CN110643560A - Engineering strain for recombinant expression of restriction endonuclease and construction method thereof - Google Patents
Engineering strain for recombinant expression of restriction endonuclease and construction method thereof Download PDFInfo
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Abstract
The invention discloses an engineering strain for recombinant expression of restriction enzyme and a construction method thereof, the engineering strain is Escherichia coli ER2566, and the engineering strain comprises the following conditions: compatible with exogenous methyltransferases; prokaryotic expression vector pACYC184 containing methyltransferase gene capable of protecting four-base universal sequence; a prokaryotic expression vector pBAD containing a restriction endonuclease gene whose recognition cleavage sequence comprises a four-base universal sequence; wherein, the four-base universal sequence comprises: TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC; compared with the prior art, the invention does not need to screen specific methylation protection strains for each restriction enzyme, thereby greatly reducing the number of methyltransferase protection strains required for producing restriction enzymes.
Description
Technical Field
The invention relates to biochemistry and engineering strains, in particular to an engineering strain for recombinant expression of restriction enzyme and a construction method thereof.
Background
Restriction enzymes, referred to as restriction enzymes for short, are one of the most important biomedical tool enzymes, and are widely used in various biomedical fields such as DNA recombination, gene localization and cloning, gene structure research, DNA sequence analysis and determination, and gene synthesis. The natural restriction enzyme is mainly derived from prokaryotes, and forms a 'restriction-modification' system with corresponding methyltransferases, wherein the 'restriction-modification' is mainly used for resisting the entry of exogenous DNA molecules, the biological meaning of the 'restriction-modification' is to protect a host from phage infection, in an original host, the methyltransferases can methylate specific sequences of plasmids contained in a host genome and self cells thereof, the corresponding restriction enzymes cannot recognize and cut the methylated DNA, and when the exogenous DNA enters the host, the exogenous DNA is not methylated and can be cut into fragments by the restriction enzymes, so that the exogenous DNA loses functions. Most of the early restriction enzymes were isolated from their original strains at high cost and low yield, and many of the natural microorganisms were not suitable for industrial culture and could not be used for extraction and isolation of restriction enzymes, and then scientists cloned restriction enzyme genes and transferred them into engineered strains for recombinant expression to increase their yields. However, because the restriction enzyme has the ability to cut DNA which is not protected by methylation, the restriction enzyme has strong cytotoxicity in engineering strains which are not protected by corresponding methyltransferases, so that the difficulty of recombinant expression is very high. The most effective recombinant expression strategy of restriction endonucleases at present is mainly to use a specific "restriction-modification" system: firstly, transferring a specific methyltransferase gene which is homologous with a specific restriction enzyme and identifies and modifies the same DNA sequence into a proper escherichia coli expression strain to express the methyltransferase; then screening out a protective strain which is protected by methylation modification of a specific site of host DNA and cannot be cut by corresponding restriction enzyme; finally, transferring the corresponding restriction enzyme gene into the protective strain to realize recombinant expression.
Because each restriction enzyme has corresponding methyltransferase, if more than 3000 restriction enzymes which are found at present are recombined and expressed by adopting the strategy, more than 3000 methyltransferases are required to be screened to protect strains, the workload is huge, and the efficiency is low; recently, researchers have discovered a broad spectrum of methyltransferases such as m.sssi derived from the spiroplama sp.mq1 strain, i.e., capable of methylating all cytosine residues in the recognition sequence 5 '-CG-3', and m.cvipi derived from Chlorella (Chlorella) virus NYs-1, i.e., capable of methylating all cytosine residues in the recognition sequence 5 '-GC-3', etc.; however, it is known that more than 70 restriction enzymes contain CG and more than 50 restriction enzymes contain GC. Theoretically, if the broad-spectrum methyltransferase gene is transferred into an engineering strain, CG or GC sequences on DNA can also resist restriction enzyme cutting, so that the broad-spectrum methyltransferase gene can be used for recombinant expression of restriction enzymes, and cases of successfully expressing restriction enzymes such as NotI, PstI and the like by using the strategy are reported at present. However, CG or GC is subjected to extensive methylation sites on the genome, which often causes excessive metabolic burden on a host and also has lethal effect, so that the restriction enzyme is greatly restricted by using the strategy for recombinant expression, therefore, the invention aims to obtain a recombinant expression strain for expressing and recognizing various restriction enzymes with short homologous sequences by using the methyltransferase for modifying the short homologous sequences, so as to produce the restriction enzyme economically and efficiently.
Disclosure of Invention
The invention establishes a restriction enzyme containing a short homologous sequence by screening and expressing universal four-base sequence methyltransferase, and particularly relates to a restriction enzyme expression method for greatly improving the efficiency and reducing the cost by utilizing four-base universal sequence methylation protection, wherein the specific scheme is as follows:
an engineered strain for recombinant expression of a restriction enzyme, characterized in that: taking Escherichia coli ER2566 as a basis for engineering strain transformation, wherein the engineering strain comprises the following conditions:
(4) compatible with exogenous methyltransferases;
(5) prokaryotic expression vector pACYC184 containing methyltransferase gene capable of protecting four-base universal sequence;
(6) a prokaryotic expression vector pBAD containing a restriction endonuclease gene whose recognition cleavage sequence comprises a four-base universal sequence;
wherein, the four-base universal sequence comprises: TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC.
The construction method of the engineering strain for recombinant expression of the restriction enzyme is characterized in that: the construction method specifically comprises the following steps:
s1: screening engineering strains: the Escherichia coli ER2566 is used as the engineering strain modification basis, and the engineering strain is compatible with exogenous methyltransferase. The vector also comprises a prokaryotic expression vector of a methyltransferase gene capable of protecting a four-base universal sequence and a prokaryotic expression vector of a restriction endonuclease gene containing a recognition cutting sequence containing the four-base universal sequence, wherein the four-base universal sequence is TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC and GTAC.
S2: selection of expression plasmids: selecting pACYC184 plasmid with Amp promoter as methyltransferase expression plasmid, using resistance as chloramphenicol, selecting pBAD plasmid with arabinose operon and promoter as restriction enzyme expression plasmid, using resistance as ampicillin;
s3: obtaining an intermediate strain: selecting a specific methyltransferase gene according to the four-base universal sequence combination and methylation pattern in S1, connecting the selected specific methyltransferase gene with pACYC184 in S2 to obtain a recombinant plasmid, and transforming the recombinant plasmid into an Escherichia coli ER2566 strain to obtain an intermediate strain;
s4: screening strains: selecting a restriction enzyme whose recognition sequence contains the four-base universal sequence and whose methylation sensitivity is the same as the action site of methyltransferase according to the methylation sequence and methylation site pattern of the methyltransferase selected in S3, cleaving the DNA of the intermediate strain obtained in step S3, selecting a methylation-protected strain that cannot be cleaved,
s5: construction of an expression strain: connecting the gene corresponding to the restriction enzyme in S4 with pBAD in S2 to obtain recombinant plasmid, and transforming the recombinant plasmid into S4 screening strains to obtain engineering strains capable of expressing the corresponding restriction enzyme;
s6: expression of the engineered strain: and (4) inducing and expressing the restriction endonuclease by using the engineering strain of the restriction endonuclease obtained in the step S5.
Further, the four base universal sequence methyltransferase comprises: (1) TCGA: M.TaqI; (2) TGCA: HpyCH4V; (3) TTAA: m.esadix5i, m.msei; (4) AATT: M.MlucI; (5) ACGT: m.hpych4iv, m.taii; (6) AGCT: CvikI-1, AluI; (7) CATG: m.faei, m.cviaii, m.nlaiiii; (8) CCGG: m.mspi, m.hpaii; (9) CGCG: bstui; (10) CTAG: M.BfaI; (11) GATC: m.sau3ai, m.bstkti, m.dpni, 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 enzyme which recognizes that the cleavage sequence comprises a four-base universal sequence includes: (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, bshii, 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, TatI
Further, the S3 intermediate strain obtaining steps are as follows:
(1) synthesizing a methyltransferase gene containing a short homologous recognition sequence onto a pUC57 vector, performing enzyme digestion by NdeI and XhoI, and performing DNA band purification by agarose gel electrophoresis;
(2) the purified methyltransferase gene was ligated to the NdeI and XhoI digested pACYC184 expression vector using T4 Ligase, the ligation product was transformed into E.coli DH 5. alpha. strain and inoculated onto 50. mu.g/mL chloramphenicol resistant plates grown overnight at 37 ℃, individual clones were picked and grown overnight in LB medium containing 50. mu.g/mL chloramphenicol, plasmids were extracted with the kit and sequenced;
(3) the expression vector pACYC184 plasmid, which was correctly sequenced and had the methyltransferase of interest, was transformed into the E.coli ER2566 strain and plated on chloramphenicol-resistant culture plates to obtain intermediate strains.
Further, the step of screening strains in S4 is as follows:
(1) extracting plasmids of each transformant in an S3 plate, detecting the protection of methyltransferase by using restriction endonuclease, wherein the enzyme activity test system is 20 mu l, 50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate and 100 mu g/ml BSA (pH7.9@25 ℃) are used as a 1XFlashOne buffer solution, and 1U of corresponding commercial restriction endonuclease reacts with 200ng plasmid at 37 ℃ for 30 min;
(2) after the reaction is finished, agarose gel with the concentration of 1x TBE of 0.8% is used for carrying out electrophoresis detection, the plasmid cannot be cut by the corresponding restriction enzyme, and the methyltransferase expressed by the transformant containing the plasmid can protect DNA in the strain from being cut by the corresponding restriction enzyme;
(3) transformants which could not be digested by the plasmid were selected as host strains for expression of restriction enzymes and used as restriction enzyme expression strains in the subsequent steps.
Further, the construction steps of the expression strain in S5 are as follows:
(1) synthesizing a restriction endonuclease gene, the recognition sequence of which contains the methylation sequence and is sensitive to the methylation pattern, into a pUC57 vector, performing enzyme digestion using NdeI and XhoI, and performing band purification by agarose gel electrophoresis;
(2) the purified restriction endonuclease gene was ligated to NdeI and XhoI digested expression vector pBAD using T4 Ligase, and the ligation product was transformed into E.coli DH 5. alpha. strain and inoculated onto 50. mu.g/mL ampicillin-containing culture plate, which was grown overnight at 37 ℃, and single clones were picked up to LB medium containing 50. mu.g/mL ampicillin and grown overnight, and plasmids were extracted by kit and sequenced;
(3) the expression vector pBAD plasmid with correct sequencing and the target restriction enzyme was transformed into the methyltransferase protective strain using the short homology recognition sequence selected in the previous step and inoculated onto a culture plate containing 50. mu.g/mL chloramphenicol and 50. mu.g/mL ampicillin to construct an expression strain.
Further, the expression steps of the engineering strain in S6 are as follows:
(1) inoculating a transformant obtained by transforming S5 into an LB culture medium containing 50 mug/mL of chloramphenicol and 50 mug/mL of ampicillin for culture, and adding L-arabinose with a final concentration of 0.2% (m/v) at 16 ℃ to induce for 16 hours when the culture reaches the late logarithmic phase, namely the expression of the engineering strain for recombining and expressing the restriction enzyme;
(2) after induction, centrifugally collecting thalli, crushing by using ultrasonic waves, centrifugally obtaining an enzyme crude extract again, detecting the activity of the restriction enzyme in the enzyme crude extract to test the activity of the restriction enzyme in a transformant, wherein an enzyme activity test system is 50 mu l, and a final concentration of 1xFlashOne buffer solution is prepared by adding 5 mu l of crude enzyme solution and 1 mu g of substrate DNA at an optimal reaction temperature for 60min by using 50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate and 100 mu g/ml BSA (pH7.9@25 ℃);
(3) and after the reaction is finished, adding 20 mu g of proteinase K, treating for 15min at 37 ℃, performing electrophoresis detection by using agarose gel with the concentration of 1x TBE of 0.8%, comparing the electrophoresis detection result with a substrate DNA band digested by using commercial restriction endonuclease to judge the activity of the restriction endonuclease in the enzyme crude extract, and completely and correctly digesting a transformant corresponding to the enzyme crude extract of the substrate DNA, namely, the transformant can be used as an engineering strain for large-scale expression of the restriction endonuclease in the subsequent step.
Compared with the prior art, the invention has the following beneficial effects:
(1) the difficulty of searching for a specific methyl transferase gene is reduced;
(2) the number of construction of specific methyltransferase protective strains is reduced;
(3) the methylation protection strain prepared by using methyltransferase for recognizing universal four-base sequence can be used for producing various restriction endonucleases of which the recognition sequence comprises the four-base sequence and is sensitive to the methylation mode.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the technical descriptions of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a diagram showing a four-base universal sequence and restriction enzyme map recognition relationship;
FIG. 2 shows a technical scheme for constructing a methyltransferase protective strain and a restriction enzyme recombinant expression strain.
FIG. 3 is an agarose gel electrophoresis image of the screening of methyltransferase protected strains.
FIG. 4 is an agarose gel electrophoresis image of screening restriction enzyme crude enzyme activity from the methyltransferase protective strain transformed into a restriction enzyme expression vector.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention.
To achieve the objects of the present invention, as shown in figures 1-4,
(1) screening engineering strains: because an exogenous 'restriction-modification' system needs to be introduced into an escherichia coli engineering strain for expression, the compatibility problem of the escherichia coli engineering strain to methyltransferases needs to be considered, and by screening ER2566, HB101, K802, BL21(DE3), BL21(DE3) pLysS, Top10, JM109, Rosetta (DE3) and XL1-Blue strains, exogenous methyltransferases can be compatible when the escherichia coli engineering strain genotype is mcrC-mrr deletion type, and the escherichia coli ER2566 strain can meet the requirements after screening.
(2) Selection of expression plasmids: when an exogenous 'restriction-modification' system is expressed, specific methylation needs to be carried out on DNA in an escherichia coli engineering strain, when a methylation transferase expression plasmid is constructed, a constitutive expression plasmid is selected, so that the escherichia coli engineering strain can carry out methylation modification on the DNA before induction expression of a restriction enzyme, the DNA in the escherichia coli engineering strain after the methylation modification cannot be cut by the specific restriction enzyme, the cytotoxicity of the restriction enzyme is removed, a large amount of expression of the restriction enzyme can be carried out, a strict operon and a promoter are selected for carrying out restriction enzyme expression, and arabinose without background expression is selected according to the property of the promoter; meanwhile, the propagation and expression of the plasmid in an escherichia coli engineering strain can be influenced by the overlarge plasmid, so 2 plasmids are selected to respectively express methyltransferase and restriction endonuclease, and the problem of plasmid compatibility needs to be considered when a plurality of plasmids are transformed into the escherichia coli engineering strain, namely the plasmids need to have different ori of origin of replication; through screening, pACYC184 plasmid with Amp promoter is selected as methyltransferase expression plasmid, resistance is chloramphenicol, pBAD plasmid with arabinose operon and promoter is selected as restriction enzyme expression plasmid, and resistance is ampicillin.
(3) Determination of four base universal sequence methylation protocol: designing all reasonable permutation and combination according to the permutation and combination of the quadruple base sequence and the recognition or cutting base sequence of the existing restriction endonuclease: TTAA, AATT, ACGT, AGCT, ATAT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC, TATA, TCGA, TGCA; searching for methyltransferases using these four-base sequences as methylated sequences, searching for restriction endonucleases having recognition sequences or cleavage sequences comprising these four-base sequences based on these four-base sequences after the methylation sequences, and sorting and summarizing the four-base sequences and their corresponding methyltransferases and restriction endonucleases, wherein the four-base universal sequence methyltransferases have the following relationship:
TCGA: M.TaqI; TGCA: 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.nlaiiii; CCGG: m.mspi, m.hpaii; CGCG: bstui; CTAG: M.BfaI; GATC: m.sau3ai, m.bstkti, m.dpni, m.bfuci, m.mboi; GCGC: m.hinpii, m.hhai; GGCC: m.haeiii, m.cviki-1, m.phoi; GTAC: m.csp6i, m.cviqi, m.rsai; the restriction enzyme corresponding relation for recognizing that the cutting sequence comprises a four-base universal sequence is as follows:
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, bshii, 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 FIG. 1, the four-base sequence in the inner circle represents the four-base sequence selected and screened for use as a universal four-base sequence, the restriction enzyme in the inner circle is a restriction enzyme that recognizes the four-base sequence in the inner circle, and the restriction enzyme in the outer circle recognizes more than four-base sequences, such as a six-base sequence or an eight-base sequence, but the four-base sequence in the inner circle in another sequence.
(4) Constructing a four-base universal sequence methyltransferase expression strain: the methyltransferase gene containing the short homology recognition sequence was synthesized into pUC57 vector, digested with NdeI and XhoI, DNA band purification by agarose gel electrophoresis, ligation of the purified methyltransferase gene using T4 Ligase into the pACYC184 expression vector cut with NdeI and XhoI, the ligation products were transformed into E.coli DH 5. alpha. strain and inoculated on a 50. mu.g/mL chloramphenicol-resistant culture plate for propagation culture, the plate was grown overnight at 37 ℃, individual clones were picked and grown overnight in LB medium containing 50. mu.g/mL chloramphenicol, and finally, transforming the expression vector pACYC184 plasmid with correct sequencing and target methyltransferase into an Escherichia coli ER2566 strain and coating the Escherichia coli ER2566 strain on a chloramphenicol-resistant culture plate to construct a four-base universal sequence methyltransferase expression strain.
(5) Screening four-base universal sequence methyltransferase expression strains: selecting a restriction enzyme which has a recognition sequence containing the methylation sequence and is sensitive to the methylation mode as a DNA in a methyltransferase expression strain according to the methylation sequence and the methylation mode of the selected methyltransferase to judge the methylation protection effect, extracting a plasmid of each transformant in the plate, detecting the protection effect of the methyltransferase by using the restriction enzyme, and reacting 1U of the corresponding commercial restriction enzyme with 200ng of the plasmid at 37 ℃ for 30min in an enzyme activity test system of 20 mu l in a final concentration of 1XFlashOne buffer (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mu g/ml BSA (pH7.9@25 ℃). And (3) carrying out electrophoresis detection by using agarose gel with the concentration of 1x TBE of 0.8% after the reaction is finished, if the plasmid is not cut by the corresponding restriction enzyme, protecting DNA in the strain from the cutting of the corresponding restriction enzyme by methyltransferase expressed by a transformant containing the plasmid, and using the transformant with the plasmid incapable of being digested as a host strain for expressing the restriction enzyme in the subsequent step.
(6) Constructing a restriction enzyme recombinant expression strain: restriction endonuclease genes whose recognition sequences contain the methylation sequence and are sensitive to the methylation pattern were synthesized into pUC57 vector, digested with NdeI and XhoI, band-purified by agarose gel electrophoresis, the purified restriction endonuclease genes were ligated to the expression vector pBAD digested with NdeI and XhoI using T4 Ligase, and the ligation products were transformed into E.coli DH 5. alpha. strain and inoculated onto 50. mu.g/mL ampicillin-containing culture plates, which were grown overnight at 37 ℃. Single clones were picked up and grown overnight in LB medium containing 50. mu.g/mL ampicillin, plasmids were extracted by kit and sequenced. Finally, the expression vector pBAD plasmid with correct sequencing and the target restriction enzyme is transformed into the methyltransferase protective strain using the short homologous recognition sequence screened in the previous step and inoculated onto a culture plate containing 50. mu.g/mL chloramphenicol and 50. mu.g/mL ampicillin to construct a recombinant expression strain of the restriction enzyme.
(7) Screening of recombinant expression strains of restriction enzymes: inoculating the transformant obtained by the last step of transformation into LB culture medium containing 50 mug/mL of chloramphenicol and 50 mug/mL of ampicillin, culturing until the late logarithmic phase, adding L-arabinose with the final concentration of 0.2% (m/v) at 16 ℃ for induction for 16 hours, centrifugally collecting thalli after induction is finished, crushing the thalli by using ultrasonic waves, centrifugally obtaining enzyme crude extract again, detecting the activity of restriction enzyme in the enzyme crude extract to test the activity of the restriction enzyme in the transformant, testing the activity of the enzyme with 50 mug, reacting 1XFlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mug/mL BSA (pH7.9@25 ℃) and 5 mug of crude enzyme solution with 1 mug of substrate DNA at the optimum reaction temperature for 60min, adding 20 mug of protease K after the reaction is finished, and (3) carrying out electrophoresis detection by using agarose gel with the concentration of 1x TBE of 0.8% after treatment for 15min at 37 ℃, comparing the electrophoresis detection result with a substrate DNA band digested by commercial restriction enzyme to judge the activity of the restriction enzyme in an enzyme crude extract, and completely and correctly digesting transformants corresponding to an enzyme crude extract of the substrate DNA, wherein the transformants can be used for mass expression of the restriction enzyme in the subsequent step.
Example (b):
(I) strain and plasmid:
the strains are all from the American Type Culture Collection (ATCC); escherichia coli DH5 alpha strain was purchased from Beijing Quanjin Biotechnology Co., Ltd, and is a commercially available product; coli ER2566 strain was purchased from seimer feishel Scientific ltd (thermo fisher Scientific), a commercially available product; the pBAD, pACYC184, pUC19 vector plasmids were purchased from the vast ling bioplasmid platform; enzyme activity detection substrate lambda DNA (HindIII digest) was purchased from Thermo Fisher scientific, Inc., commercially available from Saimer Feishel technologies, Inc.;
(II) instruments and reagents:
cell high pressure disruptor: the JN-02C low-temperature ultrahigh-pressure continuous flow cell disruption instrument is purchased from Guangzhou energy-gathering nano biotechnology, Inc.; commercial restriction enzymes were purchased from New England Biolabs (NEB), antibiotics from Monad Biotech, Inc. (Monad Biotech)
(III) methyltransferase Gene sequence and restriction endonuclease sequence:
all gene sequences were from the REBASE database (http:// base. neb. com).
Example 1: restriction enzyme recombinant expression strain constructed by using methyltransferase M
(1) Construction of methyltransferase M. AluI recombinant expression Strain
S1: constructing a methyltransferase m. alui recombinant expression plasmid: the methyltransferase M.AluI gene was synthesized into a pUC57 vector, digested with NdeI and XhoI, DNA band purification by agarose gel electrophoresis, methyltransferase M.AluI gene using T4 Ligase connected to NdeI and XhoI cut pACYC184 expression vector, the ligation products were transformed into the strain Escherichia coli DH5 a and inoculated onto a plate containing 50. mu.g/mL chloramphenicol, the plate was grown overnight at 37 ℃, individual clones were picked and grown overnight in LB medium containing 50. mu.g/mL chloramphenicol, and finally, transforming the expression vector pACYC18-M.AluI plasmid with correct sequencing and methyl transferase M.AluI into an Escherichia coli ER2566 engineering strain, coating the engineering strain on a culture plate containing 50 mu g/mL chloramphenicol resistance, and culturing to construct a methyl transferase M.AluI recombinant expression plasmid.
S2: screening methyltransferase m. alui recombinant expression strain: the recognition sequence of methyltransferase M.AluI is AGCT, methylation site and mode are C position m5, so that methylation protection effect can be screened by restriction enzymes AluI, SacI, HindIII and PvuII, plasmid of each transformant in the plate can be extracted, the protection of methyltransferase is detected by restriction enzymes, the enzyme activity test system is 20 mul, and enzyme digestion verification is carried out by using 1XFlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mug/ml BSA (pH7.9@25 ℃)), 1U restriction enzymes AluI, SacI, HindIII and PvuII at final concentration; reacted with 200ng plasmid at 37 ℃ for 30 min; after the reaction is finished, agarose gel with the concentration of 1x TBE of 0.8% is used for electrophoresis detection; if the plasmid is not cut by the corresponding restriction enzyme, the methyltransferase expressed by the transformant containing the plasmid can protect the DNA in the strain from being cut by the corresponding restriction enzyme; the transformants in which the plasmid was not digested were used as methyltransferase protective strains for the expression of the restriction enzymes AluI, SacI, HindIII, PvuII, as shown in A in FIG. 3, for the expression of the restriction enzymes in the subsequent steps.
(2) Construction of recombinant expression strains of restriction enzymes AluI, HindIII and PvuII
S1: constructing recombinant expression plasmids of restriction enzymes AluI, HindIII and PvuII: synthesizing restriction endonuclease AluI, HindIII and PvuII genes onto a pUC57 vector, carrying out enzyme digestion by NdeI and XhoI, and carrying out band purification by agarose gel electrophoresis; the restriction enzymes AluI, HindIII and PvuII genes were ligated to the NdeI and XhoI digested expression vector pBAD using T4 Ligase, and the ligation product was transformed into E.coli DH 5. alpha. strain and inoculated onto 50. mu.g/mL ampicillin-containing culture plates, which were grown overnight at 37 ℃; selecting a single clone and growing in an LB culture medium containing 50 mu g/mL ampicillin, extracting plasmids through a kit and sequencing; finally, the expression vectors pBAD-AluI, pBAD-HindIII, pBAD-PvuII, which were correctly sequenced and which had the restriction enzyme of interest, were transformed into the methyltransferase AluI protective strain selected in the previous step and inoculated onto a culture plate containing 50. mu.g/mL chloramphenicol and 50. mu.g/mL ampicillin.
S2: screening recombinant expression strains of restriction enzymes AluI, HindIII and PvuII: inoculating transformants obtained by transforming the expression vectors pBAD-AluI, pBAD-HindIII and pBAD-PvuII into LB culture medium of 50 mug/mL ampicillin and 50 mug/mL chloramphenicol, culturing to a late logarithmic phase, preparing a crude extract after inducing with L-arabinose at a final concentration of 0.2% (m/v) at 16 ℃ for 16 hours to test the activity of restriction enzymes in the transformants; the enzyme activity test system is 50 mu l, and 5 mu l of crude enzyme solution and 1 mu g of substrate DNA are reacted for 60min at the optimal reaction temperature by adding 1x FlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mu g/ml BSA (pH7.9@25 ℃)) with the final concentration; after the reaction was completed, 20. mu.g of proteinase K was added, and after treatment at 37 ℃ for 15min, detection was performed by electrophoresis using agarose gel with a 1 XTBE concentration of 0.8%, as shown in A in FIG. 4.
Example 2: strain constructed by using methyltransferase M.EsaDix5I
(1) Construction of methyltransferase M.EsaDix5I recombinant expression strain
S1: constructing a methyltransferase M.EsaDix5I recombinant expression plasmid: synthesizing a methyltransferase m.esadix5i gene into a pUC57 vector, performing enzyme digestion by NdeI and XhoI, and performing DNA band purification by agarose gel electrophoresis; the methyltransferase m.esadix5i gene was ligated using T4 Ligase to the pcyc 184 expression vector after digestion with NdeI and XhoI, the ligation product was transformed into the strain escherichia coli DH5 α and inoculated onto a culture plate containing 50 μ g/mL chloramphenicol, which was grown overnight at 37 ℃; picking single clone and growing in LB culture medium containing 50 mug/mL chloramphenicol overnight, extracting plasmid through a kit and sequencing; finally, the expression vector pACYC18-M.EsaDix5I plasmid, which was correctly sequenced and had methyltransferase M.EsaDix5I, was transformed into the E.coli ER2566 engineered strain and plated onto a culture plate containing 50. mu.g/mL chloramphenicol resistance.
S2: screening methyltransferase m.esadix5i recombinant expression strains: the recognition sequence of methyltransferase M.EsaDix5I is TTAA, and the methylation site and mode are the fourth A site m6, so that the methylation protection effect can be screened by using restriction enzymes AseI, HpaI, DraI, PacI and MseI; extracting plasmids of each transformant in the plate, and detecting the protection of methyltransferase by using restriction enzyme; the enzyme activity test system is 20 mu l, enzyme digestion verification is carried out by using 1xFlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mu g/ml BSA (pH7.9@25 ℃)), and 1U recognition sequence containing restriction endonucleases HpaI, DraI, AseI, PacI and MseI for recognizing the methyltransferase M.EsaDix5I homologous sequence; reacted with 200ng plasmid at 37 ℃ for 30 min; after the reaction is finished, agarose gel with the concentration of 1x TBE of 0.8% is used for electrophoresis detection; if the plasmid is not cut by the corresponding restriction enzyme, 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 in which the plasmid was not cut were used as methyltransferase protective strains for the expression of the restriction enzymes HpaI, DraI, AseI, PacI, MseI, see section B of FIG. 3, for restriction enzyme expression in the subsequent steps.
(2) Construction of recombinant expression strains of restriction endonucleases HpaI, DraI, AseI, PacI and MseI
S1: constructing recombinant expression plasmids of restriction enzymes HpaI, DraI, AseI, PacI and MseI: synthesizing restriction endonuclease HpaI, DraI, AseI, PacI and MseI genes into a pUC57 vector, carrying out enzyme digestion by using NdeI and XhoI, and carrying out band purification by agarose gel electrophoresis; the restriction enzymes HpaI, DraI, AseI, PacI and MseI genes were ligated to the NdeI and XhoI digested expression vector pBAD using T4 Ligase, and the ligation product was transformed into E.coli DH5 α strain and inoculated onto 50 μ g/mL ampicillin-containing culture plates grown overnight at 37 ℃; selecting a single clone and growing in an LB culture medium containing 50 mu g/mL ampicillin, extracting plasmids through a kit and sequencing; finally, expression vectors pBAD-HpaI, pBAD-DraI, pBAD-AseI, pBAD-PacI, pBAD-MseI with correct sequencing and the target restriction enzymes were transformed into the methyltransferase EsaDix5I protective strain screened in the previous step and inoculated onto a culture plate containing 50. mu.g/mL chloramphenicol and 50. mu.g/mL ampicillin.
S2: screening recombinant expression strains of restriction enzymes HpaI, DraI, AseI, PacI and MseI: inoculating transformants obtained by transforming the expression vectors pBAD-HpaI, pBAD-DraI, pBAD-AseI, pBAD-PacI and pBAD-MseI into LB culture medium containing 50 ug/mL ampicillin and 50 ug/mL chloramphenicol, and preparing a crude extract to test the activity of restriction enzymes in the transformants after inducing with L-arabinose at a final concentration of 0.2% (m/v) at 16 ℃ for 16 hours at a stage from the culture to the late logarithmic phase; the enzyme activity test system is 50 mu l, and 5 mu l of crude enzyme solution and 1 mu g of substrate DNA are reacted for 60min at the optimal reaction temperature by adding 1x FlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mu g/ml BSA (pH7.9@25 ℃)) with the final concentration; after the reaction was completed, 20. mu.g of proteinase K was added, and after 15min at 37 ℃ the detection was carried out by electrophoresis using agarose gel with a 1 XTBE concentration of 0.8%, see part B of FIG. 4.
Example 3: bacterial strain constructed by using methyltransferase M.HhaI
(1) Construction of methyltransferase M.HhaI recombinant expression strain
S1: constructing a methyltransferase M.HhaI recombinant expression plasmid: synthesizing a methyltransferase M.HhaI gene on a pUC57 vector, carrying out enzyme digestion by NdeI and XhoI, and carrying out DNA band purification by agarose gel electrophoresis; the methyltransferase m.hhal gene was ligated using T4 Ligase into the pcyc 184 expression vector after digestion with NdeI and XhoI, the ligation product was transformed into the strain escherichia coli DH5 α and inoculated onto a culture plate containing 50 μ g/mL chloramphenicol, which was grown overnight at 37 ℃; selecting a single clone and growing in an LB culture medium containing 50 mu g/mL chloramphenicol, extracting plasmids through a kit and sequencing; finally, the expression vector pACYC18-M.HhaI plasmid with correct sequencing and methyltransferase HhaI was transformed into E.coli ER2566 engineered strain and plated onto chloramphenicol resistant 50. mu.g/mL plates.
S2: screening a methyltransferase M.HhaI recombinant expression strain: the recognition sequence of methyltransferase M.HhaI is GCGC, and the methylation site and mode are the second C position m5, so the methylation protection effect can be screened by using restriction enzymes HhaI, SfoI, FspI, AfoI, HinPII, NarI and PluTI; extracting plasmids of each transformant in the plate, and detecting the protection of methyltransferase by using restriction enzyme; the enzyme activity test system is 20 mul, enzyme digestion verification is carried out by using 1XFlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mu g/ml BSA (pH7.9@25 ℃)), and 1U recognition sequence containing restriction endonuclease HhaI, SfoI, FspI, AfoI, HinPII, NarI and PluTI for recognizing the homologous sequence of methyltransferase M.HhaI; reacted with 200ng plasmid at 37 ℃ for 30 min; after the reaction is finished, agarose gel with the concentration of 1x TBE of 0.8% is used for electrophoresis detection; if the plasmid is not cut by the corresponding restriction enzyme, 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 in which the plasmid was not digested were used as methyltransferase protected strains for the expression of the restriction enzymes HhaI, SfoI, FspI, AfoI, HinPII, NarI, PluTI (see FIG. 3C) for the subsequent step of restriction enzyme expression.
(2) Construction of recombinant expression strains of restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, and PluTI
S1: constructing recombinant expression plasmids of restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI and PluTI: synthesizing restriction endonuclease HhaI, SfoI, AfoI, HinPII, NarI and PluTI genes onto a pUC57 vector, carrying out enzyme digestion by NdeI and XhoI, and carrying out band purification by agarose gel electrophoresis; the restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI, and PluTI genes were ligated to the expression vector pBAD digested with NdeI and XhoI using T4 Ligase, and the ligation product was transformed into E.coli DH 5. alpha. strain and inoculated onto a culture plate containing 50. mu.g/mL ampicillin, which was grown overnight at 37 ℃; selecting a single clone and growing in an LB culture medium containing 50 mu g/mL ampicillin, extracting plasmids through a kit and sequencing; finally, expression vectors pBAD-HhaI, pBAD-SfoI, pBAD-AfoI, pBAD-HinpII, pBAD-NarI, pBAD-PluTI, which were correctly sequenced and which had the restriction enzyme of interest, were transformed into the methyltransferase HhaI protective strain selected in the previous step and inoculated onto culture plates containing 50. mu.g/mL chloramphenicol and 50. mu.g/mL ampicillin.
S2: screening recombinant expression strains of restriction enzymes HhaI, SfoI, AfoI, HinPII, NarI and PluTI: inoculating transformants obtained by the plasmids of the transformation expression vectors pBAD-HhaI, pBAD-SfoI, pBAD-AfoI, pBAD-HinpII, pBAD-NarI and pBAD-PluTI into an LB culture medium containing 50 mu g/mL ampicillin and 50 mu g/mL chloramphenicol for culture, preparing a crude extract after inducing with L-arabinose at a final concentration of 0.2% (m/v) at 16 ℃ for 16 hours in a stage from the culture to a late logarithmic phase to test the activity of restriction enzymes in the transformants; the enzyme activity test system is 50 mu l, and 5 mu l of crude enzyme solution and 1 mu g of substrate DNA are reacted for 60min at the optimal reaction temperature by adding 1x FlashOne buffer solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100 mu g/ml BSA (pH7.9@25 ℃)) with the final concentration; after the reaction is finished, 20 mu g of proteinase K is added, and after the reaction is processed for 15min at 37 ℃, agarose gel with 1x TBE concentration of 0.8% is used for electrophoresis detection; see section C in fig. 4.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (7)
1. An engineered strain for recombinant expression of a restriction enzyme, characterized in that: taking Escherichia coli ER2566 as a modified basis of an engineering strain, wherein the engineering strain comprises the following conditions:
(1) compatible with exogenous methyltransferases;
(2) prokaryotic expression vector pACYC184 containing methyltransferase gene capable of protecting four-base universal sequence;
(3) a prokaryotic expression vector pBAD containing a restriction endonuclease gene whose recognition cleavage sequence comprises a four-base universal sequence; wherein, the four-base universal sequence comprises: TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC, GTAC.
2. The method of constructing an engineered strain for recombinant expression of a restriction enzyme according to claim 1, wherein: the construction method specifically comprises the following steps:
s1: screening engineering strains: determining that Escherichia coli ER2566 is taken as a basis for engineering strain modification, the engineering strain is compatible with exogenous methyltransferases, and simultaneously contains a prokaryotic expression vector of a methyltransferase gene capable of protecting a four-base universal sequence and a prokaryotic expression vector of a restriction endonuclease gene containing a recognition cutting sequence containing the four-base universal sequence, wherein the four-base universal sequence is TCGA, TGCA, TTAA, AATT, ACGT, AGCT, CATG, CCGG, CGCG, CTAG, GATC, GCGC, GGCC and GTAC.
S2: selection of expression plasmids: selecting pACYC184 plasmid with Amp promoter as methyltransferase expression plasmid, using resistance as chloramphenicol, selecting pBAD plasmid with arabinose operon and promoter as restriction enzyme expression plasmid, using resistance as ampicillin;
s3: obtaining an intermediate strain: selecting a specific methyltransferase gene according to the four-base universal sequence combination and methylation pattern in S1, connecting the selected specific methyltransferase gene with pACYC184 in S2 to obtain a recombinant plasmid, and transforming the recombinant plasmid into an Escherichia coli ER2566 strain to obtain an intermediate strain;
s4: screening strains: selecting a restriction enzyme whose recognition sequence contains the four-base universal sequence and whose methylation sensitivity is the same as the action site of methyltransferase according to the methylation sequence and methylation site pattern of the methyltransferase selected in S3, cleaving the DNA of the intermediate strain obtained in step S3, selecting a methylation-protected strain that cannot be cleaved,
s5: construction of an expression strain: connecting the gene corresponding to the restriction enzyme in S4 with pBAD in S2 to obtain recombinant plasmid, and transforming the recombinant plasmid into S4 screening strains to obtain engineering strains capable of expressing the corresponding restriction enzyme;
s6: expression of the engineered strain: and (4) inducing and expressing the restriction enzyme by using the engineering strain of the restriction enzyme obtained in the step S5.
3. The strain for recombinant expression of engineering restriction enzymes according to claim 1 or 2, wherein: the four base universal sequence methyltransferase comprises: (1) TCGA: M.TaqI; (2) TGCA: 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.nlaiiii; (8) CCGG: m.mspi, m.hpaii; (9) CGCG: bstui; (10) CTAG: M.BfaI; (11) GATC: m.sau3ai, m.bstkti, m.dpni, 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 enzyme which recognizes that the cleavage sequence comprises a four-base universal sequence includes: (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, bshii, 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, TatI.
4. The method for constructing an engineered strain for recombinant expression of restriction enzymes according to claim 2, wherein: the S3 intermediate strain is obtained by the following steps:
(1) synthesizing a methyltransferase gene containing a short homologous recognition sequence onto a pUC57 vector, performing enzyme digestion by NdeI and XhoI, and performing DNA band purification by agarose gel electrophoresis;
(2) the purified methyltransferase gene was ligated to the NdeI and XhoI digested pACYC184 expression vector using T4 Ligase, the ligation product was transformed into E.coli DH 5. alpha. strain and inoculated onto 50. mu.g/mL chloramphenicol resistant plates grown overnight at 37 ℃, individual clones were picked and grown overnight in LB medium containing 50. mu.g/mL chloramphenicol, plasmids were extracted with the kit and sequenced;
(3) the expression vector pACYC184 plasmid, which was correctly sequenced and had the methyltransferase of interest, was transformed into the E.coli ER2566 strain and plated on chloramphenicol-resistant culture plates to obtain intermediate strains.
5. The method for constructing an engineered strain for recombinant expression of restriction enzymes according to claim 2, wherein: the steps for screening the strain in the S4 are as follows:
(1) extracting plasmids of each transformant in an S3 plate, detecting the protection of methyltransferase by using restriction endonuclease, wherein the enzyme activity test system is 20 mu l, 50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate and 100 mu g/ml BSA (pH7.9@25 ℃) are used as a 1XFlashOne buffer solution, and 1U of corresponding commercial restriction endonuclease reacts with 200ng plasmid at 37 ℃ for 30 min;
(2) after the reaction is finished, agarose gel with the concentration of 1x TBE of 0.8% is used for carrying out electrophoresis detection, the plasmid cannot be cut by the corresponding restriction enzyme, and the methyltransferase expressed by the transformant containing the plasmid can protect DNA in the strain from being cut by the corresponding restriction enzyme;
(3) transformants which could not be digested by the plasmid were selected as host strains for expression of restriction enzymes and used as restriction enzyme expression strains in the subsequent steps.
6. The method of constructing an engineered strain for recombinant expression of a restriction enzyme according to claim 2, wherein: the construction steps of the expression strain in the S5 are as follows:
(1) synthesizing a restriction endonuclease gene, the recognition sequence of which contains the methylation sequence and is sensitive to the methylation pattern, into a pUC57 vector, performing enzyme digestion using NdeI and XhoI, and performing band purification by agarose gel electrophoresis;
(2) the purified restriction endonuclease gene was ligated to NdeI and XhoI digested expression vector pBAD using T4 Ligase, and the ligation product was transformed into E.coli DH 5. alpha. strain and inoculated onto 50. mu.g/mL ampicillin-containing culture plate, which was grown overnight at 37 ℃, and single clones were picked up to LB medium containing 50. mu.g/mL ampicillin and grown overnight, and plasmids were extracted by kit and sequenced;
(3) the expression vector pBAD plasmid with correct sequencing and the target restriction enzyme was transformed into the methyltransferase protective strain using the short homology recognition sequence selected in the previous step and inoculated onto a culture plate containing 50. mu.g/mL chloramphenicol and 50. mu.g/mL ampicillin to construct an expression strain.
7. The method for constructing an engineered strain for recombinant expression of restriction enzymes according to claim 2, wherein: the expression steps of the engineering strain in S6 are as follows:
(1) inoculating a transformant obtained by transforming S5 into an LB culture medium containing 50 mug/mL of chloramphenicol and 50 mug/mL of ampicillin for culture, and adding L-arabinose with a final concentration of 0.2% (m/v) at 16 ℃ to induce for 16 hours when the culture reaches the late logarithmic phase, namely the expression of the engineering strain for recombining and expressing the restriction enzyme;
(2) after induction, centrifugally collecting thalli, crushing by using ultrasonic waves, centrifugally obtaining an enzyme crude extract again, detecting the activity of the restriction enzyme in the enzyme crude extract to test the activity of the restriction enzyme in a transformant, wherein an enzyme activity test system is 50 mu l, and a final concentration of 1xFlashOne buffer solution is prepared by adding 5 mu l of crude enzyme solution and 1 mu g of substrate DNA at an optimal reaction temperature for 60min by using 50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate and 100 mu g/ml BSA (pH7.9@25 ℃);
(3) and after the reaction is finished, adding 20 mu g of proteinase K, treating for 15min at 37 ℃, performing electrophoresis detection by using agarose gel with the concentration of 1x TBE of 0.8%, comparing the electrophoresis detection result with a substrate DNA band digested by using commercial restriction endonuclease to judge the activity of the restriction endonuclease in the enzyme crude extract, and completely and correctly digesting a transformant corresponding to the enzyme crude extract of the substrate DNA, namely, the transformant can be used as an engineering strain for large-scale expression of the restriction endonuclease in the subsequent step.
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| US20110151450A1 (en) * | 2007-07-12 | 2011-06-23 | New England Biolabs, Inc. | High Fidelity Restriction Endonucleases |
| US20120088237A1 (en) * | 2009-03-09 | 2012-04-12 | New England Biolabs, Inc. | Engineering a Novel Methylation-Specific Restriction Endonuclease |
| CN107557372A (en) * | 2017-08-30 | 2018-01-09 | 江苏愚公生命科技有限公司 | For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI |
| CN107760697A (en) * | 2017-08-30 | 2018-03-06 | 江苏愚公生命科技有限公司 | For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme FspI |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110151450A1 (en) * | 2007-07-12 | 2011-06-23 | New England Biolabs, Inc. | High Fidelity Restriction Endonucleases |
| US20120088237A1 (en) * | 2009-03-09 | 2012-04-12 | New England Biolabs, Inc. | Engineering a Novel Methylation-Specific Restriction Endonuclease |
| CN107557372A (en) * | 2017-08-30 | 2018-01-09 | 江苏愚公生命科技有限公司 | For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI |
| CN107760697A (en) * | 2017-08-30 | 2018-03-06 | 江苏愚公生命科技有限公司 | For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme FspI |
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