; then screen out a specific site of the host DNA that is protected by methylation modification and cannot be cut by the corresponding restriction enzyme; finally, the corresponding restriction The enzyme gene was transferred into the protection strain to realize recombinant expression.

Since each restriction enzyme has its corresponding methyltransferase, if the above strategy is used to recombinantly express more than 3,000 restriction enzymes that have been discovered so far, more than 3,000 methyltransferase-protected strains need to be screened, and the workload is very large. Low efficiency; recently, some researchers have discovered some broad-spectrum methyltransferases, such as M.SssI from the Spiroplasma sp.MQ1 strain, which can recognize all cytosine residues in the 5'-CG-3' sequence. , and M.CviPI derived from Chlorella virus NYs-1, which can methylate all cytosine residues in the 5'-GC-3' recognition sequence, etc.; and there are more than 70 known The recognition sequences for one restriction enzyme contained CG, and the recognition sequences for more than 50 restriction enzymes contained GC. In theory, if the above-mentioned broad-spectrum methyltransferase gene is transferred into the engineering strain, the CG or GC sequence on the DNA can be methylated, and it can also resist the cleavage of restriction enzymes, so as to be used for recombinant expression of restriction enzymes. Cases in which restriction enzymes such as NotI and PstI were successfully expressed by this strategy. However, CG or GC broad-spectrum methylation has too many methylation sites on the genome, which often brings excessive metabolic burden to the host and also has a lethal effect. Therefore, the use of this strategy for recombinant expression of restriction enzymes is greatly restricted Therefore, the present invention aims to utilize the methyltransferases of modified short homologous sequences to obtain recombinant expression strains for expressing and recognizing various short homologous sequence restriction enzymes for economical and efficient production of restriction endonucleases .

SUMMARY OF THE INVENTION

The present invention establishes a restriction endonuclease that uses universal four-base sequence methyltransferase to screen and express a short homologous sequence, and specifically uses four-base universal sequence methylation protection for expressing restriction endonucleases. To greatly improve its efficiency and reduce costs, the specific solutions are as follows:

The engineering strain for recombinant expression restriction endonuclease is characterized in that: taking Escherichia coli ER2566 as the engineering strain transformation basis, the engineering strain comprises the following conditions:

(4) Compatible with exogenous methyltransferases;

(5) a prokaryotic expression vector pACYC184 containing a methyltransferase gene that can protect the four-base universal sequence;

(6) a prokaryotic expression vector pBAD containing a restriction endonuclease gene that recognizes a cleavage sequence comprising a four-base universal sequence;

The four-base universal sequences include: TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, and GTAC.

The construction method of the engineering strain used for recombinant expression restriction endonuclease is characterized in that: the construction method specifically comprises the following steps:

S1: Screening of engineering strains: It is determined that Escherichia coli ER2566 is used as the basis for the transformation of engineering strains, which are compatible with exogenous methyltransferases. , a prokaryotic expression vector containing a methyltransferase gene that can protect a four-base universal sequence, and a prokaryotic expression vector containing a restriction endonuclease gene that recognizes the cleavage sequence and includes a four-base universal sequence, wherein the four-base universal sequence The sequences are TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC.

S2: Select expression plasmid: select pACYC184 plasmid with Amp promoter as methyltransferase expression plasmid, use resistance to chloramphenicol, select pBAD plasmid with arabinose operon and promoter as restriction endonuclease Expression plasmid, using ampicillin resistance;

S3: Intermediate strain acquisition: According to the four-base universal sequence combination and methylation pattern in S1, a specific methyltransferase gene is selected, and the selected specific methyltransferase gene is connected with pACYC184 in S2 to obtain a recombinant plasmid. The recombinant plasmid Transform into Escherichia coli ER2566 strain to obtain an intermediate strain;

S4: Screening strains: According to the methylation sequence and methylation site mode of the methyltransferase selected by S3, the identification sequence is selected to include the four-base universal sequence and the methylation sensitivity is the same as that of the methyltransferase action site The restriction endonuclease, cutting the DNA of the intermediate strain obtained in step S3, and screening out the methylation protection strain that cannot be cut,

S5: Construction of expression strain: connect the gene corresponding to the restriction endonuclease in S4 with pBAD in S2 to obtain a recombinant plasmid, and transform the recombinant plasmid into the S4 screening strain to obtain an engineered strain that can express the corresponding restriction endonuclease;

S6: Expression of the engineered strain: The engineered strain of the restriction endonuclease obtained in step S5 is used to induce the expression of the restriction endonuclease.

Further, the four-base universal sequence methyltransferase includes: (1) TCGA: M.TaqI; (2) TGCA: M.HpyCH4V; (3) TTAA: M.EsaDix5I, M.MseI; (4) ) AATT: M.MluCI; (5) ACGT: M.HpyCH4IV, M.TaiI; (6) AGCT: M.CvikI-1, AluI; (7) CATG: M.FaeI, M.CviAII, M.NlaIII; (8) CCGG: M.MspI, M.HpaII; (9) CGCG: M.BstuI; (10) CTAG: M.BfaI; (11) GATC: M.Sau3AI, M.BstKTI, M.DpnII, M. BfuCI, M.MboI; (12) GCGC: M.HinPII, M.HhaI; (13) GGCC: M.HaeIII, M.CvikI-1, M.PhoI; (14) GTAC: M.Csp6I, M.CviQI , M.RsaI; the restriction endonucleases that recognize the cleavage sequence comprising a four-base universal sequence include: (1) TCGA: AccI, BstBI, SalI, XhoII, TliI, PaeR7I, BspDI, ClaI; (2) TGCA: ApaII, PstI, SfcI, NsiI; (3) TTAA: AseI, HincII, HpaI, SmII, AflII; (4) AATT: ApoI, EcoRI, MfeI; (5) ACGT: SnaBI, AatII, ZraI, BsaHI, BsaAI, PmII , AcII; (6) AGCT: SacI, Eco53kI, Ecll36II, MspAII, PvuII, HindIII; (7) CATG: SphI, BsaJI, BtgI, StyI, NspI, AffIII, PciI; (8) CCGG: SmaI, AvaI, AcoI, XmaI, TspMI, BsaWI, BsrFI, AgeI; (9) CGCG: NruI, BssHII, SacII, AflIII, MluI; (10) CTAG: XbaI, BmtI, NheI, AvrII, SpeI; (11) GATC: NlaIV, BstYI, BsiEI , BclI, BmaHI, PvuI, BgIII; (12) GCGC: FspI, SfoI, KasI, NarI, BanI, KasI, HacII, AfeI; (13) GGCC: MscI, BanII, ApaI, PspOMI, EaeI, EagI, StuI; ( 14) GTAC: BsrGI, KpnI, Acc65I, BsiWI, ScaI, Ta tI

Further, the step of obtaining the S3 intermediate strain is as follows:

(1) Synthesize the methyltransferase gene containing the short homologous recognition sequence on the pUC57 vector, use NdeI and XhoI to carry out enzyme digestion, and carry out DNA band purification by agarose gel electrophoresis;

(2) The above purified methyltransferase gene was ligated into the pACYC184 expression vector digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into E. coli DH5α strain and inoculated into 50 μg/mL chloramphenicol The plates were grown overnight at 37°C, and single clones were picked and grown overnight in LB medium containing 50 μg/mL chloramphenicol, and the plasmids were extracted and sequenced by the kit;

(3) The expression vector pACYC184 plasmid with correct sequencing and target methyltransferase was transformed into Escherichia coli ER2566 strain and spread on a culture plate containing chloramphenicol resistance to obtain an intermediate strain.

Further, the steps of screening strains in the S4 are as follows:

(1) Extract the plasmid of each transformant in the S3 plate, use restriction endonucleases to detect the protection of methyltransferase, the enzyme activity test system is 20 μl, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM acetic acid Magnesium, 100μg/ml BSA (pH7.9@25°C) with final concentration of 1x FlashOne buffer, 1U corresponding commercial restriction endonuclease and 200ng plasmid were reacted at 37°C for 30min;

(2) After the reaction, use agarose gel with 1x TBE concentration of 0.8% for electrophoresis detection. If the plasmid will not be digested by the corresponding restriction endonuclease, the methyltransferase expressed by the transformant containing the plasmid will be detected. The DNA in the strain can be protected from the cleavage of the corresponding restriction endonuclease;

(3) The transformants whose plasmids cannot be digested are screened out as host strains expressing restriction endonucleases and used as restriction endonuclease expressing strains in subsequent steps.

Further, the construction steps of the expression strain in the S5 are as follows:

(1) Synthesize the restriction endonuclease gene whose recognition sequence contains the methylation sequence and is sensitive to the methylation pattern into the pUC57 vector, use NdeI and XhoI for restriction enzyme digestion, and conduct banding by agarose gel electrophoresis purification;

(2) The purified restriction endonuclease gene was ligated to the expression vector pBAD digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into Escherichia coli DH5α strain, and inoculated into a 50 μg/mL On the culture plates of ampicillin, the plates were grown overnight at 37°C, and single clones were picked and grown overnight in LB medium containing 50 μg/mL ampicillin, and the plasmids were extracted and sequenced by the kit;

(3) Transform the expression vector pBAD plasmid with the correct sequencing and target restriction enzyme into the methyltransferase protection strain with short homologous recognition sequence screened in the previous step and inoculate it into the chloramphenicol containing 50 μg/mL The expression strains were constructed on a culture plate containing 50 μg/mL ampicillin and ampicillin.

Further, the expression steps of the engineering strain in the S6 are as follows:

(1) The transformants obtained by S5 transformation were inoculated into LB medium containing 50 μg/mL chloramphenicol and 50 μg/mL ampicillin for culture, and when cultured to late logarithmic stage, the final concentration of 0.2% (m /v) L-arabinose induction for 16 hours is the expression of the engineered strain recombinantly expressing restriction endonucleases;

(2) After the induction, the cells were collected by centrifugation and crushed by sonication, and the crude enzyme extract was obtained by centrifugation again. The test system is 50 μl, with 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA (pH 7.9@25°C) final concentration of 1x FlashOne buffer, plus 5 μl of crude enzyme solution and 1 μg base The biological DNA was reacted at the optimum reaction temperature for 60 min;

(3) After the reaction, 20 μg of proteinase K was added, and after treatment at 37°C for 15 minutes, electrophoresis was performed on agarose gel with 1× TBE concentration of 0.8%. The DNA bands are compared to determine the restriction endonuclease activity in the crude enzyme extract, and the transformant corresponding to the crude enzyme extract that can completely and correctly cut the substrate DNA can be used as the restriction endonuclease in the subsequent steps. Engineered strains that express Dicer in large quantities.

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

(1) Reduce the difficulty of finding specific methyltransferase genes;

(2) The number of constructing specific methyltransferase protection strains is reduced;

(3) The methylation-protected strain prepared by the methyltransferase that recognizes the universal four-base sequence can be used to produce a variety of restriction endonucleases whose recognition sequences include the four-base sequence and are sensitive to the methylation mode .

Description of drawings

In order to explain the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the technical description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. , for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is a four-base universal sequence and a restriction endonuclease map identification diagram;

Fig. 2 shows the technical circuit diagram of constructing methyltransferase protection strain and restriction endonuclease recombinant expression strain.

Figure 3 is an agarose gel electrophoresis image for screening methyltransferase-protected strains.

Fig. 4 is an agarose gel electrophoresis image for screening crude enzyme activity of restriction endonucleases from methyltransferase-protected strains transformed into restriction endonuclease expression vectors.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

In order to achieve the purpose of the present invention, as shown in Figures 1-4,

(1) Screening of engineering strains: Since the exogenous "restriction-modification" system needs to be introduced into E. coli engineering strains for expression, it is necessary to consider the compatibility of E. coli engineering strains with methyltransferases. By screening ER2566, HB101 , K802, BL21(DE3), BL21(DE3)pLysS, Top10, JM109, Rosetta(DE3), XL1-Blue strains, found that when the genotype of the E. coli engineering strain is mcrC-mrr deletion type, it can be compatible with exogenous Methyltransferase, after screening, it was determined that the Escherichia coli ER2566 strain could meet the requirements.

(2) Selection of expression plasmid: When expressing an exogenous "restriction-modification" system, it is necessary to perform specific methylation on the DNA in the E. coli engineering strain. When constructing a methyltransferase expression plasmid, select the composition type expression plasmid, so that the E. coli engineering strain can methylate the DNA before the restriction endonuclease induces expression, and the DNA in the E. coli engineering strain after methylation modification will not be specifically modified. Restriction endonuclease digestion, so as to achieve the effect of removing the cytotoxicity of the restriction endonuclease, and then a large amount of restriction endonuclease expression can be carried out, therefore, strict operons and promoters are selected for restriction endonuclease expression, According to the nature of the promoter, the arabinose without background expression was selected; at the same time, the oversized plasmid would affect its propagation and expression in E. coli engineering strains, so two plasmids were selected to express methyltransferase and restriction endonuclease respectively. In order to transform multiple plasmids into E. coli engineering strains, it is necessary to consider the issue of plasmid compatibility, that is, multiple plasmids need to have different origins of replication ori; after screening, the pACYC184 plasmid with the Amp promoter was selected as the methyltransferase For the expression plasmid, the resistance was chloramphenicol, the pBAD plasmid with the arabinose operon and the promoter was selected as the restriction endonuclease expression plasmid, and the resistance was ampicillin.

(3) Determine the four-base universal sequence methylation scheme: According to the arrangement and combination of the quadruple base sequence, and according to the recognition or cutting base sequence of the existing restriction endonucleases, all reasonable arrangements and combinations are designed: TTAA, AATT, ACGT, AGCT, ATAT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC, TATA, TCGA, TGCA; find methyltransferases that use these four-base sequences as methylation sequences, After the methylation sequence is based on these four-base sequences, the restriction endonucleases whose recognition sequence or cleavage sequence contains these four-base sequences are searched, and the four-base sequence and its corresponding methyltransferase and restriction endonuclease are identified. For classification and summarization, the methyltransferase correspondence of the four-base universal sequence is as follows:

TCGA: M.TaqI; TGCA: M.HpyCH4V; TTAA: M.EsaDix5I, M.MseI; AATT: M.MluCI; ACGT: M.HpyCH4IV, M.TaiI; AGCT: M.CvikI-1, M.AluI; CATG: M.FaeI, M.CviAII, M.NlaIII; CCGG: M.MspI, M.HpaII; CGCG: M.BstuI; CTAG: M.BfaI; GATC: M.Sau3AI, M.BstKTI, M.DpnII, M.BfuCI, M.MboI; GCGC: M.HinPII, M.HhaI; GGCC: M.HaeIII, M.CvikI-1, M.PhoI; GTAC: M.Csp6I, M.CviQI, M.RsaI; recognition cleavage The corresponding relation of the restriction endonucleases whose sequence contains the universal sequence of four bases is:

TCGA: AccI, BstBI, SalI, XhoII, TliI, PaeR7I, BspDI, ClaI; TGCA: ApaII, PstI, SfcI, NsiI; TTAA: AseI, HincII, HpaI, SmII, AflII; AATT: ApoI, EcoRI, MfeI; ACGT: SnaBI, AatII, ZraI, BsaHI, BsaAI, PmII, AcII; AGCT: SacI, Eco53kI, Ecll36II, MspAII, PvuII, HindIII; CATG: SphI, BsaJI, BtgI, StyI, NspI, AffIII, PciI; CCGG: SmaI, AvaI, AcoI, XmaI, TspMI, BsaWI, BsrFI, AgeI; CGCG: NruI, BssHII, SacII, AflIII, MluI; CTAG: XbaI, BmtI, NheI, AvrII, SpeI; GATC: NlaIV, BstYI, BsiEI, BclI, BmaHI, PvuI, BgIII; GCGC: FspI, SfoI, KasI, NarI, BanI, KasI, HacII, AfeI; GGCC: MscI, BanII, ApaI, PspOMI, EaeI, EagI, StuI; GTAC: BsrGI, KpnI, Acc65I, BsiWI, ScaI, TatI; In Figure 1, the four-base sequence in the inner circle indicates that the selected and screened four-base sequence can be used as a general-purpose four-base sequence, and the restriction endonuclease in the inner circle is the restriction endonuclease that recognizes the four-base sequence in the inner circle, The outer circle of restriction enzymes recognizes more sequences than four-base sequences, such as six-base sequences or eight-base sequences, but not the inner circle of four-base sequences in the sequence.

(4) Construction of a four-base universal sequence methyltransferase expression strain: The methyltransferase gene containing a short homologous recognition sequence was synthesized into the pUC57 vector, digested with NdeI and XhoI, and subjected to agarose gel electrophoresis. The DNA band was purified, and the purified methyltransferase gene was ligated into the pACYC184 expression vector digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into E. coli DH5α strain and inoculated with 50 μg/mL chlorine Propagation culture on a culture plate with 50 μg/mL chloramphenicol, the plate was grown overnight at 37°C, a single clone was picked and grown overnight in LB medium containing 50 μg/mL chloramphenicol, the plasmid was extracted and sequenced by the kit, Finally, the expression vector pACYC184 plasmid with correct sequencing and target methyltransferase was transformed into Escherichia coli ER2566 strain and spread on a culture plate containing chloramphenicol resistance to construct a four-base universal sequence methyltransferase expression strains.

(5) Screening four-base universal sequence methyltransferase expressing strains: According to the methylation sequence and methylation pattern of the selected methyltransferase, the identification sequence is selected to include the methylation sequence and the methylation pattern Sensitive restriction endonucleases were used to judge the effect of methylation protection on the DNA in the methyltransferase expressing strain. The plasmids of each transformant in the above plate were extracted, and the protection of methyltransferases was detected by restriction endonucleases. , the enzyme activity test system is 20μl, the final concentration is 1x FlashOne buffer (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100μg/ml BSA (pH7.9@25℃)), 1U corresponding commercial 200 ng of plasmid was reacted with the restriction endonuclease at 37 °C for 30 min. After the reaction, use 1x TBE agarose gel with a concentration of 0.8% for electrophoresis detection. If the plasmid will not be digested by the corresponding restriction enzyme, the methyltransferase expressed by the transformant containing the plasmid can be protected. The DNA in this strain is protected from the cleavage of the corresponding restriction endonuclease, and the transformant whose plasmid cannot be digested is used as the host strain expressing the restriction endonuclease, which is used for the expression of the restriction endonuclease in the subsequent steps.

(6) Construction of restriction endonuclease recombinant expression strain: The restriction endonuclease gene whose recognition sequence contains the methylation sequence and is sensitive to the methylation mode is synthesized into the pUC57 vector, and digested with NdeI and XhoI , the band was purified by agarose gel electrophoresis, the purified restriction endonuclease gene was ligated to the expression vector pBAD digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into Escherichia coli DH5α strain and inoculated onto culture plates containing 50 μg/mL ampicillin, and the plates were grown overnight at 37°C. A single clone was picked and grown overnight in LB medium containing 50 μg/mL ampicillin, and the plasmid was extracted and sequenced by the kit. Finally, the expression vector pBAD plasmid with correct sequencing and target restriction endonuclease was transformed into the methyltransferase protection strain with short homologous recognition sequence screened in the previous step and inoculated with 50 μg/mL chloramphenicol On the culture plate with 50 μg/mL ampicillin, construct the recombinant expression strain of restriction endonuclease.

(7) Screening of recombinant expression strains of restriction endonucleases: inoculate the transformants obtained in the previous step into LB medium containing 50 μg/mL chloramphenicol and 50 μg/mL ampicillin for culture, and when cultured to late logarithmic stage, Add L-arabinose with a final concentration of 0.2% (m/v) to induce 16 hours at 16°C. After the induction, the cells were collected by centrifugation and sonicated. The crude enzyme extract was obtained by centrifugation again. The endonuclease activity was used to test the activity of restriction endonucleases in the transformants. The enzyme activity test system was 50 μl, and the final concentration was 1x FlashOne buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ ml BSA (pH7.9@25℃)) plus 5μl of crude enzyme solution and 1μg of substrate DNA to react at the optimum reaction temperature for 60min, after the reaction, 20μg of proteinase K was added and treated at 37℃ for 15min, using 1x TBE with a concentration of 0.8 % agarose gel for electrophoresis detection, and compare the electrophoresis detection results with the substrate DNA bands digested with commercial restriction endonucleases to determine the restriction endonuclease activity in the crude enzyme extract, which can be completely And the transformant corresponding to the crude enzyme extract of the substrate DNA that is correctly digested can be used as the restriction endonuclease in the subsequent steps to express in large quantities.

Example:

(1) Strain and plasmid:

The strains were all from the American Culture Collection Center (ATCC); the Escherichia coli DH5α strain was purchased from Beijing Quanshijin Biotechnology Co., Ltd., a commercially available product; the Escherichia coli ER2566 strain was purchased from ThermoFisher Scientific, Commercially available products; pBAD, pACYC184, pUC19 vector plasmids were purchased from Miaoling biological plasmid platform; enzyme activity detection substrate λDNA (HindIII digest) was purchased from Thermo Fisher Scientific, commercially available products;

(2) Instruments and reagents:

High-pressure cell disruptor: JN-02C low temperature and ultra-high pressure continuous flow cell disruptor was purchased from Guangzhou Juneng Nano Biotechnology Co., Ltd.; commercial restriction enzymes were purchased from New England Biolabs (NEB), and antibiotics were purchased from Mona Biotechnology Ltd. (Monad Biotech)

(3) Methyltransferase gene sequence and restriction endonuclease sequence:

All gene sequences were obtained from the REBASE database (http://rebase.neb.com).

Example 1: Recombinant expression strain of restriction endonuclease constructed by methyltransferase M.AluI

(1) Construction of recombinant expression strain of methyltransferase M.AluI

S1: Construction of methyltransferase M.AluI recombinant expression plasmid: The methyltransferase M.AluI gene was synthesized into the pUC57 vector, digested with NdeI and XhoI, and the DNA band was purified by agarose gel electrophoresis. The methyltransferase M.AluI gene was ligated into the pACYC184 expression vector digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into strain E. coli DH5α and inoculated onto a culture plate containing 50 μg/mL chloramphenicol. Plates were grown overnight at 37°C, single clones were picked and grown overnight in LB medium containing 50 μg/mL chloramphenicol, plasmids were extracted by the kit and sequenced, and finally, the sequencing was correct and had methyltransferase M .AluI expression vector pACYC18-M.AluI plasmid was transformed into Escherichia coli ER2566 engineering strain and spread on a culture plate containing 50 μg/mL chloramphenicol resistance, cultured to construct a methyltransferase M.AluI recombinant expression plasmid .

S2: Screen the recombinant expression strain of methyltransferase M.AluI: the recognition sequence of methyltransferase M.AluI is AGCT, and the methylation site and method are C-position m5, so the restriction endonucleases AluI, SacI, HindIII and PvuII were used to screen the effect of methylation protection, and the plasmid of each transformant in the above plate was extracted, and the protection of methyltransferase was detected by restriction endonuclease. The enzyme activity test system was 20 μl, and the final concentration was 1x FlashOne Buffer (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100μg/ml BSA (pH7.9@25℃)), 1U restriction endonucleases AluI, SacI, HindIII, PvuII for verification of digestion; React with 200ng of plasmid at 37℃ for 30min; after the reaction, use 1x TBE concentration 0.8% agarose gel for electrophoresis detection; if the plasmid will not be digested by the corresponding restriction endonuclease, the transformant containing the plasmid The expressed methyltransferase can protect the DNA in the strain from being cut by the corresponding restriction endonucleases; the transformants whose plasmids will not be cut by the restriction enzymes will be used to express the restriction endonucleases AluI, SacI, HindIII , the methyltransferase-protected strain of PvuII, as shown in A in Figure 3, was used as restriction endonuclease expression for the subsequent steps.

(2) Construction of recombinant expression strains of restriction enzymes AluI, HindIII and PvuII

S1: Construction of recombinant expression plasmids of restriction enzymes AluI, HindIII, PvuII: The restriction enzymes AluI, HindIII, and PvuII genes were synthesized into pUC57 vector, digested with NdeI and XhoI, and subjected to agarose gel electrophoresis Purify the band; the restriction endonucleases AluI, HindIII, and PvuII genes were ligated to the expression vector pBAD digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into E. coli DH5α strain and inoculated into a On a culture plate containing 50 μg/mL ampicillin, the plate was grown overnight at 37°C; a single clone was picked and grown in LB medium containing 50 μg/mL ampicillin, and the plasmid was extracted and sequenced by the kit; finally, the sequenced The correct expression vectors pBAD-AluI, pBAD-HindIII, pBAD-PvuII plasmids with target restriction endonucleases were transformed into the methyltransferase AluI protection strain screened in the previous step and inoculated with 50 μg/mL chloramphenicol and 50 μg/mL ampicillin on culture plates.

S2: Screen for recombinant expression strains of restriction enzymes AluI, HindIII, PvuII: Inoculate the transformants obtained by transforming the expression vectors pBAD-AluI, pBAD-HindIII, pBAD-PvuII plasmids to 50 μg/mL ampicillin and 50 μg/mL chlorine Cultivation in LB medium of pyridoxine, and after culturing to late log phase and induction with a final concentration of 0.2% (m/v) L-arabinose at 16°C for 16 hours, crude extracts were prepared to test for restriction in transformants. Dicer activity; the enzyme activity test system is 50 μl, and the final concentration is 1x FlashOne buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA (pH7.9@25℃)) plus 5 μl of crude enzyme solution and 1 μg of substrate DNA were reacted at the optimum reaction temperature for 60 min; after the reaction, 20 μg of proteinase K was added, and after treatment at 37°C for 15 min, electrophoresis was performed on agarose gel with 1x TBE concentration of 0.8%, as shown in the figure shown in A in 4.

Example 2: Strains constructed with methyltransferase M.EsaDix5I

(1) Construction of recombinant expression strain of methyltransferase M.EsaDix5I

S1: Construction of methyltransferase M.EsaDix5I recombinant expression plasmid: The methyltransferase M.EsaDix5I gene was synthesized into pUC57 vector, digested with NdeI and XhoI, and the DNA band was purified by agarose gel electrophoresis; The methyltransferase M.EsaDix5I gene was ligated into the pACYC184 expression vector digested with NdeI and XhoI using T4 Ligase, and the ligation product was transformed into strain E. coli DH5α and inoculated onto a culture plate containing 50 μg/mL chloramphenicol. Plates were grown overnight at 37°C; single clones were picked and grown overnight in LB medium containing 50 μg/mL chloramphenicol, plasmids were extracted by the kit and sequenced; finally, the sequencing was correct and had methyltransferase M .EsaDix5I expression vector pACYC18-M.EsaDix5I plasmid was transformed into E. coli ER2566 engineered strain and spread on a culture plate containing 50 μg/mL chloramphenicol resistance.

S2: Screening the recombinant expression strain of methyltransferase M.EsaDix5I: the recognition sequence of methyltransferase M.EsaDix5I is TTAA, and the methylation site and method are the fourth A-position m6, so the restriction endonuclease AseI can be used , HpaI, DraI, PacI, MseI to screen the methylation protection effect; extract the plasmid of each transformant in the above plate, and use restriction endonuclease to detect the protection of methyltransferase; the enzyme activity test system is 20 μl, with Final concentration of 1x FlashOne buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA (pH 7.9 @ 25°C)), 1 U recognition sequence containing recognition methyltransferase M.EsaDix5I The restriction endonucleases HpaI, DraI, AseI, PacI, MseI of the homologous sequence were verified by enzyme digestion; reacted with 200ng of plasmid at 37℃ for 30min; after the reaction was completed, use 1x TBE concentration of 0.8% agarose gel for electrophoresis detection ; If the plasmid will not be cut by the corresponding restriction enzyme, then the methyltransferase expressed by the transformant containing the plasmid can protect the DNA in the strain from being cut by the corresponding restriction enzyme; Transformants that will not be digested will be used as methyltransferase-protected strains expressing restriction enzymes HpaI, DraI, AseI, PacI, MseI, see part B in Figure 3, as restriction endonucleases for subsequent steps. Dicer expression.

(2) Construction of recombinant expression strains of restriction enzymes HpaI, DraI, AseI, PacI, MseI

S1: Construction of recombinant expression plasmids of restriction enzymes HpaI, DraI, AseI, PacI, MseI: The restriction enzymes HpaI, DraI, AseI, PacI, MseI genes were synthesized into the pUC57 vector, and NdeI and XhoI were used for the enzyme Then, the bands were purified by agarose gel electrophoresis; the restriction endonucleases HpaI, DraI, AseI, PacI, MseI genes were ligated to the expression vector pBAD digested with NdeI and XhoI using T4Ligase, and the ligated products were Transformed into Escherichia coli DH5α strain and inoculated onto culture plates containing 50 μg/mL ampicillin, the plates were grown overnight at 37°C; single clones were picked and grown in LB medium containing 50 μg/mL ampicillin, by The plasmids were extracted and sequenced by the kit; finally, the expression vectors pBAD-HpaI, pBAD-DraI, pBAD-AseI, pBAD-PacI, pBAD-MseI plasmids with correct sequencing and target restriction enzymes were transformed into the plasmids screened in the previous step. Methyltransferase EsaDix5I protected strains and inoculated onto culture plates containing 50 μg/mL chloramphenicol and 50 μg/mL ampicillin.

S2: Screen the recombinant expression strains of restriction enzymes HpaI, DraI, AseI, PacI, MseI: transform the transformants obtained from the expression vectors pBAD-HpaI, pBAD-DraI, pBAD-AseI, pBAD-PacI, pBAD-MseI plasmids It was inoculated into LB medium containing 50 μg/mL ampicillin and 50 μg/mL chloramphenicol for culture, and when cultured to the late logarithmic stage, induced with a final concentration of 0.2% (m/v) L-arabinose at 16°C for 16 hours Afterwards, crude extracts were prepared to test the activity of restriction endonucleases in transformants; the enzyme activity test system was 50 μl, and the final concentration was 1x FlashOne buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg /ml BSA (pH7.9@25℃)) plus 5μl of crude enzyme solution and 1μg of substrate DNA to react at the optimal reaction temperature for 60min; after the reaction, 20μg of proteinase K was added, and after treatment at 37℃ for 15min, the concentration of 1x TBE was used as 0.8% agarose gel for electrophoresis detection, see part B in Figure 4.

Example 3: Strains constructed with methyltransferase M.Hhal

(1) Construction of recombinant expression strain of methyltransferase M.HhaI

S1: Construction of methyltransferase M.HhaI recombinant expression plasmid: The methyltransferase M.HhaI gene was synthesized into the pUC57 vector, digested with NdeI and XhoI, and the DNA band was purified by agarose gel electrophoresis; The methyltransferase M.HhaI gene was ligated into the pACYC184 expression vector digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into strain E. coli DH5α and inoculated onto a culture plate containing 50 μg/mL chloramphenicol. Plates were grown overnight at 37°C; single clones were picked and grown in LB medium containing 50 μg/mL chloramphenicol, and plasmids were extracted and sequenced by the kit; The expression vector pACYC18-M.HhaI plasmid was transformed into Escherichia coli ER2566 engineering strain and spread on a culture plate containing 50 μg/mL chloramphenicol resistance.

S2: Screening the recombinant expression strain of methyltransferase M.HhaI: the recognition sequence of methyltransferase M.HhaI is GCGC, and the methylation site and method are the second C-position m5, so the restriction endonuclease HhaI can be used , SfoI, FspI, AfoI, HinPII, NarI, PluTI to screen the methylation protection effect; extract the plasmid of each transformant in the above plate, and use restriction endonuclease to detect the protection of methyltransferase; enzyme activity test system 20 μl at a final concentration of 1x FlashOne buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA (pH 7.9 @ 25°C)), 1 U recognition sequence containing recognition methyl transfer Restriction endonucleases HhaI, SfoI, FspI, AfoI, HinPII, NarI, and PluTI of the homologous sequence of enzyme M.HhaI were verified by digestion; reacted with 200ng of plasmid at 37℃ for 30min; after the reaction, 1x TBE was used at a concentration of 0.8% If the plasmid will not be cut by the corresponding restriction endonuclease, the methyltransferase expressed by the transformant containing the plasmid can protect the DNA in the strain from the corresponding restriction Endonuclease cleavage; transformants whose plasmids will not be digested will be used as methyltransferase-protected strains expressing the restriction enzymes Hhal, SfoI, FspI, AfoI, HinPII, NarI, and PluTI (Figure 3C). ), used as restriction endonuclease expression for subsequent steps.

(2) Construction of recombinant expression strains of restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, and PluTI

S1: Construction of recombinant expression plasmids of restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, PluTI: The restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, and PluTI genes were synthesized into pUC57 vector, using NdeI Digested with XhoI, and purified the band by agarose gel electrophoresis; the restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, and PluTI genes were connected to the expression vector digested with NdeI and XhoI using T4 Ligase. pBAD, and the ligation product was transformed into Escherichia coli DH5α strain and inoculated on a culture plate containing 50 μg/mL ampicillin, and the plate was grown overnight at 37 °C; Grow in LB medium, extract the plasmids by the kit and sequence; finally, the expression vectors pBAD-HhaI, pBAD-SfoI, pBAD-AfoI, pBAD-HinpII, pBAD-NarI with correct sequencing and target restriction endonucleases, The pBAD-PluTI plasmid was transformed into the methyltransferase HhaI protection strain screened in the previous step and inoculated onto a culture plate containing 50 μg/mL chloramphenicol and 50 μg/mL ampicillin.

S2: Screen the recombinant expression strains of restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, PluTI: transform the expression vectors pBAD-HhaI, pBAD-SfoI, pBAD-AfoI, pBAD-HinpII, pBAD-NarI, pBAD- The transformants obtained from the PluTI plasmid were inoculated into LB medium containing 50 μg/mL ampicillin and 50 μg/mL chloramphenicol, and were cultured to the late logarithmic stage at 16°C with a final concentration of 0.2% (m/v) L - Crude extracts were prepared after 16 hours of arabinose induction to test the activity of restriction enzymes in transformants; the enzyme activity assay system was 50 μl at a final concentration of 1x FlashOne buffer (50 mM potassium acetate, 20 mM Tris-acetate) , 10mM magnesium acetate, 100μg/ml BSA (pH7.9@25℃)) plus 5μl of crude enzyme solution and 1μg of substrate DNA to react at the optimum reaction temperature for 60min; after the reaction, 20μg of proteinase K was added and treated at 37℃ for 15min This was followed by electrophoresis using a 1x TBE 0.8% agarose gel; see Figure 4, part C.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (7)

1. the engineering strain for recombinant expression restriction endonuclease, is characterized in that: take Escherichia coli ER2566 as the engineering strain transformation basis, and this engineering strain comprises the following conditions: (1) Compatible with exogenous methyltransferases; (2) The prokaryotic expression vector pACYC184 containing the methyltransferase gene that can protect the universal sequence of four bases; (3) The prokaryotic expression vector pBAD containing the restriction endonuclease gene whose recognition cleavage sequence comprises a four-base universal sequence; wherein, the four-base universal sequence includes: TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG , CGCG, CTAG, GATC, GCGC, GGCC, GTAC. 2. the construction method of the engineering strain for recombinant expression restriction endonuclease as claimed in claim 1, is characterized in that: described construction method specifically comprises the steps: S1: Screening of engineering strains: It is determined that E. coli ER2566 is used as the basis for the transformation of engineering strains. The engineering strains are compatible with exogenous methyltransferases and contain prokaryotic expression vectors that can protect the methyltransferase gene of the four-base universal sequence. A prokaryotic expression vector that recognizes a restriction endonuclease gene whose cleavage sequence comprises a four-base universal sequence, wherein the four-base universal sequence is TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC , GCGC, GGCC, GTAC. S2: Select expression plasmid: select pACYC184 plasmid with Amp promoter as methyltransferase expression plasmid, use resistance to chloramphenicol, select pBAD plasmid with arabinose operon and promoter as restriction endonuclease Expression plasmid, using ampicillin resistance; S3: Intermediate strain acquisition: According to the four-base universal sequence combination and methylation pattern in S1, a specific methyltransferase gene is selected, and the selected specific methyltransferase gene is connected with pACYC184 in S2 to obtain a recombinant plasmid. The recombinant plasmid Transform into Escherichia coli ER2566 strain to obtain an intermediate strain; S4: Screening strains: According to the methylation sequence and methylation site mode of the methyltransferase selected by S3, the identification sequence is selected to include the four-base universal sequence and the methylation sensitivity is the same as that of the methyltransferase action site The restriction endonuclease, cutting the DNA of the intermediate strain obtained in step S3, and screening out the methylation protection strain that cannot be cut, S5: Construction of expression strain: connect the gene corresponding to the restriction endonuclease in S4 with pBAD in S2 to obtain a recombinant plasmid, and transform the recombinant plasmid into the S4 screening strain to obtain an engineered strain that can express the corresponding restriction endonuclease; S6: Expression of the engineered strain: The engineered strain of the restriction endonuclease obtained in step S5 is used to induce the expression of the restriction endonuclease. 3. The engineering strain for recombinant expression restriction endonuclease according to claim 1 or 2, wherein: the methyltransferase of the four-base universal sequence comprises: (1) TCGA: M.TaqI; (2) TGCA: M.HpyCH4V; (3) TTAA: M.EsaDix5I, M.MseI; (4) AATT: M.MluCI; (5) ACGT: M.HpyCH4IV, M.TaiI; (6) AGCT: M .CvikI-1, M.AluI; (7) CATG: M.FaeI, M.CviAII, M.NlaIII; (8) CCGG: M.MspI, M.HpaII; (9) CGCG: M.BstuI; (10 ) CTAG: M.BfaI; (11) GATC: M.Sau3AI, M.BstKTI, M.DpnII, M.BfuCI, M.MboI; (12) GCGC: M.HinPII, M.HhaI; (13) GGCC: M.HaeIII, M.CvikI-1, M.PhoI; (14) GTAC: M.Csp6I, M.CviQI, M.RsaI; the restriction endonucleases whose recognition cleavage sequences comprise four-base universal sequences include: (1) TCGA: AccI, BstBI, SalI, XhoII, TliI, PaeR7I, BspDI, ClaI; (2) TGCA: ApaII, PstI, SfcI, NsiI; (3) TTAA: AseI, HincII, HpaI, SmII, AflII; ( 4) AATT: ApoI, EcoRI, MfeI; (5) ACGT: SnaBI, AatII, ZraI, BsaHI, BsaAI, PmII, AcII; (6) AGCT: SacI, Eco53kI, Ecll36II, MspAII, PvuII, HindIII; (7) CATG : SphI, BsaJI, BtgI, StyI, NspI, AffIII, PciI; (8) CCGG: SmaI, AvaI, AcoI, XmaI, TspMI, BsaWI, BsrFI, AgeI; (9) CGCG: NruI, BssHII, SacII, AflIII, MluI ; (10) CTAG: XbaI, BmtI, NheI, AvrII, SpeI; (11) GATC: NlaIV, BstYI, BsiEI, BclI, BmaHI, PvuI, BgIII; (12) GCGC: FspI, SfoI, KasI, NarI, BanI, KasI, HacII, AfeI; (13) GGCC: MscI, BanII, ApaI, PspOMI, EaeI, EagI, StuI; (14) GT AC: BsrGI, KpnI, Acc65I, BsiWI, ScaI, TatI. 4. the construction method of the engineering strain for recombinant expression restriction endonuclease according to claim 2, is characterized in that: described S3 intermediate strain acquisition step is as follows: (1) Synthesize the methyltransferase gene containing the short homologous recognition sequence on the pUC57 vector, use NdeI and XhoI to carry out enzyme digestion, and carry out DNA band purification by agarose gel electrophoresis; (2) The above purified methyltransferase gene was ligated into the pACYC184 expression vector digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into E. coli DH5α strain and inoculated into 50 μg/mL chloramphenicol The plates were grown overnight at 37°C, and single clones were picked and grown overnight in LB medium containing 50 μg/mL chloramphenicol, and the plasmids were extracted and sequenced by the kit; (3) The expression vector pACYC184 plasmid with correct sequencing and target methyltransferase was transformed into Escherichia coli ER2566 strain and spread on a culture plate containing chloramphenicol resistance to obtain an intermediate strain. 5. the construction method of the engineering strain for recombinant expression restriction endonuclease according to claim 2, is characterized in that: screening strain step in described S4 is as follows: (1) Extract the plasmid of each transformant in the S3 plate, use restriction endonucleases to detect the protection of methyltransferase, the enzyme activity test system is 20 μl, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM acetic acid Magnesium, 100μg/ml BSA (pH7.9@25°C) with final concentration of 1x FlashOne buffer, 1U corresponding commercial restriction endonuclease and 200ng plasmid were reacted at 37°C for 30min; (2) After the reaction, use agarose gel with 1x TBE concentration of 0.8% for electrophoresis detection. If the plasmid will not be digested by the corresponding restriction endonuclease, the methyltransferase expressed by the transformant containing the plasmid will be detected. The DNA in the strain can be protected from the cleavage of the corresponding restriction endonuclease; (3) The transformants whose plasmids cannot be digested are screened out as host strains expressing restriction endonucleases and used as restriction endonuclease expressing strains in subsequent steps. 6. the construction method of the engineering strain that is used for recombinant expression restriction endonuclease as claimed in claim 2, is characterized in that: in described S5, expression strain construction step is as follows: (1) Synthesize the restriction endonuclease gene whose recognition sequence contains the methylation sequence and is sensitive to the methylation pattern into the pUC57 vector, use NdeI and XhoI for restriction enzyme digestion, and conduct banding by agarose gel electrophoresis purification; (2) The purified restriction endonuclease gene was ligated to the expression vector pBAD digested with NdeI and XhoI using T4 Ligase, and the ligated product was transformed into Escherichia coli DH5α strain, and inoculated into a 50 μg/mL On the culture plates of ampicillin, the plates were grown overnight at 37°C, and single clones were picked and grown overnight in LB medium containing 50 μg/mL ampicillin, and the plasmids were extracted and sequenced by the kit; (3) Transform the expression vector pBAD plasmid with the correct sequencing and target restriction enzyme into the methyltransferase protection strain with short homologous recognition sequence screened in the previous step and inoculate it into the chloramphenicol containing 50 μg/mL The expression strains were constructed on a culture plate containing 50 μg/mL ampicillin and ampicillin. 7. the construction method of the engineering strain for recombinant expression restriction endonuclease according to claim 2, is characterized in that: the expression step of engineering strain in described S6 is as follows: (1) The transformants obtained by S5 transformation were inoculated into LB medium containing 50 μg/mL chloramphenicol and 50 μg/mL ampicillin for culture, and when cultured to late logarithmic stage, the final concentration of 0.2% (m /v) L-arabinose induction for 16 hours is the expression of the engineered strain recombinantly expressing restriction endonucleases; (2) After the induction, the cells were collected by centrifugation and crushed by sonication, and the crude enzyme extract was obtained by centrifugation again. The test system is 50 μl, with 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA (pH 7.9@25°C) final concentration of 1x FlashOne buffer, plus 5 μl of crude enzyme solution and 1 μg base The biological DNA was reacted at the optimum reaction temperature for 60 min; (3) After the reaction, 20 μg of proteinase K was added, and after treatment at 37°C for 15 minutes, electrophoresis was performed on agarose gel with 1× TBE concentration of 0.8%. The DNA bands are compared to determine the restriction endonuclease activity in the crude enzyme extract, and the transformant corresponding to the crude enzyme extract that can completely and correctly cut the substrate DNA can be used as the restriction endonuclease in the subsequent steps. Engineered strains that express Dicer in large quantities.

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