CN103146739B - Establishing method of bifidobacterium functional gene no-trace knockout method - Google Patents
Establishing method of bifidobacterium functional gene no-trace knockout method Download PDFInfo
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Abstract
The invention discloses an establishing method of a bifidobacterium functional gene no-trace knockout method. The establishing method comprises the following steps of: constructing a serpin gene suicide type targeting vector UPD; loading a promoter of the bifidobacterium by using pDG7 as a framework to construct a bifidobacterium-escherichia coli shuttle expression vector pDG-Hup; constructing a bifidobacterium-escherichia coli shuttle expression vector Pdg-hup-cre of a Cre enzyme gene based on pDG-Hup; transferring the targeting vector into the bifidobacterium and obtaining a bifidobacterium serpin gene defective strain through homologous recombination; and removing the spectinomycin spc gene introduced in the bifidobacterium genome by utilizing a site-specific recombination Cre-loxp system to realize no-trace knockout of the bifidobacterium resistance gene. The establishing method of the bifidobacterium functional gene no-trace knockout method disclosed by the invention is used for obtaining the defective strain of the bifidobacterium functional gene by utilizing a homologous recombination principle, can be applied to the knockout of the bifidobacterium functional gene and can be used for improving the strains.
Description
Technical field
The invention belongs to the application in bifidus bacillus genetically engineered, specifically relate to the seamless knockout technique of a kind of bifidus bacillus functional gene.
Background technology
Gene knockout is a kind of mode of modifying native gene of utilizing DNA homology restructuring principle to carry out, make to recombinate between the target gene homologous sequence on foreign DNA and recipient cell genomic dna, foreign vector DNA site-directed integration is entered on the predetermined site of target gene, or replace with a certain DNA fragmentation of target gene, complete accurate modification and transformation to genomic dna target gene, cause the structure of target gene or the sudden change of composition, cause gene function to be lost, and the gene of modified and transformation can be with copying of genomic dna and stable copying.It has, and polarization is strong, target gene can overcome blindness and the randomness of the random integration of mutagenesis with the feature of genomic dna genetic stability after knocking out, and is a kind of method of desirable modification, transformation microorganism hereditary material.In Research for Industrial Microbial Germ improvement, the pathways metabolism and the metabolism that by knocking out of target gene, change microorganism are flowed, can not only improve the output of leavened prod and produce new meta-bolites, can also improve the artificial regulatory of fermentation industry fermentation approach and process, be the most promising microbial strains improved means of 21 century.
The application of Cre-loxp system in gene targeting can be said to gene target position technology and bring a revolution.Cre recombinase is a kind of activated protein of molecular weight 38kd that derives from the recombinase family of pl phage.It not only has catalytic activity, and similar with restriction enzyme, identifies special DNA sequence dna, as: Loxp site, causes restructuring.Loxp site is the specificity recombination site of Cre recombinase effect, by 34 bp, formed, that is: and ATAACTTCGTATAGCATACATTATACGAAGTTAT, the interval region of the foldback sequence of two 13 bp and 8 bp forms.Cre recombinase has 70% recombination efficiency, not by any cofactor, acts on the DNA substrate of various structures, as linear, ring-type, and superhelix (being become linear structure by the uncoiling of Cre recombinase) even.Cre recombinase, by the combination with the upper Loxp of DNA site, is brought into play its recombination.Cre/Loxp system is short and pithy, the effect of Cre enzyme is single-minded, and do not affect normal expression and the regulation and control of gene, this feature makes it in gene targeting operation, have very important effect, and this system is successfully for the gene knockout of multiple eukaryotic cell and bacterium at present.
Bifidus bacillus (
bifidobacterium)can maintain the microecological balance of host's enteron aisle, improve human body to lactose tolerance, there is the blood fat of reduction, antineoplastic action, and can synthesize multivitamin, promote body's immunity.In recent years, domestic preparation and food development containing bifidobacteria viable bacteria is rapid, and bifidus bacillus product puts goods on the market.Abroad, also all the fashion containing the food of bifidus bacillus, particularly in Japan, Europe and North America.Yet bifidus bacillus is as a kind of gram-positive microorganism, and cell walls is thicker, it is carried out to genetic manipulation is a difficult point always, has limited the progress in bifidus bacillus genetically engineered field.
The present invention is by the conversion of bifidus bacillus, targeting vector and shuttle expression carrier are imported to bifidus bacillus step by step, and first by Cre-loxp system applies in the genetically engineered of gram-positive microorganism bifidus bacillus, successfully realize the seamless of bifidus bacillus functional gene and knocked out, for studying its gene function, provide means.
Summary of the invention
The object of this invention is to provide the seamless knockout technique of a kind of bifidus bacillus functional gene; The method first by Cre-loxp system applies in the research of gram-positive microorganism bifidus bacillus, obtained the features such as the seamless gene-deleted strain of bifidus bacillus functional gene.
The concrete technical scheme of the present invention is as follows:
An establishment method for the seamless gene-deleted strain of bifidus bacillus functional gene, comprises the following steps:
(1) SEQ ID.NO.1 is passed through
ecor V site and
psti enzyme is cut and is connected on pUC57 carrier, obtains pUC57-LoxP carrier;
(2) take pER8 plasmid as template, by primer SEQ. ID.NO.2 and SEQ. ID.NO.3, increase, amplified production is passed through
xhoi and
apatwo the cutting afterwards of I is connected with pUC57-LoxP carrier, builds and obtains carrier pUC-Spc;
(3) using the genome of bifidus bacillus as template, by primer SEQ. ID.NO.4 and SEQ. ID.NO.5, increase, amplified production is passed through
ecor I and
spetwo the cutting afterwards of I is connected with pUC-Spc carrier, builds and obtains carrier pUC-spc-serpin
up800;
(4) using the genome of bifidus bacillus as template, by primer SEQ. ID.NO.6 and SEQ. ID.NO.7, increase, amplified production is passed through
bgliI and
hindiII pair is cut rear and pUC-spc-serpin
up800carrier connects, and builds and obtains targeting vector plasmid pUC-spc-serpin
up+down, i.e. UPD;
(5) SEQ. ID.NO.8 sequence warp
ecorI and
sali double digestion is connected to pDG7, builds shuttle expression carrier pDG-Hup;
(6) take the pCrepA plasmid that contains Cre enzyme gene is template, by primer SEQ. ID.NO.9 and SEQ. ID.NO.10 amplification Cre enzyme gene; Amplified production process
xhoi and
sali is two cut after, is connected with pDG-Hup, structure obtains pDG-Hup-Cre;
(7) conversion of targeting vector: add targeting vector plasmid pUC-spc-serpin in bifidus bacillus protoplastis
up+down, merge and transform; Washing, recovery, be coated on bacterium liquid on the regeneration culture medium with spectinomycin and cultivate 2-3 d after recovery, obtains
⊿ serpinstrain;
(8) will
⊿ serpinprotoplastis competent cell is made in strain, and shuttle expression carrier pDG-Hup-Cre is imported
⊿ serpinstrain, merges and transforms, and bacterium colony after transforming is coated on the regeneration culture medium of amicillin resistance, screens the bifidus bacillus of no longer carrying spectinomycin resistance gene
serpinthe seamless gene-deleted strain of gene
⊿ serpin ⊿ spcstrain.
The condition that fusion described in step (7) and step (8) transforms is: what join preheating is in the solution of 20 ± 1mM/L, 40 ± 1% (v/v) PEG6000 containing magnesium ion concentration, merges conversion.
In step (7), being prepared as of described bifidus bacillus protoplastis: the bifidus bacillus of preservation is transferred on MRS flat board, and 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; From flat board, choose a single bacterium colony, be inoculated in MRS liquid nutrient medium, 37 ± 1 ℃ of anaerobism are cultivated 20-26h; With 3 ± 1%(v/v) inoculum size transfer in fresh MRS liquid nutrient medium, 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; Centrifugal collection thalline, resuspended; Adding mutanolysin to final concentration is 4.5-5.5 mg/L, 36-38 ℃ of enzymolysis 25-35 minute; Be resuspended in SMM solution, obtain bifidus bacillus protoplastis.
Preferably, described bifidus bacillus is bifidus longum bb.
Another object of the present invention is to provide a kind of seamless gene-deleted strain of bifidus bacillus functional gene that does not carry spectinomycin resistance gene.
Preferably, the described seamless gene-deleted strain of bifidus bacillus functional gene, it is to be prepared by above-mentioned method.
Advantage of the present invention is: present method substep imports bifidus bacillus by targeting vector and shuttle expression carrier, utilize the principle of homologous recombination, screening has obtained gene-deleted strain, after importing shuttle expression carrier, express Cre recombinase, removed resistant gene, thereby having realized the seamless of bifidus bacillus functional gene knocks out, utilize this method to carry out gene knockout, the recombinant bacterium finally obtaining does not carry any resistance screening mark, in its use procedure, can avoid antibiotic application, for the strain improvement of bifidus bacillus provides means.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of targeting vector UPD.
Fig. 2 is recombinant vectors pUC-Spc double digestion the result, M:1 K marker; 1:pUC-spc carrier; 2,3:pUC-spc carrier is used respectively
xhoi,
apai double digestion result.
Fig. 3 is recombinant vectors pUC-spc-serpin
up800double digestion the result, M:1 K marker; 1,2:pUC-spc-serpin
up800carrier; 3,4: pUC-spc-serpin
up800carrier is used respectively
ecor I,
spei double digestion result.
Fig. 4 is targeting vector pUC-spc-serpin
up+downdouble digestion checking electrophoresis result, 1 K marker; 1:pUC-spc-serpin
up+downcarrier is used respectively
bgliI,
hindiII double digestion result; 2:pUC-spc-serpin
up+downcarrier is used respectively
xhoi,
hindiII double digestion result.
Fig. 5 is Cre-loxP system schematic.
Fig. 6 is recombinant plasmid pDG-Hup-Cre structural representation.
Fig. 7 is that pDG-Hup enzyme is cut the result, pDG-HUP transformant double digestion checking 1: γ Hind III 2:pDG7 double digestion (
bamh I with
sali) 3:pDG-HUP double digestion (
bamh I with
sali) 4:pDG7 not enzyme cut 5:pDG-HUP not enzyme cut 6:pDG-HUP double digestion (
bgliI with
ecor I) 7:pDG7 double digestion (
bgliI with
ecor I) 8:DL2000.
Fig. 8 is that pDG-Hup-Cre enzyme is cut the result, from left to right 1:DL2000; 2:pDG-Hup-Cre; 3:pDG-Hup-Cre; 4: λ Hind III.
Fig. 9 is that pDG-Hup-Cre transforms
⊿ serpinthe dull and stereotyped result of bifidus bacillus.
Figure 10 is that PCR verifies positive transformant collection of illustrative plates, 1:500-15000 bp Wide range maker; 2:
spcpositive control; 3/4:
spcprimer PCR detects
⊿ serpinstrain; 5:
spcprimer PCR detects
⊿ serpin ⊿ spcstrain; 6:
serpinpositive control 7/8:
serpinprimer PCR detects
⊿ serpinstrain; 9:
serpinprimer PCR detects
⊿ serpin ⊿ spcstrain.
Figure 11 is that PCR checking and enzyme are cut checking pDG-Hup-Cre conversion
⊿ serpinbifidus bacillus electrophoretogram.PCR verifies electrophoretogram (left side), and enzyme is cut the recombinant plasmid electrophoretogram (right side) transforming in rear bifidus bacillus body.
Figure 12 bifidus bacillus genome dna electrophoresis figure, 1/2:NCC2705 prime strain genome; 3/4:
⊿ serpinpnca gene group; 5/6:
⊿ serpin ⊿ spcpnca gene group; M:Wide range marker 500-15000 bp.
The positive transformant dotting of Figure 13 proof diagram A:T-spc is probe, A1: blank, and A2:
⊿ serpin ⊿ spc, A3
⊿ serpinstrain, A4
⊿ serpinstrain, A5 Bifidobacterium longum NCC2705 genome (negative control), A6
spc+ (positive control), B:T-
serpinfor probe; B1: blank B2:
⊿ serpin ⊿ spcstrain, B3:
⊿ serpinstrain, B 4:
⊿ serpinstrain, B5: Bifidobacterium longum NCC2705 genome (positive control), B6:
serpin+ (positive control).
Figure 14 is Southern hybridization analysis figure, A: the Southern hybridization figure that the T-serpin of take is probe, B: the Southern hybridization figure that the T-spc of take is probe; A1:
⊿ serpinstrain; A2:
⊿ serpin ⊿ spcstrain; A3:NCC2705 bifidus longum bb genome; A0: negative control; B1:
⊿ serpinstrain; B2:
⊿ serpin ⊿ spcstrain; B3: Bifidobacterium longum NCC2705 genome; B0: negative control.
Figure 15 promotor.
Embodiment
Embodiment 1: the structure of bifidus bacillus targeting vector and shuttle expression carrier:
1. in view of the feature of LoxP site sequence, directly the identical LoxP site of both direction is synthesized on pUC57 carrier, sequence (totally 108 bp) from
ecor V site and
pstinsert in I site, afterwards spectinomycin resistance gene is inserted respectively in the centre from two LoxP sites and two ends
spchomology arm sequence with upstream and downstream.
This step carrier construction pUC57-LoxP.Two synthetic LoxP site sequences are:
5 '-CGG
aCTAGTaTAACTTCGTATAATGTATGCTATACGAAGTTAT
cTCGAGaACGATCGGTT
gGGCCCaTAACTTCGTATAATGTATGCTATACGAAGTTAT
aGATCTcC-3 ' (SEQ ID.NO.1), restriction endonuclease is followed successively by
spei,
xhoi,
apai,
bgliI.
2. take pER8 plasmid as template, design primer P-Spc-F:5 '-CCG
cTCGAGtGGTCCAGAACCTTGACCG-3 ' (
xhoi), (SEQ. ID.NO.2)
P-Spc-R:5 '-CCC
gGGCCCtTATTTGCCGACTACCTTGGTG-3 ' (
apai),, (SEQ. ID.NO.3) spectinomycin resistance gene that increases, amplification system (40 μ L: comprise pER8 plasmid dilution template 4 μ L, 2 * Taq mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2o 12 μ L), pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 50 seconds, 60 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
xhoi and
apatwo the cutting afterwards of I is connected with pUC57-LoxP carrier, carrier construction pUC-Spc.Fig. 1 is targeting vector building process schematic diagram, and Fig. 2 is pUC-Spc double digestion the result.
3. design primer Serpin-up800-F:5 '-CCG
gAATTCaACCAGCACGTCAGGCC-3 ' (
ecor I), (SEQ. ID.NO.4), Serpin-up800-R:5 '-CGG
aCTAGTgGTTGGCCCCTTTGCTTG-3 ' (
spei), (SEQ. ID.NO.5) usings the genome of Bifidobacterium longum NCC2705 as masterplate, amplification
serpinupstream 800 bp fragments, amplification system (40 μ L: comprise the genome dilution template 4 μ L of NCC2705,2 * Taq mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2o 12 μ L), pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 57 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
ecor I and
spetwo the cutting afterwards of I is connected with pUC-Spc, successful carrier construction pUC-spc-serpin
up800.Fig. 3 is pUC-spc-serpin
up800double digestion the result.
4. design primer Serpin-down800-F:5 '-GGA
aGATCTtACTTTCCTGGCAGTCCGTC-3 ' (
bgliI) (SEQ. ID.NO.6)
Serpin-down800-R:5 '-CCC
aAGCTTcGTACACATACCAGGCGCG-3 ' (
hindiII), (SEQ. ID.NO.7) usings the genome of Bifidobacterium longum NCC2705 as masterplate, amplification
serpindownstream 800 bp gene fragments, amplification system 40 μ L: comprise the genome dilution template 4 μ L of Bifidobacterium longum NCC2705,2 * Taq mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2o 12 μ L, pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 56 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
bgliI and
hindiII pair is cut rear and pUC-spc-serpin
up800connect successful carrier construction pUC-spc-serpin
up+downbe UPD.Fig. 4 is targeting vector pUC-spc-serpin
up+downit is UPD double digestion checking electrophoresis result.
5. the recombinant plasmid pDG7 that contains bifidus bacillus endogenous replicon of take is skeleton, 200 bp(of synthetic Hup gene start codon and upstream thereof comprise Hu promoter sequence), and after its sequence, add multiple clone site (MCS), in its two ends, add respectively restriction enzyme site
ecorI and
sali, double digestion is connected to pDG7, successfully builds shuttle expression carrier pDG-Hup.Fig. 7 is that pDG-Hup enzyme is cut the result.
Synthetic promoter sequence (totally 283 bp) (as Figure 15)
6. design primer Cre-F:5 '-CCGCTCGAGATGTCCAATTTACTGACCGTACACC-3 '
xhoi, (SEQ. ID.NO.9)
Cre-R:5 '-CGCGTCGACCTAATCGCCATCTTCCAGCAG-3 '
sali, (SEQ. ID.NO.10)
the pCrepA plasmid (containing Cre enzyme gene) of take is template, amplification Cre enzyme gene.Amplification system 40 μ L: comprise pCrepA plasmid dilution template 4 μ L, 2 * Pfu mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2o 12 μ L, pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 62 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
xhoi and
sali is two cut after, be connected with pDG-Hup, successfully build pDG-Hup-Cre.Fig. 5 is Cre-loxP system schematic, and Fig. 6 is recombinant plasmid pDG-Hup-Cre collection of illustrative plates.
Embodiment 2: the preparation of bifidus longum bb protoplastis and conversion:
1. the conversion of targeting vector: Bifidobacterium longum NCC2705 is transferred to (MRS nutrient agar: peptone 10 g, extractum carnis 10 g, yeast extract paste 5 g, Tween-80 1 g, K MRS flat board from the glycerine pipe of-70 ℃ of preservations
2hPO
42 g, CH
3cOONa3H
2o 5 g, Citric Acid three ammonium 2 g, MgSO
47H
2o 0.2 g, MnSO
4h
2o 0.05 g, Agar 15 g, glucose 20 g, adding distil water to 1000 mL, adjusts 6.4,121 ℃ of autoclaving 15 min of pH 6.2-.MRS broth culture: the same agar that do not add of composition), 37 ℃ of anaerobism are cultivated 24 h.From flat board, choose a single bacterium colony, be inoculated in 5 mL MRS liquid nutrient mediums, 37 ℃ of anaerobism are cultivated 24 h; Then with the inoculum size of 3% (V/V), transfer in 25 fresh mL MRS liquid nutrient mediums, 37 ℃ of anaerobism are cultivated 24 h; Centrifugal collection thalline, resuspended; Adding mutanolysin to final concentration is 4.5-5.5 mg/L, 36-38 ℃ of enzymolysis 25-35 minute; Be resuspended in SMM(Hepes (4-hydroxyethyl piperazine ethanesulfonic acid) 0.02 M/L, magnesium chloride 1 mM/L, raffinose 0.5 M/L) in solution, obtain bifidus bacillus protoplastis.The targeting vector plasmid pUC-spc-serpin that adds 50 μ L precoolings in the protoplastis of 500 μ L bifidus longum bbs
up+down(plasmid DNA amount <5 μ g), adds 40% (v/v) PEG6000 solution, 1.5 mL(of preheating containing magnesium ion concentration 20 mM/L) fusion conversion.After conversion, add SMMP(liquid regeneration culture medium: SMM=3:1) solution washing is 2 times.Add 1 mLSMMP, 1 h that recovers.After recovery, bacterium liquid is coated on to spectinomycin (final concentration 100 μ g/mL) regeneration solid medium (tryptone 5.0 g, yeast extract 5.0 g, aminothiopropionic acid hydrochloride 0.4 g, glucose 5.0 g, KH
2pO
42.5 g, CH
3cOONa 10.0 g, CaCl
22H
2o 5.0 g, MgCl
26H
2o 5.0 g, agar 15.0 g, raffinose 0.2-0.3M/L, adjusts 6.5,115 ℃ of autoclaving 20 min of pH) the upper 2-3 of cultivation d; Obtain
⊿ serpinstrain;
2. picking
⊿ serpinbacterium colony, is activated in the MRS liquid nutrient medium of 100 μ g/mL spectinomycin resistances.Extract
⊿ serpinstrain bifidus bacillus genomic dna, as template, design primer serpin-F:5 '-ACC AAT CGC TGC TAA GTT CG-3 ' (SEQ. ID.NO.11), R:5 '-GAA CCG GTG TCG ATC ATC TT-3 ' (SEQ. ID.NO.12) amplification serpin gene, amplification system (10 μ L: the genome dilution template 1 μ L that comprises transformant, 2 * Taq mix, 5 μ L, 10 μ M primer pair 1 μ L, ddH
2o 3 μ L), pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 60 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, detect before and after restructuring
serpinthe variation of gene signal; With primer spc-F/R(SEQ. ID.NO.2 and SEQ. ID.NO.3) amplification
spcgene, amplification system, with amplification system and the response procedures of 2 steps in embodiment 1, detects after restructuring
spcgene signal.Be verified as positive
⊿ serpinstrain, is activated in the MRS liquid nutrient medium of 100 μ g/mL spectinomycin resistances.
3. will
⊿ serpinprotoplastis competent cell is made in strain, and shuttle expression carrier pDG-Hup-Cre is imported
⊿ serpinstrain, adds 40% (v/v) PEG6000 solution, 1.5 mL(of preheating containing magnesium ion concentration 20 mM/L) fusion conversion.Bacterium colony after transforming is coated on to regeneration solid medium (tryptone 5.0 g, yeast extract 5.0 g, aminothiopropionic acid hydrochloride 0.4 g, glucose 5.0 g, the KH of 25 μ g/mL amicillin resistances
2pO
42.5 g, CH
3cOONa 10.0 g, CaCl
22H
2o 5.0 g, MgCl
26H
2o 5.0 g, agar 15.0 g, raffinose 0.2-0.3M/L, adjusts 6.5,115 ℃ of autoclaving 20 min of pH) upper, obtain possible
⊿ serpin ⊿ spcstrain.
4. picking
⊿ serpin ⊿ spcbacterium colony, is activated in 25 μ g/mL amicillin resistance MRS liquid nutrient mediums.Extract
⊿ serpinstrain bifidus bacillus genomic dna, as template, with primer spc-F/R(SEQ. ID.NO.2 and SEQ. ID.NO.3) amplification
spcgene, amplification system, with amplification system and the response procedures of 2 steps in embodiment 1, detects after restructuring
spcgene signal.The bacterial strain that no longer carries spectinomycin resistance gene is positive
⊿ serpin ⊿ spcstrain.Screening obtains
⊿ serpin ⊿ spcstrain is activated to is not with the MRS substratum of resistance to upload culture, further checking.
Embodiment 3: bifidus bacillus
serpinthe checking of gene-deleted strain:
With
⊿ serpin ⊿ spcpnca gene group DNA is template, with primer serpin-F/R(SEQ. ID.NO.11 and SEQ. ID.NO.12) amplification
serpingene (amplification system and response procedures are with step 2 in embodiment 2), detects in regrouping process
serpinthe variation of gene signal; With primer SEQ. ID.NO.2 and SEQ. ID.NO.3 amplification
spcgene, detects shuttle expression carrier and transforms front and back
spcgene signal.Figure 10 is the PCR the result after substep transforms, warp
serpinthe amplification of genetically deficient fragment PCR and 1% agarose gel electrophoresis, show
⊿ serpinstrain can amplify 1000 bp, shows to contain in transformant genome
spcgene;
⊿ serpinstrain fail the to increase fragment of 1400 bp, shows that homologous recombination has occurred the homologous fragment of practicing shooting on fragment and karyomit(e),
serpingene quilt
spcgene substitution, has built
sepingene-deleted strain; Yet
⊿ serpin ⊿ spcstrain is failed to increase and is arrived
serpingene, has also lost
spcthe signal of gene, this illustrates that, under the help of shuttle expression carrier, Cre recombinase is by the resistant gene between the identical LoxP site of both direction
spcfragment has been deleted.
With
⊿ serpin ⊿ spcthe genomic dna of strain is template, and with primer Cre-F/R amplification cre gene, PCR verifies pDG-Hup-Cre positive transformant (amplification system is with 6 step implementation steps in embodiment 1), and extracts recombinant plasmid enzyme and cut checking.Fig. 9 is the dull and stereotyped photo of conversion, the positive transformant PCR of Figure 10 proof diagram, and Figure 11 is that pDG-Hup-Cre transforms PCR checking and enzyme is cut proof diagram.Result shows, shuttle expression carrier pDG-Hup-Cre has successfully imported bifidus bacillus.
3. Figure 12 is that glass bead method is extracted bifidus longum bb genomic dna, and electrophoresis result shows.Electrophoretic band concentrates on 15000 bp, and the genome effect of extraction is better, can be for further dotting or Southern hybridization check.The spectinomycin spc digoxigenin labeled fragment T-spc of take is probe,
serpinlabeled fragment T-serpin is that probe carries out respectively dotting checking.Figure 13 shows
⊿ serpinstrain
he ⊿ serpin ⊿ spcstrain pair
serpingene is all revealed as feminine gender,
⊿ serpinstrain pair
spcspectinomycin gene is aobvious positive, and
⊿ serpin ⊿ spcstrain pair
spcspectinomycin gene is revealed as feminine gender, result demonstration, and Cre recombinase successfully will
spcgene has removed from the genome of bifidus bacillus recombinant bacterial strain.
4. will screen
⊿ serpinstrain and
⊿ serpin ⊿ spcsouthern hybridization checking is carried out in strain.Figure 14 shows that positive control A3 presents single band when hybridizing with T-serpin probe, A1/A2 is all without band,
⊿ serpinstrain and
⊿ serpin ⊿ spcin strain without
serpingene signal; When hybridizing with T-spc probe, B1
⊿ serpinstrain presents single band, and B3 is that bifidus bacillus prime strain does not present band,
⊿ serpinthe spectinomycin resistance gene of having introduced external source in strain transformant genome, there is not band in B2, explanation
⊿ serpin ⊿ spcin pnca gene group
spcresistant gene has been removed by the restructuring of Cre recombinase, has successfully realized the seamless of bifidus bacillus and has knocked out.
These are only specific embodiments of the invention, with this, do not limit protection scope of the present invention; Not violating any replacement and the improvement of doing on the basis of the present invention's design, all belong to protection scope of the present invention.
Sequence table
<110> University Of Nanchang
The foundation of the seamless knockout technique of <120> bifidus bacillus functional gene
<160> 12
<170> PatentIn version 3.1
<210> 1
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acgatcggtt gggcccataa cttcgtataa tgtatgctat acgaagttat 100
agatctcc 108
<210> 2
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<400> 3
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<213> artificial sequence
<400> 8
cgcggatccc attccgctgg gcgcggcggc catgaagtgg cttgacaagc 50
ataatcttgt ctgattcgtc tattttcata cccccttcgg ggaaatagat 100
gtgaaaaccc ttataaaacg cgggttttcg cagaaacatg cgctagtatc 150
attgatgaca acatggacta agcaaaagtg cttgtcccct gacccaagaa 200
ggatgcttta tgctcgagga gctcgggccc ggtacctacg taagatctgc 250
ggccgccctg caggactagt gtcgacgtcg gcc 283
<210> 9
<211> 34
<212> DNA
<213> artificial sequence
<400> 9
ccgctcgaga tgtccaattt actgaccgta cacc 34
<210> 10
<211> 30
<212> DNA
<213> artificial sequence
<400> 10
cgcgtcgacc taatcgccat cttccagcag 30
<210> 11
<211> 20
<212> DNA
<213> artificial sequence
<400> 11
accaatcgct gctaagttcg 20
<210> 12
<211> 20
<212> DNA
<213> artificial sequence
<400> 12
gaaccggtgt cgatcatctt 20
Claims (4)
1. an establishment method for the seamless gene-deleted strain of bifidus bacillus functional gene, is characterized in that, comprises the following steps:
(1) SEQ ID.NO.1 is cut and is connected on pUC57 carrier by EcoR V site and Pst I enzyme, obtain pUC57-LoxP carrier;
(2) take pER8 plasmid as template, by primer SEQ. ID.NO.2 and SEQ. ID.NO.3 amplification spectinomycin resistance gene spc, by amplified production, through Xho I with Apa I is two is connected with pUC57-LoxP carrier after cutting, structure obtains carrier pUC-Spc;
(3) using the genome of bifidus bacillus as template, by primer SEQ. ID.NO.4 and SEQ. ID.NO.5, increase, by amplified production, through EcoR I with Spe I is two is connected with pUC-Spc carrier after cutting, structure obtains carrier pUC-spc-serpin
up800;
(4) using the genome of bifidus bacillus as template, by primer SEQ. ID.NO.6 and SEQ. ID.NO.7, increase, by amplified production through Bgl II and Hind III is two cut after with pUC-spc-serpin
up800carrier connects, and builds and obtains targeting vector plasmid pUC-spc-serpin
up+down, i.e. UPD;
(5) SEQ. ID.NO.8 sequence is connected to pDG7 through EcoR I and Sal I double digestion, builds shuttle expression carrier pDG-Hup;
(6) take the pCrepA plasmid that contains Cre enzyme gene is template, by primer SEQ. ID.NO.9 and SEQ. ID.NO.10 amplification Cre enzyme gene; Amplified production through Xho I and Sal I are two cut after, is connected with pDG-Hup, structure obtains pDG-Hup-Cre;
(7) conversion of targeting vector: by targeting vector plasmid pUC-spc-serpin
up+downjoin in bifidus bacillus protoplastis, merge and transform; Washing, recovery, be coated on bacterium liquid on the regeneration culture medium with spectinomycin and cultivate 2-3 d after recovery, and get is Dao ⊿ serpin strain;
(8) make protoplastis competent cell Jiang ⊿ serpin strain, by shuttle expression carrier pDG-Hup-Cre Dao Ru ⊿ serpin strain, merge and transform, bacterium colony after transforming is coated on the regeneration culture medium of amicillin resistance, screens the seamless disappearance of the bifidus bacillus serpin gene Zhu ⊿ serpin ⊿ spc strain of no longer carrying spectinomycin resistance gene; Described bifidus bacillus is bifidus longum bb.
2. establishment method according to claim 1, it is characterized in that, the condition that fusion described in step (7) and step (8) transforms is: what join preheating is in the solution of 20 ± 1mM/L, 40 ± 1% (v/v) PEG6000 containing magnesium ion concentration, merges conversion.
3. establishment method according to claim 1, is characterized in that, in step (7), and being prepared as of described bifidus bacillus protoplastis: the bifidus bacillus of preservation is transferred on MRS flat board, and 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; From flat board, choose a single bacterium colony, be inoculated in MRS liquid nutrient medium, 37 ± 1 ℃ of anaerobism are cultivated 20-26h; With 3 ± 1%(v/v) inoculum size transfer in fresh MRS liquid nutrient medium, 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; Centrifugal collection thalline, resuspended; Adding mutanolysin to final concentration is 4.5-5.5 mg/L, 36-38 ℃ of enzymolysis 25-35 minute; Be resuspended in SMM solution, obtain bifidus bacillus protoplastis.
4. do not carry the seamless gene-deleted strain of bifidus bacillus functional gene of spectinomycin resistance gene, it is to be prepared by the method described in claim 1-3 any one.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429520A (en) * | 2008-11-10 | 2009-05-13 | 天津科技大学 | Epiphyte genome conformity plasmid pUNFIN without selection mark, construction method and uses thereof |
| CN101812475A (en) * | 2009-11-19 | 2010-08-25 | 无锡亚克生物科技有限公司 | Recombinant vector pGAprEHS for expressing Harpin protein and engineering bacteria thereof |
| CN102094036A (en) * | 2010-12-13 | 2011-06-15 | 西北农林科技大学 | Allele double knockout targeting vector system and construction method thereof |
| WO2011100250A1 (en) * | 2010-02-09 | 2011-08-18 | The Trustees Of Columbia University In The City Of New York | In vivo gene regulation by the combination of knock- in-teto sequence into the genome and tetracycline-controlled trans-suppressor (tts) protein |
| CN102250937A (en) * | 2011-05-30 | 2011-11-23 | 黑龙江省科学院微生物研究所 | Construction method of recombinant plasmid vector p18-kan+/- containing loxP-kanr-loxP segments |
-
2013
- 2013-03-06 CN CN201310070885.8A patent/CN103146739B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429520A (en) * | 2008-11-10 | 2009-05-13 | 天津科技大学 | Epiphyte genome conformity plasmid pUNFIN without selection mark, construction method and uses thereof |
| CN101812475A (en) * | 2009-11-19 | 2010-08-25 | 无锡亚克生物科技有限公司 | Recombinant vector pGAprEHS for expressing Harpin protein and engineering bacteria thereof |
| WO2011100250A1 (en) * | 2010-02-09 | 2011-08-18 | The Trustees Of Columbia University In The City Of New York | In vivo gene regulation by the combination of knock- in-teto sequence into the genome and tetracycline-controlled trans-suppressor (tts) protein |
| CN102094036A (en) * | 2010-12-13 | 2011-06-15 | 西北农林科技大学 | Allele double knockout targeting vector system and construction method thereof |
| CN102250937A (en) * | 2011-05-30 | 2011-11-23 | 黑龙江省科学院微生物研究所 | Construction method of recombinant plasmid vector p18-kan+/- containing loxP-kanr-loxP segments |
Non-Patent Citations (4)
| Title |
|---|
| alpha 1,3-galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences andcre-expressing adenovirus;Sendai, Y et al.;《TRANSPLANTATION》;20060315;第81卷(第5期);第760-766页，尤其是摘要、材料方法和结论部分 * |
| Paul A. Beare et al..Two Systems for Targeted Gene Deletion in Coxiella burnetii.《Applied and Environmental Microbiology》.2012,第78卷(第13期),4580-4589. * |
| Sendai Y et al..alpha 1 * |
| Two Systems for Targeted Gene Deletion in Coxiella burnetii;Paul A. Beare et al.;《Applied and Environmental Microbiology》;20120420;第78卷(第13期);第5980-5989页 * |
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