CN103589744B - Suicide plasmid pYTRLRRT with prcR gene knockout effect and construction method thereof - Google Patents

Suicide plasmid pYTRLRRT with prcR gene knockout effect and construction method thereof Download PDF

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CN103589744B
CN103589744B CN201310572456.0A CN201310572456A CN103589744B CN 103589744 B CN103589744 B CN 103589744B CN 201310572456 A CN201310572456 A CN 201310572456A CN 103589744 B CN103589744 B CN 103589744B
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prcrr
prcrl
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CN103589744A (en
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葛菁萍
孙育新
王洋
由田
李兴霖
孙艳阳
平文祥
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Heilongjiang University
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Abstract

The invention provides a suicide plasmid pYTRLRRT with a prcR gene knockout effect and a construction method thereof, and relates to a suicide plasmid and a construction method thereof. The suicide plasmid pYTRLRRT with the prcR gene knockout effect is formed by a prcR gene left fragment prcRL, a prcR gene right fragment prcRR, a tetracycline resistant gene and a plasmid pUC18. The construction method comprises the steps as follows: 1, amplifying the fragment prcRL; 2, obtaining a plasmid pUC18-RL; 3, amplifying the fragment prcRR; 4, obtaining a plasmid pUC18-RR; 5, amplifying the tetracycline resistant gene Tet; and 6, obtaining the suicide plasmid pYTRLRRT with the prcR gene knockout effect. The invention is applied to the field of the bioengineering research.

Description

A kind of suicide plasmid pYTRLRRT and construction process thereof knocking out prcR gene
Technical field
The present invention relates to a kind of suicide plasmid and construction process thereof.
Background technology
In Recent study, researchist finds at some G +aIP in bacterium QS system is not only signaling molecule, and it also has antibacterial ability, as the nisin of Lactococcus lactis, and Plantaricin by L. plantarum of plant lactobacillus etc.These antibacterial peptides (antimicrobialpeptide, AMP) except containing except abc transport body encoding gene in gene cluster, also have accessory protein (accessoryprotein, an AP) encoding gene, their product constitutes AMP and exports and system for handling.Before the structure gene of AMP is all positioned at its corresponding immune protein gene, the AMP great majority of generation are rich in halfcystine, have hydrophobicity, and find after deliberation, the maximum output of AMP is relevant with the non-optimal growth conditions of producing strains.
According to the different in kind of AMP, AMP can be divided into two large classes: a class is I type AMPs or lantibiotic, they are some heat stable peptide classes, can be carried out the posttranslational modification of height, as nisin, subtilyne before secreted; Another kind of is II type AMPs or heat-staple AMPs, and they have distinctive double-glycine motif, there is not modification before secretion, but after secretion, the N end of precursor peptide has a fragment and is removed, as Plantaricin by L. plantarum, lactobacillin P etc.
Lactobacillus paraceasi (Lactobacillusparacasei) HD1.7, gram-positive microorganism, is separated and obtains for 2003 from the rich derived food company limited in Qiqihar lactate Chinese sauerkraut fermented liquid.Its maximum feature can produce to suppress multiple G in its fermented liquid +, G-and yeast saccharomyces cerevisiae growth peptide matters, molecular weight is at about 10kD, thermally-stabilised, under acidic conditions, (pH2 ~ 6) are active high, the nisin that its Performance Ratio is conventional on the market is at present more superior, because nisin can effectively suppress gram-positive microorganism as some spoilage organism, pathogenic bacterium and sporeformer, and also little to Gram-negative bacteria role, even do not work.Also find, when by high-density thalline fermented liquid (>10 through research simultaneously 11individual/mL) add low density thalline fermented liquid (<10 to 5individual/mL) in time, the generation of this AMP in low density thalline can be promoted; The thalline fermented liquid of fermentation 24h and 48h, its bacteriostasis difference is larger.Above phenomena illustrates, the output of this AMP that this bacterium produces may be subject to population density impact and control.
External Nakayama etc. have been cloned into lactobacillus paraceasi quasi-group Body Effect genes involved (prcA, prcK and prcR) by PCR method, but for the concrete function of these genes in quorum sensing, and whether AMP output is relevant with quorum sensing is still not clear.
At present, the research method of gene function mainly contains two kinds: one is reinforcing gene expression, then studies the expression product obtained; Another kind weakens or terminator expression, by observing the change of biological integral function, and then infers corresponding gene function.Because first method often can not reflect the truly expressed situation of gene product, and be abandoned gradually.In second method, RNA perturbation technique (RNAi) in fact just reduces the expression of gene on post-transcriptional level, do not resemble gene Knockout can completely gene be weeded out from genome, sometimes can be difficult to analyze because of the phenotype produced that totally do not cause of background yet.Therefore, gene Knockout is adopted to become method ideal and suitable at present.
Gene Knockout is different according to the object of gene targeting, and carrier has different method of design, can be divided into replaceability carrier and insertional vector.A kind of insertional vector is described in " response phase method optimizes the preliminary foundation of Paracin1.7 fermentation condition and producing strains genetic conversion system thereof ", although this carrier is also successful, and resistant gene tet is imported lactobacillus paraceasi HD1.7 cell, but resistant gene tet does not insert and is appointed as a little, not according to Design Theory generation homologous recombination, prcR gene could not be knocked out.
Summary of the invention
The present invention could not the problem of prcR gene in successful knockout lactobacillus paraceasi (Lactobacillusparacasei) HD1.7 cell in order to solve existing suicide plasmid, and a kind of suicide plasmid pYTRLRRT and the construction process thereof knocking out prcR gene provided.
The suicide plasmid pYTRLRRT that the present invention knocks out prcR gene is made up of prcR gene left-hand fragment prcRL, prcR gene right fragment prcRR, tetracycline resistance gene and plasmid pUC18; The nucleotide sequence of fragment prcRL is as shown in SEQ ID NO:1; The nucleotide sequence of fragment prcRR is as shown in SEQ ID NO:2.
The above-mentioned suicide plasmid pYTRLRRT knocking out prcR gene builds according to the following steps:
One, the upstream primer being template PCR amplifications prcR gene left-hand fragment prcRL, amplified fragments prcRL with L.paracasei HD1.7 genomic dna is that the nucleotides sequence of prcRL-up, prcRL-up is classified as 5 '-CCG gAGCTCthe downstream primer of CCTACTCAGCATTCAGAGGTCAACT-3 ', amplified fragments prcRL is that the nucleotides sequence of prcRL-down, prcRL-down is classified as 5 '-GTC gGTACCthe reaction system of ATCTTCAGCATCGTTTGGTGGTTGG-3 ', pcr amplified fragment prcRL is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRL-up that is 1pmol/ μ L, the 10 μ L concentration prcRL-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRL is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 61 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Two, with restriction enzyme Sac I and Kpn I, respectively double digestion is carried out to the fragment prcRL that plasmid pUC18 and step one obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRL double digestion fragments, 5 μ l plasmid pUC18 double digestion fragments, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RL;
Three, the upstream primer being template PCR amplifications prcR gene right fragment prcRR, amplified fragments prcRR with L.paracasei HD1.7 genomic dna is that the nucleotides sequence of prcRR-up, prcRR-up is classified as 5 '-GTC gGTACCthe downstream primer of GGTCAGCATTCGTAGAGTGTCGGCC-3 ', amplified fragments prcRR is that the nucleotides sequence of prcRR-down, prcRR-down is classified as 5 '-CCG cTGCAGthe reaction system of GCAGTGACCAGAGATAGCTCGGCGT-3 ', pcr amplified fragment prcRR is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRR-up that is 1pmol/ μ L, the 10 μ L concentration prcRR-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRR is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 69 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Four, with restriction enzyme Pst I and Kpn I, respectively double digestion is carried out to the fragment prcRR that plasmid pUC18-RL and step 3 obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRR double digestion fragments, 5 μ l plasmid pUC18-RL double digestion fragments, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RLRR;
Five, with pBR322 plasmid for template PCR amplifications tetracycline resistance gene Tet, the upstream primer of amplification Tet is that the nucleotides sequence of Tet-up, Tet-up is classified as 5 '-CCG gGTACCthe downstream primer of TCTCATGTTTGACAGCTT-3 ', amplification Tet is that the nucleotides sequence of Tet-down, Tet-down is classified as 5 '-GTC gGTACCthe reaction system of TAATAGATATGTTCTGCCAAGGGT-3 ', pcr amplification Tet is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration pBR322 plasmid that is 25ng/ μ L, the 10 μ L concentration Tet-up that is 1pmol/ μ L, the 10 μ L concentration Tet-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplification Tet is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 46 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Six, carry out enzyme with restriction enzyme Kpn I respectively to the tetracycline resistance gene Tet that plasmid pUC18-RLRR and step 5 obtain to cut, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l tetracycline resistance gene Tet endonuclease bamhis, 5 μ l plasmid pUC18-RLRR endonuclease bamhis, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, namely obtain the suicide plasmid pYTRLRRT knocking out prcR gene.
Build suicide plasmid and must select suitable carrier, vector plasmid not reproducible in recipient bacterium, vector plasmid must with one being incorporated into alternative resistance marker after in karyomit(e), and vector plasmid is with the multiple clone site being easy to clone; Sufficiently long homology arm must be selected, the more long generation being more conducive to homologous recombination of homology arm, can reduce the incidence of mistake displacement, and homology arm must meet again the unicity of restriction enzyme site, the restriction enzyme site used when homology arm inside can not comprise insertion; Must select suitable target gene, target gene fragment is identical with the fragment length of replacement, and not containing the restriction enzyme site used when inserting, can not introduce sudden change.So the difficulty that will meet above-mentioned all conditions structure suicide plasmid is very large simultaneously.
In suicide plasmid pYTRLRRT of the present invention, the length of homology arm reaches 2.7kb, homology brachium improves homologous recombination rate, avoid the generation of faulty gene displacement, successfully knock out the prcR gene in lactobacillus paraceasi (Lactobacillusparacasei) HD1.7 cell.And the homology arm that the present invention selects is not containing the restriction enzyme site used when inserting, and not containing mutational site, has biological safety.
Present invention utilizes tetracycline resistance gene and replace prcR gene as target gene, not only make positive recipient bacterium easily screen, and prove that prcR gene is successfully knocked out by the expression of tetracyclin resistance.
The present invention is for research prcR gene and lactobacillus paraceasi quasi-group Body Effect have established basic substance afterwards.
Accompanying drawing explanation
Fig. 1 is the digestion verification result figure of the plasmid pUC18-RL that in embodiment 1, step 2 obtains.
Fig. 2 is the digestion verification result figure of the plasmid pUC18-RLRR that in embodiment 1, step 2 obtains.
Fig. 3 is the digestion verification result figure of the plasmid pYTRLRRT that in embodiment 1, step 2 obtains.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the suicide plasmid pYTRLRRT that present embodiment knocks out prcR gene is made up of prcR gene left-hand fragment prcRL, prcR gene right fragment prcRR, tetracycline resistance gene and plasmid pUC18; The nucleotide sequence of fragment prcRL is as shown in SEQ ID NO:1; The nucleotide sequence of fragment prcRR is as shown in SEQ ID NO:2.
Embodiment two: the suicide plasmid pYTRLRRT that present embodiment knocks out prcR gene builds according to the following steps:
One, the upstream primer being template PCR amplifications prcR gene left-hand fragment prcRL, amplified fragments prcRL with L.paracasei HD1.7 genomic dna is that the nucleotides sequence of prcRL-up, prcRL-up is classified as 5 '-CCG gAGCTCthe downstream primer of CCTACTCAGCATTCAGAGGTCAACT-3 ', amplified fragments prcRL is that the nucleotides sequence of prcRL-down, prcRL-down is classified as 5 '-GTC gGTACCthe reaction system of ATCTTCAGCATCGTTTGGTGGTTGG-3 ', pcr amplified fragment prcRL is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRL-up that is 1pmol/ μ L, the 10 μ L concentration prcRL-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRL is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 61 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Two, with restriction enzyme Sac I and Kpn I, respectively double digestion is carried out to the fragment prcRL that plasmid pUC18 and step one obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRL double digestion fragments, 5 μ L plasmid pUC18 double digestion fragments, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RL;
Three, the upstream primer being template PCR amplifications prcR gene right fragment prcRR, amplified fragments prcRR with L.paracasei HD1.7 genomic dna is that the nucleotides sequence of prcRR-up, prcRR-up is classified as 5 '-GTC gGTACCthe downstream primer of GGTCAGCATTCGTAGAGTGTCGGCC-3 ', amplified fragments prcRR is that the nucleotides sequence of prcRR-down, prcRR-down is classified as 5 '-CCG cTGCAGthe reaction system of GCAGTGACCAGAGATAGCTCGGCGT-3 ', pcr amplified fragment prcRR is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRR-up that is 1pmol/ μ L, the 10 μ L concentration prcRR-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRR is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 69 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Four, with restriction enzyme Pst I and Kpn I, respectively double digestion is carried out to the fragment prcRR that plasmid pUC18-RL and step 3 obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRR double digestion fragments, 5 μ l plasmid pUC18-RL double digestion fragments, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RLRR;
Five, with pBR322 plasmid for template PCR amplifications tetracycline resistance gene Tet, the upstream primer of amplification Tet is that the nucleotides sequence of Tet-up, Tet-up is classified as 5 '-CCG gGTACCthe downstream primer of TCTCATGTTTGACAGCTT-3 ', amplification Tet is that the nucleotides sequence of Tet-down, Tet-down is classified as 5 '-GTC gGTACCthe reaction system of TAATAGATATGTTCTGCCAAGGGT-3 ', pcr amplification Tet is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration pBR322 plasmid that is 25ng/ μ L, the 10 μ L concentration Tet-up that is 1pmol/ μ L, the 10 μ L concentration Tet-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplification Tet is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 46 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Six, carry out enzyme with restriction enzyme Kpn I respectively to the tetracycline resistance gene Tet that plasmid pUC18-RLRR and step 5 obtain to cut, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l tetracycline resistance gene Tet endonuclease bamhis, 5 μ l plasmid pUC18-RLRR endonuclease bamhis, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, namely obtain the suicide plasmid pYTRLRRT knocking out prcR gene.
The part that the acceptance of the bid of present embodiment primer sequence is marked with underscore is restriction enzyme site.
The fragment prcRL length that present embodiment step one amplifies is 1349bp, and the fragment prcRR length that step 3 amplifies is 1343bp, and the fragment Tet length that step 5 amplifies is 1407bp.
Because the plasmid of G-bacterium can not at G +copy in bacterium, therefore, need add on the plasmid of G-bacterium can at G +the resistant gene of expressing in bacterium.Present embodiment have selected pUC18 as plasmid backbone, using tetracycline gene as resistant gene, builds prcR gene knockout carrier.This vector to enter after HD1.7 not reproducible, but can be incorporated on HD1.7 karyomit(e) with homologous gene generation homologous recombination, gives expression to tetracyclin resistance, thus screens.
It is key core step that enzyme is cut with being connected, and being affects recombination fraction and test complicated and simple key factor.In experiment the position of restriction enzyme site number, restriction enzyme site and the selection of restriction endonuclease kind be restriction enzyme site design in the most important thing and fundamental factor.
The length of homology arm determines homologous recombination rate, and therefore present embodiment devises the homology arm reaching 2.7kb, ensure that suicide plasmid successfully knocks out prcR gene.
Embodiment three: the difference of present embodiment and embodiment two is: in step 2 plasmid pUC18 endonuclease reaction system be 20 μ L by 1 μ L concentration be the Sac I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × BufferTango, 7 μ L concentration be 80ng/ μ L plasmid pUC18 and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.Other steps are identical with embodiment two with parameter.
Embodiment four: the difference of present embodiment and embodiment two or three is: in step 2 fragment prcRL endonuclease reaction system be 20 μ L by 1 μ L concentration be the Sac I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × Buffer Tango, 10 μ L concentration be 80ng/ μ L fragment prcRL and the aseptic ddH of 6 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.Other steps are identical with embodiment two or three with parameter.
Embodiment five: the difference of present embodiment and embodiment two, three or four is: in step 4 plasmid pUC18-RL endonuclease reaction system be 20 μ L by 1 μ L concentration be the Pst I of 10U/ μ L, 1 μ L concentration be 10U/ μ L KpnI, 2 μ L10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L plasmid pUC18-RL and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.Other steps and parameter and embodiment two, three or four identical.
Embodiment six: the difference of one of present embodiment and embodiment two to five is: in step 4 fragment prcRR endonuclease reaction system be 20 μ L by 1 μ L concentration be the Pst I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L fragment prcRR and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.Other steps are identical with one of embodiment two to five with parameter.
Embodiment seven: the difference of one of present embodiment and embodiment two to six is: in step 6 plasmid pUC18-RLRR endonuclease reaction system be 20 μ L by 1 μ L concentration be Kpn I, 2 μ L10 × Buffer Tango of 10U/ μ L, 7 μ L concentration are plasmid pUC18-RLRR and the aseptic ddH of 10 μ L of 80ng/ μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.Other steps are identical with one of embodiment two to six with parameter.
Embodiment eight: the difference of one of present embodiment and embodiment two to seven is: in step 6 tetracycline resistance gene Tet endonuclease reaction system be 20 μ L by 1 μ L concentration be Kpn I, 2 μ L10 × Buffer Tango of 10U/ μ L, 7 μ L concentration are tetracycline resistance gene Tet and the aseptic ddH of 10 μ L of 80ng/ μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.Other steps are identical with one of embodiment two to seven with parameter.
Embodiment 1
The suicide plasmid pYTRLRRT knocking out prcR gene builds according to the following steps:
One, the upstream primer being template PCR amplifications prcR gene left-hand fragment prcRL, amplified fragments prcRL with L.paracasei HD1.7 genomic dna is that the nucleotides sequence of prcRL-up, prcRL-up is classified as 5 '-CCG gAGCTCthe downstream primer of CCTACTCAGCATTCAGAGGTCAACT-3 ', amplified fragments prcRL is that the nucleotides sequence of prcRL-down, prcRL-down is classified as 5 '-GTC gGTACCthe reaction system of ATCTTCAGCATCGTTTGGTGGTTGG-3 ', pcr amplified fragment prcRL is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRL-up that is 1pmol/ μ L, the 10 μ L concentration prcRL-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRL is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 61 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Two, with restriction enzyme Sac I and Kpn I, respectively double digestion is carried out to the fragment prcRL that plasmid pUC18 and step one obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRL double digestion fragments, 5 μ l plasmid pUC18 double digestion fragments, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RL;
Three, the upstream primer being template PCR amplifications prcR gene right fragment prcRR, amplified fragments prcRR with L.paracasei HD1.7 genomic dna is that the nucleotides sequence of prcRR-up, prcRR-up is classified as 5 '-GTC gGTACCthe downstream primer of GGTCAGCATTCGTAGAGTGTCGGCC-3 ', amplified fragments prcRR is that the nucleotides sequence of prcRR-down, prcRR-down is classified as 5 '-CCG cTGCAGthe reaction system of GCAGTGACCAGAGATAGCTCGGCGT-3 ', pcr amplified fragment prcRR is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRR-up that is 1pmol/ μ L, the 10 μ L concentration prcRR-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRR is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 69 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Four, with restriction enzyme Pst I and Kpn I, respectively double digestion is carried out to the fragment prcRR that plasmid pUC18-RL and step 3 obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRR double digestion fragments, 5 μ l plasmid pUC18-RL double digestion fragments, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RLRR;
Five, with pBR322 plasmid for template PCR amplifications tetracycline resistance gene Tet, the upstream primer of amplification Tet is that the nucleotides sequence of Tet-up, Tet-up is classified as 5 '-CCG gGTACCthe downstream primer of TCTCATGTTTGACAGCTT-3 ', amplification Tet is that the nucleotides sequence of Tet-down, Tet-down is classified as 5 '-GTC gGTACCthe reaction system of TAATAGATATGTTCTGCCAAGGGT-3 ', pcr amplification Tet is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration pBR322 plasmid that is 25ng/ μ L, the 10 μ L concentration Tet-up that is 1pmol/ μ L, the 10 μ L concentration Tet-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplification Tet is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 46 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Six, carry out enzyme with restriction enzyme Kpn I respectively to the tetracycline resistance gene Tet that plasmid pUC18-RLRR and step 5 obtain to cut, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l tetracycline resistance gene Tet endonuclease bamhis, 5 μ l plasmid pUC18-RLRR endonuclease bamhis, 2.5 μ l10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, namely obtain the suicide plasmid pYTRLRRT knocking out prcR gene.
Wherein, in step 2 plasmid pUC18 endonuclease reaction system be 20 μ L by 1 μ L concentration be the Sac I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L plasmid pUC18 and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue;
In step 2 fragment prcRL endonuclease reaction system be 20 μ L by 1 μ L concentration be the Sac I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × Buffer Tango, 10 μ L concentration be 80ng/ μ L fragment prcRL and the aseptic ddH of 6 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue;
In step 4 plasmid pUC18-RL endonuclease reaction system be 20 μ L by 1 μ L concentration be the Pst I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L plasmid pUC18-RL and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue;
In step 4 fragment prcRR endonuclease reaction system be 20 μ L by 1 μ L concentration be the Pst I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L fragment prcRR and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue;
In step 6 plasmid pUC18-RLRR endonuclease reaction system be 20 μ L by 1 μ L concentration be Kpn I, 2 μ L10 × Buffer Tango of 10U/ μ L, 7 μ L concentration are plasmid pUC18-RLRR and the aseptic ddH of 10 μ L of 80ng/ μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue;
In step 6 tetracycline resistance gene Tet endonuclease reaction system be 20 μ L by 1 μ L concentration be Kpn I, 2 μ L10 × Buffer Tango of 10U/ μ L, 7 μ L concentration are tetracycline resistance gene Tet and the aseptic ddH of 10 μ L of 80ng/ μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
Plasmid pUC18-the RL that step 2 obtains is converted in E.coli DH5 α competent cell, then be seeded on screening flat board that kantlex concentration is 50 μ g/mL and cultivate, bacterium colony is positive bacteria, selects positive bacteria and carries out PCR checking (primer is prcRL-up and prcRL-down) and digestion verification (restriction enzyme Sac I and Kpn I).PCR verifies that all swimming lanes all have clear band at about 1350bp, conforms to prcRL gene fragment size; There are two bands (as shown in Figure 1) clearly in digestion verification, size respectively with plasmid pUC18 (2.6kb) and prcRL(1.35kb) conform to, illustrates that pUC18-RL plasmid construction is successfully.
Plasmid pUC18-the RLRR that step 4 obtains is converted in E.coli DH5 α competent cell, then be seeded on screening flat board that kantlex concentration is 50 μ g/mL and cultivate, bacterium colony is positive bacteria, selects positive bacteria and carries out PCR checking (primer is prcRR-up and prcRR-down) and digestion verification (restriction enzyme Kpn I and Pst I).PCR verifies that all swimming lanes all have clear band at about 1350bp, conforms to prcRR gene fragment size; There are two bands (as shown in Figure 2) clearly in digestion verification, size respectively with plasmid pUC18-RL(3.95kb) with prcRR(1.35kb) conform to, illustrates that pUC18-RLRR plasmid construction is successfully.
The suicide plasmid pYTRLRRT that step 6 obtains is converted in E.coli DH5 α competent cell, then be seeded on tetracycline concentration is 50 μ g/mL, penbritin (Ampicillin) concentration is 100 μ g/mL screening flat board and cultivate, bacterium colony is positive bacteria, selects positive bacteria and carries out PCR checking (primer is Tet-up and Tet-down) and digestion verification (restriction enzyme Kpn I).PCR verifies that all swimming lanes all have clear band at about 1400bp, conforms to Tet gene fragment size; There are two bands (as shown in Figure 3) clearly in digestion verification, size respectively with plasmid pUC18-RLRR(5.3kb) with Tet(1.4kb) conform to, illustrates that pYTRLRRT plasmid construction is successfully.
Be converted into by suicide plasmid pYTRLRRT in lactobacillus paraceasi (Lactobacillusparacasei) HD1.7 cell, electric conversion condition is voltage 1.8kV, bacterial growth concentration is A 600=0.78, lactobacillus paraceasi (Lactobacillusparacasei) HD1.7 through 1% glycine process, adopts AEBl electricity to turn damping fluid.
Lactobacillus paraceasi (Lactobacillusparacasei) the HD1.7 cell that suicide plasmid pYTRLRRT electricity transforms is seeded to that tetracycline concentration is 50 μ g/mL, penbritin (Ampicillin) concentration is on the screening flat board of 100 μ g/mL, select positive bacteria and carry out cultivating that the LB coated by bacterium liquid again containing tsiklomitsin (70 μ g/mL) and penbritin (100pg/mL) is dull and stereotyped, 37 DEG C of incubated overnight, flat board has bacterium colony grow; And on the negative control plates inoculating lactobacillus paraceasi HD1.7 cell aseptic drop out existing.Because suicide plasmid pYTRLRRT can not express in lactobacillus paraceasi HD1.7 cell, the appearance of positive bacterium colony illustrates tetracycline resistance gene tet gene fusion, the expression in lactobacillus paraceasi (Lactobacillusparacasei) HD1.7 cell in suicide plasmid pYTRLRRT.
According to L.paracasei HD1.7(lactobacillus paraceasi HD1.7) in the sequence of prcR gene both sides, primer pair (prcR gene the upstream primer 5 '-ATGACNAAYCAYCARAC-3 ' of the prcR gene that can increase for stencil design goes out with L.paracaseiHD1.7 genomic dna, prcR gene downstream primer 5 '-TGCCAGGTTATGGGAAT-3 '), then this Auele Specific Primer is utilized to carry out pcr amplification to positive lactobacillus paraceasi (Lactobacillusparacasei) the HD1.7 cell that suicide plasmid pYTRLRRT electricity transforms, again the fragment amplified is connected on plasmid pUC18, then be converted in E.coli DH5 α competent cell, then be seeded on screening flat board that kantlex concentration is 50 μ g/mL and cultivate, bacterium colony is positive bacteria, illustrate that suicide plasmid pYTRLRRT has knocked out the prcR gene in lactobacillus paraceasi (Lactobacillusparacasei) HD1.7.Select positive bacteria and carry out PCR checking (primer is Tet-up and Tet-down) and digestion verification (restriction enzyme Kpn I).PCR verifies that all swimming lanes all have clear band at about 1400bp, conforms to Tet gene fragment size; Also the clear band of about 1400bp is had to occur in digestion verification.

Claims (8)

1. knock out a suicide plasmid pYTRLRRT for prcR gene, it is characterized in that the suicide plasmid pYTRLRRT knocking out prcR gene is made up of prcR gene left-hand fragment prcRL, prcR gene right fragment prcRR, tetracycline resistance gene and plasmid pUC18; The nucleotide sequence of fragment prcRL is as shown in SEQ ID NO:1; The nucleotide sequence of fragment prcRR is as shown in SEQ IDNO:2;
The suicide plasmid pYTRLRRT knocking out prcR gene is built by following steps: one, with LactobacillusparacaseiHD1.7 genomic dna for template PCR amplifications prcR gene left-hand fragment prcRL, the upstream primer of amplified fragments prcRL is prcRL-up, the nucleotides sequence of prcRL-up is classified as 5 '-CCGGAGCTCCCTACTCAGCATTCAGAGGTCAACT-3 ', the downstream primer of amplified fragments prcRL is prcRL-down, the nucleotides sequence of prcRL-down is classified as 5 '-GTCGGTACCATCTTCAGCATCGTTTGGTGGTTGG-3 ', the reaction system of pcr amplified fragment prcRL is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRL-up that is 1pmol/ μ L, the 10 μ L concentration prcRL-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms, the reaction conditions of pcr amplified fragment prcRL is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 61 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min,
Two, with restriction enzyme Sac I and Kpn I, respectively double digestion is carried out to the fragment prcRL that plasmid pUC18 and step one obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRL double digestion fragments, 5 μ l plasmid pUC18 double digestion fragments, 2.5 μ l 10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RL;
Three, with L.paracasei HD1.7 genomic dna for template PCR amplifications prcR gene right fragment prcRR, the upstream primer of amplified fragments prcRR is prcRR-up, the nucleotides sequence of prcRR-up is classified as 5 '-GTCGGTACCGGTCAGCATTCGTAGAGTGTCGGCC-3 ', the downstream primer of amplified fragments prcRR is prcRR-down, the nucleotides sequence of prcRR-down is classified as 5 '-CCGCTGCAGGCAGTGACCAGAGATAGCTCGGCGT-3 ', and the reaction system of pcr amplified fragment prcRR is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRR-up that is 1pmol/ μ L, the 10 μ L concentration prcRR-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRR is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 69 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Four, with restriction enzyme Pst I and Kpn I, respectively double digestion is carried out to the fragment prcRR that plasmid pUC18-RL and step 3 obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRR double digestion fragments, 5 μ l plasmid pUC18-RL double digestion fragments, 2.5 μ l 10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RLRR;
Five, with pBR322 plasmid for template PCR amplifications tetracycline resistance gene Tet, the upstream primer of amplification Tet is Tet-up, the nucleotides sequence of Tet-up is classified as 5 '-CCGGGTACCTCTCATGTTTGACAGCTT-3 ', the downstream primer of amplification Tet is Tet-down, the nucleotides sequence of Tet-down is classified as 5 '-GTCGGTACCTAATAGATATGTTCTGCCAAGGGT-3 ', and the reaction system of pcr amplification Tet is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration pBR322 plasmid that is 25ng/ μ L, the 10 μ L concentration Tet-up that is 1pmol/ μ L, the 10 μ L concentration Tet-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplification Tet is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 46 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Six, carry out enzyme with restriction enzyme Kpn I respectively to the tetracycline resistance gene Tet that plasmid pUC18-RLRR and step 5 obtain to cut, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l tetracycline resistance gene Tet endonuclease bamhis, 5 μ l plasmid pUC18-RLRR endonuclease bamhis, 2.5 μ l 10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, namely obtain the suicide plasmid pYTRLRRT knocking out prcR gene.
2. knock out the construction process of the suicide plasmid pYTRLRRT of prcR gene described in claim 1, it is characterized in that the suicide plasmid pYTRLRRT knocking out prcR gene builds according to the following steps:
One, with L.paracasei HD1.7 genomic dna for template PCR amplifications prcR gene left-hand fragment prcRL, the upstream primer of amplified fragments prcRL is prcRL-up, the nucleotides sequence of prcRL-up is classified as 5 '-CCGGAGCTCCCTACTCAGCATTCAGAGGTCAACT-3 ', the downstream primer of amplified fragments prcRL is prcRL-down, the nucleotides sequence of prcRL-down is classified as 5 '-GTCGGTACCATCTTCAGCATCGTTTGGTGGTTGG-3 ', and the reaction system of pcr amplified fragment prcRL is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRL-up that is 1pmol/ μ L, the 10 μ L concentration prcRL-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRL is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 61 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Two, with restriction enzyme Sac I and Kpn I, respectively double digestion is carried out to the fragment prcRL that plasmid pUC18 and step one obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRL double digestion fragments, 5 μ l plasmid pUC18 double digestion fragments, 2.5 μ l 10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RL;
Three, with L.paracasei HD1.7 genomic dna for template PCR amplifications prcR gene right fragment prcRR, the upstream primer of amplified fragments prcRR is prcRR-up, the nucleotides sequence of prcRR-up is classified as 5 '-GTCGGTACCGGTCAGCATTCGTAGAGTGTCGGCC-3 ', the downstream primer of amplified fragments prcRR is prcRR-down, the nucleotides sequence of prcRR-down is classified as 5 '-CCGCTGCAGGCAGTGACCAGAGATAGCTCGGCGT-3 ', and the reaction system of pcr amplified fragment prcRR is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration L.paracasei HD1.7 genomic dna that is 25ng/ μ L, the 10 μ L concentration prcRR-up that is 1pmol/ μ L, the 10 μ L concentration prcRR-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplified fragment prcRR is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 69 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Four, with restriction enzyme Pst I and Kpn I, respectively double digestion is carried out to the fragment prcRR that plasmid pUC18-RL and step 3 obtain, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l prcRR double digestion fragments, 5 μ l plasmid pUC18-RL double digestion fragments, 2.5 μ l 10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, obtain plasmid pUC18-RLRR;
Five, with pBR322 plasmid for template PCR amplifications tetracycline resistance gene Tet, the upstream primer of amplification Tet is Tet-up, the nucleotides sequence of Tet-up is classified as 5 '-CCGGGTACCTCTCATGTTTGACAGCTT-3 ', the downstream primer of amplification Tet is Tet-down, the nucleotides sequence of Tet-down is classified as 5 '-GTCGGTACCTAATAGATATGTTCTGCCAAGGGT-3 ', and the reaction system of pcr amplification Tet is that 50 μ L contain Mg by 10 μ L 2+5 × PrimeSTAR Buffer, 4 μ L concentration be respectively PrimeSTAR HS archaeal dna polymerase and 5.5 μ L sterilizing ddH that the dNTP Mixture of 2.5mmol/L, the 10 μ L concentration pBR322 plasmid that is 25ng/ μ L, the 10 μ L concentration Tet-up that is 1pmol/ μ L, the 10 μ L concentration Tet-down that is 1pmol/ μ L, 0.5 μ L concentration are 2.5U/ μ L 2o forms; The reaction conditions of pcr amplification Tet is 98 DEG C of denaturation 3min, 98 DEG C of sex change 10s, 46 DEG C of annealing 15s, 72 DEG C of extensions 1.5min, totally 30 circulations, then 72 DEG C extend 10min;
Six, carry out enzyme with restriction enzyme Kpn I respectively to the tetracycline resistance gene Tet that plasmid pUC18-RLRR and step 5 obtain to cut, then double digestion fragment is connected with T4DNA ligase enzyme, enzyme disjunctor is that the T4DNA ligase enzyme that 25 μ l are 3U/ μ L by 16.5 μ l tetracycline resistance gene Tet endonuclease bamhis, 5 μ l plasmid pUC18-RLRR endonuclease bamhis, 2.5 μ l 10 × T4DNA Ligase Buffer and 1 μ l concentration forms, 16 DEG C of connections of spending the night, namely obtain the suicide plasmid pYTRLRRT knocking out prcR gene.
3. the construction process knocking out the suicide plasmid pYTRLRRT of prcR gene according to claim 2, it is characterized in that plasmid pUC18 endonuclease reaction system in step 2 be 20 μ L by 1 μ L concentration be the Sac I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L 10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L plasmid pUC18 and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
4. the construction process knocking out the suicide plasmid pYTRLRRT of prcR gene according to claim 2, it is characterized in that fragment prcRL endonuclease reaction system in step 2 be 20 μ L by 1 μ L concentration be the Sac I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L 10 × Buffer Tango, 10 μ L concentration be 80ng/ μ L fragment prcRL and the aseptic ddH of 6 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
5. the construction process knocking out the suicide plasmid pYTRLRRT of prcR gene according to claim 2, it is characterized in that plasmid pUC18-RL endonuclease reaction system in step 4 be 20 μ L by 1 μ L concentration be the Pst I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L 10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L plasmid pUC18-RL and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
6. the construction process knocking out the suicide plasmid pYTRLRRT of prcR gene according to claim 2, it is characterized in that fragment prcRR endonuclease reaction system in step 4 be 20 μ L by 1 μ L concentration be the Pst I of 10U/ μ L, 1 μ L concentration be 10U/ μ L Kpn I, 2 μ L 10 × Buffer Tango, 7 μ L concentration be 80ng/ μ L fragment prcRR and the aseptic ddH of 9 μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
7. the construction process knocking out the suicide plasmid pYTRLRRT of prcR gene according to claim 2, it is characterized in that plasmid pUC18-RLRR endonuclease reaction system in step 6 be 20 μ L by 1 μ L concentration be Kpn I, 2 μ L10 × Buffer Tango of 10U/ μ L, 7 μ L concentration are plasmid pUC18-RLRR and the aseptic ddH of 10 μ L of 80ng/ μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
8. the construction process knocking out the suicide plasmid pYTRLRRT of prcR gene according to claim 2, it is characterized in that tetracycline resistance gene Tet endonuclease reaction system in step 6 be 20 μ L by 1 μ L concentration be Kpn I, 2 μ L10 × Buffer Tango of 10U/ μ L, 7 μ L concentration are tetracycline resistance gene Tet and the aseptic ddH of 10 μ L of 80ng/ μ L 2o forms; 37 DEG C of constant temperature enzymes cut 2h, and reclaim endonuclease bamhi with digestion products glue.
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