CN103789337A - Suicide vector pGMB152 for blue-white selection and application thereof - Google Patents
Suicide vector pGMB152 for blue-white selection and application thereof Download PDFInfo
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Abstract
The invention discloses a suicide vector pGMB152 for blue-white selection and application thereof. The suicide vector pGMB152 is formed by cloning an LacZYA gene into a suicide vector pGMB151, and then salmonella pullorum hsdM gene deletion strains can be built by using the suicide vector by a recombinant bacteria blue-white selection method. On the basis of the traditional selection method and according to the characteristic that beta-galactosidase coded by the LacZYA report gene on the suicide vector pGMB152 reacts with a substrate X-gal to generate a blue substance, existence of the pGMB152 vector in the recombinant bacteria is intuitively displayed by colors, so as to distinguish generated mutant strains. By adopting the method, the workload is obviously reduced, and the efficiency of selecting the gene deletion strains is improved; furthermore, the result is intuitive, the gene deletion strains can be selected just by identifying blue-white recombinant bacteria, and the suicide vector pGMB152 is free of residue of an antibiotic gene.
Description
Technical field
The present invention relates to and build and use pGMB152 suicide vector to build the method for white dysentery salmonella S06004 Δ hsdM gene-deleted strain with the blue hickie sieve method of recombinant bacterium.
Background technology
At present, what salmonella structure gene-deleted strain was conventional has two kinds of methods, and a kind of is the homologous recombination method of efficient suicide vector system, and another kind is Red homologous recombination method.Suicide vector system homologous recombination method gene knockout, it is the chromogene modification technique growing up the eighties, its ultimate principle is the restructuring suicide vector building containing 2 homologous dna fragments, homologous recombination occurs in salmonella and carry out the exchange of allelotrope sequence and cause goal gene loss.Because copying of suicide vector needs specific π protein, and there is not the gene of expressing π albumen in the salmonella of studying, π albumen can not be provided, thereby restructuring suicide vector is after the exchange of secondary homologous recombination generation allelotrope, will from salmonella, automatically lose, complete whole homologous recombination process.But original method, screening process is not only loaded down with trivial details, and there are many uncertainties, also lack intuitive, most importantly in gene-deleted strain screening final stage, constructed gene-deleted strain and its parent plant on apparent without any difference, impalpable, when selecting gene-deleted strain and identifying, blindness is larger.For further optimizing suicide vector system, we transform on former methodical basis, use for reference the blue hickie sieve method of gene clone, have created the efficient and residual screening method of antibiotic-free gene directly perceived.
Summary of the invention
The object of the invention is further to optimize efficient suicide vector system, to suicide vector, pGMB151 transforms, and improves the efficiency of screening-gene gene-deleted strain, significantly reduces the workload in screening-gene gene-deleted strain process.Between the Sma of suicide vector pGMB151 I and Sal I restriction enzyme site, clone enters LacZYA gene (GenBank:J01636.1), forms pGMB151-LacZYA suicide vector, i.e. suicide vector pGMB152.This suicide vector pGMB152 is because of the sweet enzyme of LacZYA expression beta galactose, containing its substrate 5-Bromo-4-chloro-3-indolyl-β-D-Galactopyranoside(X-gal) in substratum, make the recombinant bacterium that contains this carrier show blueness, distinguish by the blue hickie of recombinant bacterium that intuitively whether suicide vector exists in salmonella.Lose restructuring suicide vector because of gene-deleted strain and be white in color, from different containing restructuring suicide vector generation monoallelic homologous recombination bacterium (blueness) color, while selecting gene-deleted strain evaluation, just do not have blindness, thereby improve gene-deleted strain screening efficiency.The method of screening-gene gene-deleted strain is also applicable to other bacterium.
Suicide vector pGMB152 for the screening of blue hickie of the present invention, is cloned into suicide vector pGMB151 by reporter gene LacZYA and derivative.Specifically between the Sma of suicide vector pGMB151 I and Sal I restriction enzyme site, be cloned into LacZYA gene.
The invention also discloses the application of suicide vector pGMB152 in screening-gene gene-deleted strain.
Described application, concrete grammar is: by chloramphenicol resistance gene Cm
rthe fragment that both sides are connected with the upstream and downstream homology arm of goal gene A is cloned on the restricted interior restriction enzyme site of pGMB152 carrier S al I, removes Cm
rafter gene, called after pGMB152-Δ A; This plasmid pGMB152-Δ A is proceeded in Host Strains, carry out solid-phase hybridization with Host Strains, coat microbiotic Amp, the Sm that contains carrier resistance and the LB flat board that adds X-gal, select blue colonies, with going down to posterity without the substratum of NaCl containing 10% sucrose, finally, coat the flat board containing X-gal antibiotic-free, select white colony and obtain goal gene gene-deleted strain.
To build white dysentery salmonella Δ hsdM gene-deleted strain as example: between Sma I and Sal I restriction enzyme digestion sites, LacZYA gene clone is entered to pGMB151 plasmid, then by chloramphenicol resistance gene (Cm
r) the both sides fragment that is connected with the upstream and downstream homology arm of hsdM gene is cloned on the restricted interior restriction enzyme site of pGMB152 carrier S al I, removes Cm
rafter gene, called after pGMB152-Δ hsdM.This plasmid is proceeded in E.coli χ 7213 bacterium (donor bacterium), with white dysentery salmonella S06004(recipient bacterium) carry out solid-phase hybridization, coat the microbiotic (Amp, Sm) that contains carrier resistance and the LB flat board that adds X-gal, select blue colonies, with going down to posterity without the substratum of NaCl containing 10% sucrose, last, coat the flat board containing X-gal antibiotic-free, select white colony, after PCR identifies, called after gene-deleted strain S06004 Δ hsdM.The inventive method is on the basis of original screening method, the sweet enzyme of beta galactose by LacZYA reporter gene coding on pGMB152 suicide vector reacts the characteristic that generates blue material with its substrate 5-Bromo-4-chloro-3-indolyl-β-D-Galactopyranoside (X-gal), show intuitively existing of pGMB152 carrier in recombinant bacterium with color, distinguish to give the mutant strain being produced.Containing in the salmonella of pGMB152 suicide vector, on pGMB152 suicide vector, equipotential gene fragment and bacterial genomes are after monoallelic homologous recombination (Single crossover), the sweet enzyme of beta galactose of LacZYA reporter gene coding on suicide vector, substrate X-gal in catalysis substratum, thus make recombinant bacterium present blueness.After coercing of sucrose continues to go down to posterity under pressure, part bacterium colony is transmitted white, this shows that their entrained reporter gene LacZYA lose, that is shows that suicide vector loses, and illustrate that diallele homologous recombination (double crossover) has occurred and is convenient to definite gene-deleted strain.This method and original method comparison, reduced workload significantly, improved the efficiency of screening-gene gene-deleted strain; And result is more directly perceived, as long as just can screen gene-deleted strain by identifying blue hickie recombinant bacterium, and antibiotic-free gene is residual.
Accompanying drawing explanation
Fig. 1 .pMD-Δ hsdM cuts checking and reclaims checking electrophorogram through Xho I enzyme;
M：DNA?Marker?DL2000
Lane1,2:pMD-Δ hsdM reclaim product after Xho I enzyme is cut.
Fig. 2 .pMD-Cm
rafter cutting, reclaims Xho I enzyme checking electrophorogram;
M：λ14DNA?Marker
Lane1,2:pMD-Cm
rafter cutting, Xho I enzyme reclaims product C m
r.
Fig. 3 .pMD-Δ hsdM/Cm
rcut checking electrophorogram through Sal I enzyme;
M：λ14DNA?Marker
Lane1,2:pMD-Δ hsdM/Cm
rcut product through Sal I enzyme.
Fig. 4 .pMD-Δ hsdM/Cm
rcut checking electrophorogram through Xho I enzyme; M: λ 14DNA Marker
Lane1,2,3,4:pMD-Δ hsdM/Cm
rcut product through Xho I enzyme.
Fig. 5 .pGMB152-Δ hsdM plasmid is cut checking electrophorogram through Sal I and Xho I enzyme;
M：λ14DNA?Marker
Lane1:pGMB152-Δ hsdM plasmid
Lane2:pGMB152-Δ hsdM plasmid is cut checking through Sal I enzyme
Lane3:pGMB152-Δ hsdM plasmid is cut checking through Xho I enzyme.
Fig. 6. PCR checking electrophorogram after diallele homologous recombination;
M：DNA?Marker?DL2000
Lane1: positive control (S06004PCR)
Lane2:S06004 Δ hsdM PCR verifies (white colony of diallele homologous recombination)
Lane3:S06004 Δ hsdM PCR verifies (white colony of diallele homologous recombination)
Lane4: monoallelic homologous recombination bacterium (locus coeruleus PCR).
Fig. 7 .E.coli χ 7213(pGMB152-Δ hsdM) and S06004 monoallelic homologous recombination result figure;
Fig. 8 .E.coli χ 7213(pGMB152-Δ hsdM) and S06004 diallele homologous recombination (after passing for 5 generations) result.Note: white colony is the mutant strain after homology weight group.
The gene knockout process of Fig. 9 based on pGMB152 suicide vector.
The gene knockout process of Figure 10 based on pGMD151 suicide vector.
Said S06004 in the present invention, refers to white dysentery Salmonellas S06004 strain, also claims wild strain S06004.Within 2006, be located away from Xuzhou, genome sequence (Accession:CP006575.1, GI:529190224)
The white dysentery salmonella gene gene-deleted strain S06004 Δ hsdM that the present invention obtains.
Embodiment
PFUSE plasmid:
aJ, Tsolis RM, van der Velden AWM, Stojiljkovic I, Anic S, Heffron is of a new iron regulated locus of Salmonella typhi.Gene F.1996.Identification
, 183, (1 – 2): 207 – 213.PGMB151 plasmid: Khan AQ, Zhao L, Hirose K, Miyake M, Li TM Hashimoto Y, Kawamura Y, Ezaki is typhi rpoS mutant is less cytotoxic than the parent strain but survives inside resting THP-1macrophages.FEMS Microbiology Letters T.1998.Salmonella, 161,201-208.
The structure of 1.pGMB152-Δ hsdM
1.1 LacZYA gene clone is arrived to pGMB151 plasmid
Extract in a small amount pFUSE plasmid and pGMB151 plasmid, respectively these two plasmids are carried out to single endonuclease digestion with Sal I and Sma I restriction enzyme, enzyme is cut system (20 μ L): plasmid (pFuse/pGMB151) 7 μ L, BamH I1 μ L, 10 × M Buffer2 μ L, the ddH of sterilizing
2o10 μ L, 37 ℃ of water-bath enzymes are cut 2h.Plasmid pFUSE single endonuclease digestion is two fragments (5.0kb and 4.4kb), and plasmid pGMB151 single endonuclease digestion is a fragment (7.8kb).Enzyme is cut product through 1% agarose gel electrophoresis, and test kit reclaims the 5.0kb fragment of purifying pFUSE and the 7.8kb fragment of pGMB151, by the connection of spending the night of both 16 ℃ of metal baths.Linked system (10 μ L): T4DNA ligase enzyme 1 μ L, 10 × connection Buffer1 μ L, the 5.0kb fragment 2 μ L of pFUSE, the 7.8kb fragment 6 μ L of pGMB151.Connect product Transformed E .coli SM10(and be purchased from U.S. strain library ATCC), coating LB flat board, picking list bacterium colony is cultivated, and extraction plasmid carries out enzyme and cuts evaluation.Identify correct plasmid pGMB151-LacZYA, called after pGMB152.
The amplification of 1.2hsdM gene upstream and downstream homology arm
1.2.1 design of primers is with synthetic
1.2.2 the genomic extraction of white dysentery salmonella S06004
By white dysentery salmonella S06004 at LB(Nal) solid plate recovery, 37 ℃ of condition incubated overnight.Next day, picking list bacterium colony, was inoculated in LB(Nal) liquid, 200r/min, 37 ℃ of jolting overnight incubation, extract white dysentery salmonella S06004 genome.The extraction of bacterial genomes DNA is specifically carried out with reference to Genomic DNA Extraction Kit specification sheets.
1.2.3 the amplification of upstream and downstream homology arm hsdM12, hsdM34 fragment
Take white dysentery salmonella S06004 genomic dna as template, PCR method amplification hsdM upstream and downstream homology arm hsdM12, hsdM34 fragment.The pcr amplification system (50 μ L) of hsdM12, hsdM34 is as follows:
PCR reaction conditions is as follows: 94 ℃ of 5min; 94 ℃ of 45sec, 58 ℃ of 45sec, 72 ℃ of 60sec, 30cycles; 72 ℃ of 10min.
Reaction finishes rear 1% the agarose gel electrophoresis detection that utilizes, and test kit reclaims purified pcr product, and reclaimer operation is undertaken by Fragment purification Kit specification sheets.Reclaim product and be connected linked system (10 μ L) with pMD-20T carrier (buying the precious biological company limited in Dalian): reclaim product 4.5 μ L, pMD20-T carrier 0.5 μ L, SolutionI5.0 μ L, 16 ℃ of metal baths connections are spent the night.Connection product is transformed into competence E.coli DH5 α, 37 ℃ of cultivations on the solid LB of Amp, IPTG and X-gal substratum, through blue hickie screening, the single white colony of picking is cultivated, and extracts plasmid PCR and identifies, and PCR method is the same.Positive colony bacterium is sent the precious biological company limited in Dalian gene sequencing, and the plasmid that checks order correct is called after pMD-hsdM12, pMD-hsdM34 respectively.
1.2.4 the splicing of homologous recombination fragment and evaluation
1.2.4.1hsdM12 and the splicing of hsdM34 fragment
Extract pMD-hsdM12, pMD-hsdM34 plasmid in a small amount, carry out double digestion respectively with XhoI and Hind III restriction enzyme, enzyme is cut system (20 μ L): plasmid (pMD-hsdM12/pMD-hsdM34) 8 μ L, Hind III1 μ L, XhoI1 μ L, 10 × M Buffer2 μ L, the ddH of sterilizing
2o8 μ L, 37 ℃ of water-bath enzymes are cut 2h.Plasmid pMD-hsdM12 double digestion is a fragment (about 3.7kb), and plasmid pMD-hsdM34 double digestion is two fragments (about 1.0Kb and 2.7kb).Enzyme is cut product through 1% agarose gel electrophoresis, and test kit reclaims the 3.7kb fragment of purifying pMD-hsdM12 and the 1.0Kb fragment of pMD-hsdM34, and both 16 ℃ of metal baths are connected and spent the night.Linked system (10 μ L): T4DNA ligase enzyme 1 μ L, 10 × connection Buffer1 μ L, the 3.7kb fragment 2 μ L of pMD-hsdM12, the 1.0Kb fragment 6 μ L of pMD-hsdM34.Connect product Transformed E .coli DH5 α, coating is containing Amp(100 μ g/mL) LB solid plate, picking list bacterium colony is cultivated, and extraction plasmid carries out PCR, enzyme is cut evaluation.Identify correct plasmid called after pMD-Δ hsdM.
1.3Cm resistant gene (Cm
r) with the splicing of pMD-Δ hsdM
Take pKD3 plasmid (be purchased from U.S.'s bacterial classification and preserve center) as template pcr amplification Cm
rgene, is cloned in pMD-T (being purchased from the precious biological company limited in Dalian) vector construction pMD-Cm
rplasmid, by recombinant plasmid pMD-Δ hsdM and the pMD-Cm building
rplasmid carries out respectively Xho I restriction enzyme single endonuclease digestion, agarose gel electrophoresis, approximately 4.9kb fragment and the pMD-Cm of recovery purifying pMD-Δ hsdM
rapproximately 1.1kb fragment, T4DNA ligase enzyme connects, and connects product Transformed E .coli DH5 α, containing Amp(100 μ g/mL) and Cm(100 μ g/mL) solid LB flat board on cultivate, the cultivation of picking list bacterium colony, PCR, enzyme are cut evaluation.Identify correct recombinant plasmid called after pMD-Δ hsdM/Cm
r.
1.4 suicide vector pGMB152 and pMD-Δ hsdM/Cm
rsplicing and evaluation
By suicide vector pGMB152, through Sal I restriction enzyme single endonuclease digestion, enzyme is cut product after 1% agarose gel electrophoresis, reclaims the about 12.8kb carrier segments of purifying pGMB152; Plasmid pMD-Δ hsdM/Cm
rbecome two fragments (3.3kb and 2.7kb) through Sal I digestion with restriction enzyme, after 1% agarose gel electrophoresis, reclaim the about 3.3kb fragment of purifying, reclaim fragment with about pGMB15212.8kb and be connected through T4DNA ligase enzyme.Connect product and be transformed into E.coli Spy372 competent cell, containing Amp(100 μ g/mL), Sm(100 μ g/mL), Cm(100 μ g/mL) solid LB flat board on cultivate.Picking list bacterium colony is cultivated, and extraction plasmid PCR, enzyme are cut evaluation, identify correct recombinant plasmid called after pGMB152-Δ hsdM/Cm
r.
1.5 remove restructuring suicide vector pGMB152-Δ hsdM/Cm
rcm
rgene
By pGMB152-Δ hsdM/Cm
rthrough Xho I restriction enzyme single endonuclease digestion, enzyme is cut into two fragments (15kb and 1.1kb), and enzyme is cut product after 1% agarose gel electrophoresis, reclaims the about 15kb carrier segments of purifying pGMB152, certainly connects through T4DNA ligase enzyme.Connect product and be transformed into E.coli Spy372 competent cell, containing Amp(100 μ g/mL), Sm(100 μ g/mL) solid LB flat board on cultivate.Picking list bacterium colony is cultivated, and extraction plasmid PCR, enzyme are cut evaluation, identify correct recombinant plasmid called after pGMB152-Δ hsdM.
2. screening and the evaluation of white dysentery salmonella S06004 Δ hsdM mutant strain
Recombinant plasmid pGMB152-Δ hsdM is transformed into E.coli χ 7213, at Amp(100 μ g/mL), Sm(100 μ g/mL), DAP(50mg/mL) solid LB flat board on cultivate, positive bacteria called after E.coli χ 7213(pGMB152-Δ hsdM).Take E.coli χ 7213(pGMB152-Δ hsdM) as donor bacterium, white dysentery salmonella S06004 is recipient bacterium, carries out conjugal transfer.Monoallelic homologous recombination: picking recipient bacterium S06004 is inoculated in the g/mL containing Nal(30 μ respectively) LB substratum, donor bacterium E.coli χ 7213(pGMB152-Δ hsdM) be inoculated in the g/mL containing Amp(100 μ), Sm(100 μ g/mL), DAP(50mg/mL) LB substratum, 37 ℃, after 200r/min jolting incubated overnight, bacterium liquid washs twice rear suspension with aseptic PBS, in certain proportion recipient bacterium is mixed with donor bacteria suspension, the aseptic filter membrane of 0.22 μ m is affixed on the LB solid plate containing DAP, the suspension of mixing is dripped on filter membrane, dull and stereotyped in 37 ℃ of overnight incubation (about 24h).The lawn of growing on filter membrane is washed down with aseptic PBS, is coated the L containing X-gal(40 μ), Amp(100 μ g/mL), Sm(100 μ g/mL) LB flat board, 37 ℃ of overnight incubation, the blue colonies of growing on LB culture plate (Fig. 7).
Diallele homologous recombination: by monoallelic homologous recombination gained locus coeruleus bacterium by containing repeatedly the going down to posterity (5-7 generation) without NaCl liquid LB substratum of 10% sucrose, aseptic 10mM MgSO for bacterium liquid
4after solution is washed twice, suspend again, coat containing 10% sucrose, X-gal(40 μ L) the flat board without NaCl, picking white colony (Fig. 8).Apply the above white colony of multipair primer pair and carry out PCR checking, verify correct recombinant bacterium called after S06004 Δ hsdM.(pGMB152 screening process is as Fig. 9, and efficiency is higher than the screening process of plasmid pGMB151, Figure 10).
Claims (5)
1. for a suicide vector pGMB152 for blue hickie screening, reporter gene LacZYA is cloned into suicide vector pGMB151 and derivative.
2. suicide vector pGMB152 according to claim 1, is characterized in that, is to be cloned into LacZYA gene between the Sma of suicide vector pGMB151 I and Sal I restriction enzyme site.
3. the application of suicide vector pGMB152 claimed in claim 1 in screening-gene gene-deleted strain.
4. application according to claim 3, is characterized in that concrete grammar is: by chloramphenicol resistance gene Cm
rthe fragment that both sides are connected with the upstream and downstream homology arm of goal gene A is cloned on the restricted interior restriction enzyme site of pGMB152 carrier S al I, removes Cm
rafter gene, called after pGMB152-Δ A; This plasmid pGMB152-Δ A is proceeded in Host Strains, carry out solid-phase hybridization with Host Strains, coat microbiotic Amp, the Sm that contains carrier resistance and the LB flat board that adds X-gal, select blue colonies, with going down to posterity without the substratum of NaCl containing 10% sucrose, finally, coat the flat board containing X-gal antibiotic-free, select white colony and obtain goal gene gene-deleted strain.
5. the method that builds white dysentery salmonella S06004 Δ hsdM gene-deleted strain with pGMB152 suicide vector described in claim 1, is characterized in that, by chloramphenicol resistance gene Cm
rthe fragment that both sides are connected with the upstream and downstream homology arm of hsdM gene is cloned on the restricted interior restriction enzyme site of pGMB152 carrier S al I, removes Cm
rafter gene, called after pGMB152-Δ hsdM; This plasmid pGMB152-Δ hsdM is proceeded in E.coli χ 7213 Host Strains, carry out solid-phase hybridization with white dysentery salmonella, coat microbiotic Amp, the Sm that contains carrier resistance and the LB flat board that adds X-gal, select blue colonies, with going down to posterity without the substratum of NaCl containing 10% sucrose, finally, coat the flat board containing X-gal antibiotic-free, select white colony and obtain gene-deleted strain S06004 Δ hsdM.
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| CN104357472A (en) * | 2014-11-18 | 2015-02-18 | 苏州大学 | Multifunctional TA cloning vector system for blue-white selection and application of multifunctional TA cloning vector system |
| CN104894154A (en) * | 2015-06-18 | 2015-09-09 | 福建师范大学 | Temperature-sensitive vector pBBR1MCS-2-Ts2 and application thereof |
| CN106967744A (en) * | 2017-04-28 | 2017-07-21 | 扬州大学 | A kind of method that utilization suicide vector eliminates multicopy plasmid in detection of Salmonella |
| CN111057671A (en) * | 2019-12-04 | 2020-04-24 | 扬州大学 | Salmonella pullorum single-gene traceless knockout attenuated strain and preparation method and application thereof |
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| CN103436478A (en) * | 2013-09-06 | 2013-12-11 | 扬州大学 | Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357472A (en) * | 2014-11-18 | 2015-02-18 | 苏州大学 | Multifunctional TA cloning vector system for blue-white selection and application of multifunctional TA cloning vector system |
| CN104894154A (en) * | 2015-06-18 | 2015-09-09 | 福建师范大学 | Temperature-sensitive vector pBBR1MCS-2-Ts2 and application thereof |
| CN104894154B (en) * | 2015-06-18 | 2018-03-02 | 福建师范大学 | The Ts2 of responsive to temperature type carrier pBBR1MCS 2 and its application |
| CN106967744A (en) * | 2017-04-28 | 2017-07-21 | 扬州大学 | A kind of method that utilization suicide vector eliminates multicopy plasmid in detection of Salmonella |
| CN111057671A (en) * | 2019-12-04 | 2020-04-24 | 扬州大学 | Salmonella pullorum single-gene traceless knockout attenuated strain and preparation method and application thereof |
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