CN105063073A - Universal gene-knockout suicide vector for vibrios and application thereof - Google Patents

Universal gene-knockout suicide vector for vibrios and application thereof Download PDF

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CN105063073A
CN105063073A CN201510593592.7A CN201510593592A CN105063073A CN 105063073 A CN105063073 A CN 105063073A CN 201510593592 A CN201510593592 A CN 201510593592A CN 105063073 A CN105063073 A CN 105063073A
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plp12t
suicide vector
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plp12
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罗鹏
何香燕
刘秋婷
胡超群
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses a universal gene-knockout suicide vector for vibrios and a construction method theroef and provides an application thereof in gene knockout of the vibrios. The universal gene-knockout suicide vector pLP12 is a ring-shaped vector and comprises a PBAD promoter, a repressor protein gene araC, an RP4 transferring initiation site, a chlorampenicol resistant gene, an R6K duplicating initiation site, a multiple-cloning-site area and a lethal gene vmi480; the multiple-cloning-site area at least contains two AhdI restriction enzyme digestion sites; the suicide vector pLP12 is subject to AhdI restriction enzyme digestion to form linearized suicide vector pLP12T. The universal gene-knockout suicide vector adopts entirely-new reverse selection genes vmi480 and is used for replacing the common sacB gene. Foreign fragments carried by the pLP12T are transferred to vibrio cells to be mutated by a jointing mode, under the pressure of antibiotics and reverse selection of products of lethal gene vmi480, first-time homologous recombination and second-time homologous recombination are carried out on the vibrios successively, and finally the mutant strain with deletion of target genes is generated.

Description

The gene knockout suicide vector that a kind of vibrios is general and application thereof
Technical field:
The invention belongs to microorganism hereditary field operation, be specifically related to the general gene knockout suicide vector of a kind of vibrios and application thereof.
Background technology:
Vibrios is heterotrophic organism the most common in ocean environment, has at least 12 kinds of vibrios to cause human disease, and wherein vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus are the most serious to human health risk.In addition the vibrios of many kinds is also aquatic products economic animal cause of disease, and its vibriosis caused causes a large amount of financial loss of aquaculture.Although had been found that vibrio cholerae and some key pathogenetic gene of Vibrio parahaemolyticus very early, scientist has still seriously lagged behind the pathogenic bacterium of enterobacteriaceae to the understanding of vibrios pathogenesis.Lack the major obstacle that maturation, easily Genetic Manipulative Technology are the research of vibrios molecular basis of the pathogenesis.
Gene Knockout based on bacterium recBCD homologous recombination system is the technology that can realize in various bacteria at present.In this technology, the in-frame deletion of goal gene is merged fragment and is first obtained by overlapping pcr amplification, PCR primer and suicide vector connect after adopting identical enzyme enzyme to cut purifying again, and transformation of E. coli, obtain the suicide plasmid (carrier) with exogenous sequences, this suicide plasmid engages via donor bacterium and is input in acceptor target bacteria.This plasmid can not copy due to the restriction of self in target bacteria, under favorable selection pressure (resistant gene normally on plasmid), the target bacteria only having genome to incorporate suicide plasmid by homologous recombination could be survived, and forms insertion mutation; Under Negative selection pressure, there is second time homologous recombination in insertion mutation cell, and the suicide plasmid integrating state departs from karyomit(e), forms a wild-type cell and a deletion mutant cell in theory, thus realizes knocking out of target gene.As can be seen here, suicide vector is the core of the gene Knockout based on bacterium recBCD homologous recombination system.
Also very rare based on the application of gene Knockout in vibrios of bacterium recBCD homologous recombination system at present, only there are some accidentally successfully reports.There are some obvious defects in the gene Knockout that can be used successfully to the homologous recombination of the bacteriums such as intestinal bacteria, comprising in vibrios gene knockout: (1) lacks versatility, is only limitted to the genetically deficient in some vibrios; (2) sacB is widely used as the lethal gene of suicide vector, but the lethal effect that sacB causes is very weak in vibrios, the ratio causing deletion mutantion clone correct in Negative selection flat board to occur is very low, causes a large amount of deletion mutantion screening operations; (3) need to carry out double digestion to the overlapping PCR primer of object fragment and suicide plasmid, waste time and energy; (4) suicide plasmid itself is comparatively large, and with mobI jumping gene, not only causes the efficiency of plasmid conjugal transfer low, and likely cause Non-specific integration.(5) recipient bacterium must have the resistance marker different from donor bacterium and suicide vector, limits the application of routine techniques.Therefore, when for vibrios gene knockout, existing suicide vector shows obvious limitation, in the urgent need to developing new universal suicide vector to overcome above-mentioned defect, realizes the widespread use of gene Knockout in vibrios based on bacterium recBCD homologous recombination system.
Summary of the invention:
The object of the invention is for the deficiencies in the prior art, the gene knockout suicide vector pLP12T providing a kind of vibrios general.
Gene knockout suicide vector pLP12 of the present invention, is circular vectors, it is characterized in that, comprise P bADpromotor, repressor protein gene araC, RP4 shift initiation site (oriT rP4), chloramphenicol resistance gene (cat), R6K replication origin (oriV r6K γ), multiple clone site district (MCS) and lethal gene vmi480, described multiple clone site district (MCS) at least comprises two AhdI restriction enzyme sites, the nucleotide sequence of described lethal gene vmi480 is as shown in the 21-617 bit base sequence of SEQIDNO.1, and described lethal gene vmi480 is positioned at P bADpromotor downstream is also in P bADunder promotor controls.
Preferably, described gene knockout suicide vector pLP12, its nucleotide sequence as shown in SEQIDNO.1, the base containing 3871bp.
Present invention also offers a kind of gene knockout suicide vector pLP12T, for linear carrier, it is characterized in that, be the linear carrier obtained after said gene being knocked out two AhdI restriction enzyme sites in suicide vector pLP12 AhdI Mei Qie multiple clone site district, be gene knockout suicide vector pLP12T.
Preferably, described gene knockout suicide vector pLP12T, its nucleotide sequence as shown in SEQIDNO.2, the base containing 3848bp.
Described P bADpromoter systems (comprises P bADpromotor and repressor protein gene araC) belong to tight mouth controlled expression promoter systems, glucose can close the expression of downstream lethal gene vmi480 completely, and under the induction of pectinose, vmi480 expresses and is activated.Different according to pectinose induced concentration, the expression amount of vmi480 can be modulated.OriT rP4be one section and be rich in AT sequence, provide the effect of metastasis donor bacterium under, suicide plasmid from then on site starts to form strand transfer.Chloramphenicol resistance gene provides the resistance marker of first time integrated plasmid screening.OriV r6K γfor plasmid carries out the initiation site of self-replacation.At P bADunder the control of promoter systems, vmi480 can express or suppress to express.When oppositely screening, under pectinose induction, vmi480 activates and expresses Vmi480, and it has strong toxicity.Only have the cell that second time homologous recombination loss plasmid occurs to survive, the result of second time restructuring is exactly a generation wild-type cell and a Gene Deletion cell after archeocyte division, thus realizes gene knockout.Multiple clone site district (MCS) convenient to plasmid carry out enzyme cut produce can with the same suicide vector carrying out PCR primer that enzyme cuts and connect.MCS comprises two AhdI restriction enzyme sites, can produce linear suicide type carrier T after employing AhdI enzyme is cut.In this case, do not need that enzyme is carried out to PCR primer and cut, after purifying, just directly connect this carrier.
Present invention also offers the application of gene knockout suicide vector pLP12T in gene knockout.
Preferably, the described application of gene knockout suicide vector pLP12T in vibrios gene knockout.
Further preferably, the in-frame deletion of goal gene is merged fragment be connected with gene knockout suicide vector pLP12T, the in-frame deletion obtaining carrying goal gene merges the pLP12T of fragment, called after pLP12T-X, pLP12T-X enters in donor bacterium through transforming, then this donor bacterium is mixed with recipient bacterium, through conjugation, pLP12T-X transfer is entered recipient bacterium, select through paraxin, the cell only having pLP12T-X to be incorporated into recipient bacterium genome target site could be survived, obtain the goal gene insertion mutation type bacterial strain that pLP12T-X is incorporated into recipient bacterium genome target site, then this goal gene insertion mutation type bacterial strain is under pectinose induction vmi480 gene produces the Negative selection pressure of toxin Vmi480, the cell only having generation second time homologous recombination to lost suicide vector part could be survived, after second time restructuring, former goal gene insertion mutation type bacterial strain can produce a genetically deficient cell and a normal wild-type cell, therefore the clone of Negative selection grow on plates is mainly goal gene deletion clone and normal wild-type cell clone, through screening, as detected the clone that can filter out goal gene disappearance by PCR, thus finally reach goal gene is knocked out object, obtain the bacterial strain of goal gene disappearance.
Described recipient bacterium is vibrios.
Described donor bacterium is defective escherichia coli β 2163.Adopt defective escherichia coli β 2163 as donor bacterium, do not need to consider whether recipient bacterium has antibiotics resistance, therefore greatly facilitate the application based on the gene knockout method engaged.
Compared with existing suicide vector and using method, the present invention has following beneficial effect:
(1) have employed brand-new Negative selection gene vmi480 in gene knockout suicide vector of the present invention, for substituting conventional sacB gene.Vmi480 have strong, the lethal object of lethal effect extensively, the performance of lethal effect is by the advantage that NaCl affects.This lays a good foundation for gene knockout suicide vector of the present invention is widely used in halophilic vibrio gene knockout.
(2) multiple clone site of gene knockout suicide vector of the present invention introduces two AhdI sites, and enzyme can form carrier T after cutting, after once forming carrier, and Reusability.PCR primer is cut without enzyme, also need not consider restriction enzyme site, greatly save the time.
(3) gene knockout suicide vector of the present invention obviates bulk redundancy part in general suicide plasmid, and suicide vector diminishes, and is conducive to improving the frequency of conjugal transfer and the correct probability integrated.
(4) based on P bADthe high efficiency of promotor and lethal gene, on Negative selection flat board, the ratio of deletion mutantion clone improves greatly, substantially reduce the number the work of screening and cloning.
Accompanying drawing explanation
Fig. 1 is the general gene knockout suicide vector pLP12 physical map of vibrios, can form suicide vector pLP12T after employing AhdI enzyme is cut.
Fig. 2 is that the general gene knockout suicide vector pLP12T of vibrios builds schema.
Fig. 3 amounts to 6 gene knockout results detections for application vibrios universal genetic knocks out suicide vector to 4 kinds of different vibrios.M: molecular weight Marker; 1-3: the detected result corresponding respectively to the vah gene wild strain of vibrio alginolyticus E0601, insertion mutation strain, deletion mutantion strain; 4-6: the detected result corresponding respectively to the degS gene wild strain of vibrio cholerae HN375, insertion mutation strain, deletion mutantion strain; 7-9: the detected result corresponding respectively to the vasC gene wild strain of vibrio cholerae HN375, insertion mutation strain, deletion mutantion strain; 10-12: the detected result corresponding respectively to the pilO gene wild strain of Vibrio parahaemolyticus E0680, insertion mutation strain, deletion mutantion strain; 13-15: the detected result corresponding respectively to the ascS gene wild strain of Vibrio parahaemolyticus E06135, insertion mutation strain, deletion mutantion strain; 16-18: the detected result corresponding respectively to the impB gene wild strain of Vibrio vulnificus ATCC27562, insertion mutation strain, deletion mutantion strain: N: represent the negative control detected result that use water does template.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.These embodiments are only exemplary, do not form any restriction to scope of the present invention.Those skilled in the art should understand that: can modify to the details of technical solution of the present invention and form lower without departing from the spirit and scope of the present invention or replace, but these amendments and replacement all fall within the scope of protection of the present invention.
Following examples 1 are that the general gene knockout suicide vector pLP12T of vibrios builds, and embodiment 2-7 is the application of suicide vector pLP12T in representative vibrios gene knockout.Vibrio alginolyticus is distributed widely in ocean and estuarine environment, is to be separated kind the most common in vibrios, is also the conditioned pathogen of multiple aquatic animal simultaneously, and the vibriosis outburst that it causes can cause huge financial loss.Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus are to the most serious vibrios kind of human health risk.In addition, these four kinds of vibrios also have larger hereditary difference to each other.Therefore select these four kinds of vibrios as Typical Representative, for verifying the versatility of gene knockout suicide vector pLP12T in vibrios gene knockout that vibrios is general.
In the present embodiment, all pcr amplification primers are in table 1.
The primer adopted in table 1 the present invention
* dashed part is restriction enzyme site
The structure (flow process as shown in Figure 2) of the gene knockout suicide vector pLP12T that embodiment 1 vibrios is general
(1) with plasmid pSW23T, (VincentBurrus provides, source document DemarreG, etal.ResearchinMicrobiology, 2005,156:245 – 255) be template, adopt primer pSW23T-F/pSW23T-R to carry out pcr amplification, polysaccharase is TaKaRa company high-fidelity DNA polymerase PrimeSTAR (unless stated otherwise, polysaccharase used enzyme all for this reason in the present invention).Pcr amplification conditioned reference enzyme specification sheets.Obtain PCR primer, this PCR primer comprises oriV r6K γ, oriT rP4, cat, MCS site fragment.
(2) be template with pSW25T-ccdB (VincentBurrus provides, source http://openwetware.org/wiki/Mazel/PSW25T-ccdB.docx), adopt primer pSW25T-F/pSW25T-R to carry out pcr amplification.Obtain PCR primer, this PCR primer comprises P tACpromotor, ccdB and comprise an AhdI restriction enzyme site fragment.
(3) carry out EcoRI/SphI double digestion to two kinds of PCR primer of above-mentioned steps (1) and (2) respectively, digestion products adopts PCR primer purification kit (Axygen) to carry out purifying again.
(4) two purified products connect with T4DNA ligase enzyme (TaKaRa), connect product (plasmid pLP10) transformation of E. coli DH5 α λ pir cell, and are coated with LB flat board (containing 20 μ g/ml paraxin).With primer pair pLP10L-TF1/pLP10L-TR1 and pLP10L-TF2/pLP10L-TR2, PCR screening is carried out to the clone on flat board, correct positive colony PCR primer long 1065bp, 537bp respectively.PCR fragment carries out sequence verification again, jointly determines correct clone, and this contains in correctly cloning and connects product-plasmid pLP10.Enlarged culturing is correctly cloned, and extracts plasmid pLP10.The multiple clone site AhdI-EcoRI-SacI-AhdI-NheI of plasmid pLP10, is formed respectively by the upper restriction enzyme site of introducing of the 5 ' end of primer pSW23T-F and pSW25T-F.
(5) with plasmid pLP10 for template, adopt primer pLP10-F/pLP10-R to carry out Inverse PCR amplification, obtain fragment pLP10-1, to remove ccdB gene complete in pLP10.With intestinal bacteria LP79, (construction process is with reference to contriver's article, CarraroN, MatteauD, LuoP, etal.PLOSGenetics, 2014,10 (10): e1004714) genome is template, adopt vmi480-F/vmi480-R to carry out pcr amplification, obtain complete vmi480 gene fragment, its nucleotide sequence is as shown in the 21-617 bit base sequence of SEQIDNO.1.Intestinal bacteria LP79 obtains the genomic islands MGIVmi1 from Vibrio mimicus VM573 by conjugal transfer in advance, and this genomic islands contains vmi480 lethal gene.
(6) PCR fragment pLP10-1 and vmi480 gene fragment carry out NdeI/XhoI double digestion and after purifying respectively, connect with T4DNA ligase enzyme, connect product (plasmid pLP11) transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on pLP11L-TF/pLP11L-TR primer pair flat board, the long 515bp of the positive PCR primer of correct clone, PCR fragment carries out sequence verification again, jointly determines correct clone, and this contains in correctly cloning and connects product-plasmid pLP11.Enlarged culturing is correctly cloned, and extracts plasmid pLP11.
(7) with plasmid pLP11 for template, adopt primer pLP11-F/pLP11-R to carry out Inverse PCR amplification, obtain fragment pLP11-1, to remove the complete P of pLP11 tACpromoter systems.Be template with pBAD30 plasmid (general as biological in spit of fland, NTCC1809), adopt primer pBAD30-PF/pBAD30-PR to be primer amplification, obtain P in pBAD30 plasmid bADpromotor part, the two ends of called after amplified fragments pBAD30-1, amplified fragments pBAD30-1 are respectively containing one section of sequence identical with pLP11-1 two ends.Seamless Cloning Kit (Vazyme) is adopted to carry out vitro recombination to pLP11-1 and pBAD30-1, recombinant products (plasmid pLP12) transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on pLP12L-TF/pLP12L-TR primer pair flat board, the long 1104bp of correct positive colony PCR primer, PCR fragment carries out sequence verification again, jointly determines correct clone, and this contains recombinant products-plasmid pLP12 in correctly cloning.The clone that enlarged culturing is correct, and extract plasmid pLP12, be the gene knockout suicide vector pLP12 that vibrios is general, its nucleotide sequence is as shown in SEQIDNO.1.Concrete structure as shown in Figure 1.
(8) with the general gene knockout suicide vector pLP12 of vibrios for template, then carry out pcr amplification, amplimer STVU-F/STVU-R.Amplified production carries out AhdI enzyme and cuts after PCR primer Purification Kit, enzyme is cut rear employing DNA concentrated reagent box and is carried out purifying (middle ancient cooking vessel), obtains the gene knockout suicide vector pLP12T (its nucleotide sequence is as shown in SEQIDNO.2) that linearizing vibrios is general.3 ' end of pLP12T double-strand respectively has an outstanding base T, and therefore can increase with common archaeal dna polymerase produces PCR primer and be directly connected.The two must not carry out enzyme again and cut.
Embodiment 2 is applied pLP12T and is knocked out vibrio alginolyticus hemolysin gene (vah)
(1) with overlapping PCR mode increase obtain vibrio alginolyticus E0601 vah gene in-frame deletion merge fragment.First round PCR primer pair is respectively vah-MF1/vah-MR1, vah-MF2/vah-MR2, and template is vibrio alginolyticus E0601 genomic dna.The first round produces PCR primer vah1, vah2 respectively.Vah1, vah2 are after PCR primer purification kit (Axygen) purifying, and the template of PCR is taken turns in equal-volume mixing as second.Second takes turns PCR adopts primer vah-MF1/vah-MR2, and adopt regular Taq DNA polymerase (TaKaRa), pcr amplification condition pcr amplification conditioned reference enzyme specification sheets, obtains second thus and take turns PCR primer.
(2) second take turns PCR primer purified after, directly the gene knockout suicide vector pLP12T general with above-mentioned vibrios is connected, employing T4DNA ligase enzyme.Connect product (plasmid pLP12-vah) directly transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on vah-MF1/vah-MR2 primer pair flat board, correct clone can produce long 660bp amplified band.PCR fragment carries out sequence verification again, jointly determines correct clone, and this correct clone contains connection product-plasmid pLP12-vah.Enlarged culturing is correctly cloned, and extracts plasmid pLP12-vah.
(3) pLP12-vah electricity transformation of E. coli β 2163 cell (bacterial strain of the prior art, construction process DemarreG, etal.Anewfamilyofmobilizablesuicideplasmidsbasedonbroadh ostrangeR388plasmid (IncW) andRP4plasmid (IncPalpha) conjugativemachineriesandtheircognateEscherichiacolihost strains.ResearchinMicrobiology, 2005,156:245-255).Electricity conversion condition: voltage 1800V, electric shock time 5ms.After electric shock, cell is in 37 DEG C of renewal cultivation 1hr, coating LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Incubated overnight, picking is cloned arbitrarily, and purifying is also preserved, and namely obtains the donor bacterium-intestinal bacteria β 2163 (pLP12-vah) comprising suicide plasmid pLP12-vah.
(4) incubated overnight intestinal bacteria β 2163 (pLP12-vah) and recipient bacterium vibrio alginolyticus E0601.Get 100 μ L donor bacterium to mix with 100 μ L recipient bacteriums, 8000g is centrifugal, and 2min abandons supernatant, and it is resuspended to add 400 μ L fresh LB, the careful supernatant discarded of the centrifugal 2min of 8000g again.Add 10 μ L fresh LB resuspended, re-suspension liquid is all sucked and drops on LB agar plate.After super clean bench is air-dry, is inverted for 37 DEG C and cultivates 6hr.
(5) fully blow outstanding above-mentioned culture with 1ml fresh LB, get 100 μ L and be coated with LB agar plate (containing 0.3%D-glucose, 10 μ g/ml paraxin), 37 DEG C of incubated overnight.On this flat board, only have and first time homologous recombination occurs, chromosomal integration has the insertion mutation vibrio alginolyticus cell of suicide plasmid pLP12-vah to grow.Random picked clones carries out PCR qualification, PCR primer vah-MF1/vah-MR2, and correct clone produces 1218bp and 660bp bis-bands (swimming lane 2 in Fig. 3), obtains the strain of vah insertion mutation.
(6) strain of vah insertion mutation is after LB (0.3%D-glucose) cultivates 3 hours, gradient dilution, respectively gets 100 μ L and is coated with LB agar plate (0.2%L-pectinose), 37 DEG C of incubated overnight.On this flat board, only have and second time homologous recombination occurs, the cell that chromosome elimination integrates state suicide plasmid pLP12-vah can be survived.After second time restructuring occurs bacterial cell, the cell that the cell of a vah genetically deficient and comprise wild-type vah gene can be produced.
(7) PCR detection is carried out with the clone on vah-MF1/vah-MR2 primer pair LB agar plate (0.2%L-pectinose), correct vah genetically deficient clone produces the band (swimming lane 3 in Fig. 3) of 660bp, and the Wild type clone that second time restructuring is formed produces 1218bp band (swimming lane 1 in Fig. 3).Correct vah genetically deficient clone, again through PCR primer sequence verification, namely obtains vah gene-deleted strain, thus completes vibrio alginolyticus vah gene knockout.In the present embodiment, Negative selection flat board (10 -1dilution) upper random picking 16 clones in, have 12 for correct vah genetically deficient clone.
Embodiment 3 is applied pLP12T and is knocked out vibrio cholerae pericentral siphon serine peptidases gene (degS)
(1) the degS gene in-frame deletion fusion fragment of vibrio cholerae HN375 is obtained with overlapping pcr amplification.First round PCR primer pair is respectively degs-MF1/degs-MR1, degs-MF2/degs-MR2, and template is vibrio cholerae HN375 genomic dna.Purifying after two PCR primer equal-volume mixing, takes turns the template of PCR as second.Second takes turns PCR adopts primer degs-MF1/degs-MR2, adopts regular Taq DNA polymerase, obtains second and take turns PCR primer.
(2) above-mentioned second take turns PCR primer, be directly connected with suicide vector pLP12T.Connect product (plasmid pLP12-degS) transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on degs-MF1/degs-MR2 primer pair flat board, correct clone can produce long 926bp amplified band.PCR fragment carries out sequence verification again, jointly determines correct clone, containing connecting product-plasmid pLP12-degS in this correct clone.Enlarged culturing is correctly cloned, and extracts plasmid pLP12-degS.
(3) pLP12-degS electricity transformation of E. coli β 2163 cell.Electricity conversion condition and the rear cell culture condition of electric shock are with embodiment 2.Picking is cloned arbitrarily, and purifying is also preserved, and namely obtains the donor bacterium-intestinal bacteria β 2163 (pLP12-degS) comprising suicide plasmid pLP12-degS.
(4) incubated overnight intestinal bacteria β 2163 (pLP12-degS) and recipient bacterium vibrio cholerae HN375.Get 100 μ L donor bacterium to mix with 100 μ L recipient bacteriums, 8000g is centrifugal, and 3min abandons supernatant, and it is resuspended to add 500 μ L fresh LB, the careful supernatant discarded of the centrifugal 2min of 8000g again.Add 10 μ L fresh LB resuspended, re-suspension liquid is all sucked and drops on LB agar plate.After super clean bench is air-dry, is inverted for 37 DEG C and cultivates 7hr.
(5) fully blow outstanding above-mentioned culture with 1ml fresh LB, get 100 μ L and be coated with LB agar plate (containing 10 μ g/ml paraxin, 0.3%D-glucose), 37 DEG C of incubated overnight.On this flat board, only have and first time homologous recombination occurs, chromosomal integration has the insertion mutation vibrio cholerae cell of suicide plasmid pLP12-degS just can grow.Random picked clones carries out PCR qualification, PCR primer degS-MF1/degS-MR2, and correct clone produces 1496bp and 926bp bis-bands (swimming lane 5 in Fig. 3), namely obtains the strain of degS insertion mutation.
(6) strain of degS insertion mutation is after LB (0.3%D-glucose) cultivates 3 hours, gradient dilution, respectively gets 100 μ L and is coated with LB agar plate (0.2%L-pectinose), 37 DEG C of incubated overnight.On this flat board, only have and second time homologous recombination occurs, the cell that chromosome elimination integrates state suicide plasmid pLP12-degS can be survived.After second time restructuring occurs bacterial cell, the cell that the cell of a degS genetically deficient and comprise wild-type degS gene can be produced.
(7) PCR detection is carried out with the clone on degS-MF1/degS-MR2 primer pair LB agar plate (0.2%L-pectinose), correct degS genetically deficient clone produces the band (swimming lane 6 in Fig. 3) of 926bp, and the Wild type clone that second time restructuring is formed produces 1496bp band (swimming lane 4 in Fig. 3).Correct degS genetically deficient clone, again through PCR primer sequence verification, namely obtains degS gene-deleted strain, thus completes vibrio cholerae degS gene knockout.In the present embodiment, Negative selection flat board (10 -1dilution) upper random picking 16 clones in, have 8 for correct degS genetically deficient clone.
Embodiment 4 is applied pLP12T and is knocked out vibrio cholerae VI type excretory system gene (vasC)
(1) the vasC gene in-frame deletion fusion fragment of vibrio cholerae HN375 is obtained with overlapping pcr amplification.First round PCR primer pair is respectively vasC-MF1/vasC-MR1, vasC-MF2/vasC-MR2, and template is vibrio cholerae HN375 genomic dna.After two PCR primer purifying, the template of PCR is taken turns in equal-volume mixing as second.Second takes turns PCR primer vasC-MF1/vasC-MR2, adopts regular Taq DNA polymerase, obtains second and take turns PCR primer.
(2) second take turns PCR primer purified after, be directly connected with gene knockout suicide vector pLP12T prepared by embodiment 1.Connect product (plasmid pLP12-vasC) transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on vasC-MF1/vasC-MR2 primer pair flat board, correct clone can produce long 925bp amplified band.PCR fragment carries out sequence verification again, jointly determines correct clone, and this is correctly cloned containing connecting product (plasmid pLP12-vasC).Enlarged culturing is correctly cloned, and extracts plasmid pLP12-vasC.
(3) pLP12-vasC electricity transformation of E. coli β 2163 cell.Electricity conversion condition and the rear cell culture condition of electric shock are with embodiment 2.Picking is cloned arbitrarily, and purifying is also preserved, and namely obtains the donor bacterium-intestinal bacteria β 2163 (pLP12-vasC) comprising suicide plasmid pLP12-vasC.
(4) incubated overnight intestinal bacteria β 2163 (pLP12-vasC) and recipient bacterium vibrio cholerae HN375.Get 100 μ L donor bacterium to mix with 100 μ L recipient bacteriums, 8000g is centrifugal, and 2min abandons supernatant, and it is resuspended to add 600 μ L fresh LB, the centrifugal 2min of 8000g again, careful supernatant discarded.Add 10 μ L fresh LB resuspended, re-suspension liquid is all sucked and drops on LB agar plate.After super clean bench is air-dry, is inverted for 37 DEG C and cultivates 8hr.
(5) fully blow outstanding above-mentioned culture with 1ml fresh LB, get 100 μ L and be coated with LB agar plate (containing 0.3%D-glucose, 10 μ g/ml paraxin), 37 DEG C of incubated overnight.On this flat board, only have and first time homologous recombination occurs, chromosomal integration has the insertion mutation vibrio cholerae cell of suicide plasmid pLP12-vasC to grow.Random picked clones carries out PCR qualification, PCR primer vasC-MF1/vasC-MR2, and correct clone produces 1630bp and 925bp bis-bands (swimming lane 8 in Fig. 3), obtains the strain of vasC insertion mutation.
(6) strain of vasC insertion mutation is after LB (0.3%D-glucose) cultivates 3 hours, gradient dilution, respectively gets 100 μ L and is coated with LB agar plate (0.2%L-pectinose), 37 DEG C of incubated overnight.On this flat board, only have and second time homologous recombination occurs, the cell that chromosome elimination integrates state suicide plasmid pLP12-vasC can be survived.Bacterial cell comprises the cell of wild-type vasC gene through the cell of a second time restructuring generation vasC genetically deficient and one.
(7) PCR detection is carried out with the clone on vasC-MF1/vasC-MR2 primer pair LB agar plate (0.2%L-pectinose), correct vasC genetically deficient clone produces the band (swimming lane 9 in Fig. 3) of 925bp, and the Wild type clone that second time restructuring is formed then produces 1630bp band (swimming lane 7 in Fig. 3).Correct vasC genetically deficient clone, again through PCR primer sequence verification, namely obtains vasC gene-deleted strain, thus completes vibrio cholerae vasC gene knockout.In the present embodiment, Negative selection flat board (10 -1dilution) upper random picking 16 clones in, have 7 for correct vasC genetically deficient clone.
Embodiment 5 is applied pLP12T and is knocked out Vibrio parahaemolyticus IV type flagellar production gene (pilO)
(1) the pilO gene in-frame deletion fusion fragment of Vibrio parahaemolyticus E0680 is obtained with overlapping pcr amplification.First round PCR primer pair is respectively pilO-MF1/pilO-MR1, pilO-MF2/pilO-MR2, and template is Vibrio parahaemolyticus E0680 genomic dna.After two PCR primer purifying, equal-volume mixing takes turns pcr template as second.Second takes turns PCR primer pilO-MF1/pilO – MR2, adopts regular Taq DNA polymerase.Obtain second and take turns PCR primer.
(2) second take turns PCR primer purified after, directly the gene knockout suicide vector pLP12T general with vibrios is connected.Connect product (plasmid pLP12-pilO) transformation of E. coli DH5 α λ pir cell, coating LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on pilO-MF1/pilO – MR2 primer pair flat board, correct clone can produce the amplified band that size is 815bp.PCR fragment carries out sequence verification again, jointly determines correct clone, and this is correctly cloned containing connecting product-plasmid pLP12-pilO.Enlarged culturing is correctly cloned, and extracts plasmid pLP12-pilO.
(3) pLP12-pilO electricity transformation of E. coli β 2163 cell.Electricity conversion condition and the rear cell culture condition of electric shock are with embodiment 2.Picking is cloned arbitrarily, and purifying is also preserved, and namely obtains the donor bacterium-intestinal bacteria β 2163 (pLP12-pilO) comprising suicide plasmid pLP12-pilO.
(4) incubated overnight intestinal bacteria β 2163 (pLP12-pilO) and recipient bacterium Vibrio parahaemolyticus E0680.Get 100 μ L donor bacterium to mix with 100 μ L recipient bacteriums, 8000g is centrifugal, and 2min abandons supernatant, and it is resuspended to add 400 μ L fresh LB, the centrifugal 2min of 8000g again, careful supernatant discarded.Add 10 μ L fresh LB resuspended, re-suspension liquid is all sucked and drops on LB agar plate.After super clean bench is air-dry, is inverted for 37 DEG C and cultivates 8hr.
(5) fully blow outstanding above-mentioned culture with 1ml fresh LB, get 100 μ L and be coated with LB agar plate (containing 0.3%D-glucose, 10 μ g/ml paraxin), 37 DEG C of incubated overnight.On this flat board, only have and first time homologous recombination occurs, chromosomal integration has the insertion mutation Vibrio parahaemolyticus cell of suicide plasmid pLP12-pilO just can grow.Random picked clones carries out PCR qualification, PCR primer pilO-MF1/pilO – MR2, and correct clone produces 1214bp and 815bp bis-bands (swimming lane 11 in Fig. 3), obtains the strain of pilO insertion mutation.
(6) strain of pilO insertion mutation is after LB (0.3%D-glucose) cultivates 3 hours, gradient dilution, respectively gets 100 μ L and is coated with LB agar plate (0.2%L-pectinose), 37 DEG C of incubated overnight.On this flat board, only have and second time homologous recombination occurs, the cell that chromosome elimination integrates state suicide plasmid pLP12-pilO can be survived.Second time restructuring occurs bacterial cell can produce the cell that the cell of a pilO genetically deficient and comprises wild-type pilO gene.
(7) PCR detection is carried out with the clone on pilO-MF1/pilO-MR2 primer pair LB agar plate (0.2%L-pectinose), correct pilO genetically deficient clone produces the band (swimming lane 12 in Fig. 3) of 815bp, and the Wild type clone that second time restructuring is formed produces 1214bp band (swimming lane 10 in Fig. 3).Correct pilO genetically deficient clone, again through PCR primer sequence verification, namely obtains pilO gene-deleted strain, thus completes Vibrio parahaemolyticus pilO gene knockout.In the present embodiment, Negative selection flat board (10 -1dilution) upper random picking 16 clones in, have 7 for correct pilO genetically deficient clone.
Embodiment 6 is applied pLP12T and is knocked out albumen translocase gene (ascS) before Vibrio parahaemolyticus
(1) with the ascS gene of overlapping pcr amplification Vibrio parahaemolyticus E06135, obtain ascS in-frame deletion and merge fragment.First round PCR primer pair is respectively ascS-MF1/ascS-MR1, ascS-MF2/ascS-MR2, and template is Vibrio parahaemolyticus E06135 genomic dna.Two PCR primer equal-volumes mix and take turns the template of PCR after purifying as second.Second takes turns PCR primer ascS-MF1/ascS – MR2, adopts regular Taq DNA polymerase, obtains second and take turns PCR primer.
(2) take turns second PCR primer purified after, directly the gene knockout suicide vector pLP12T general with above-mentioned vibrios is connected.Connect product (plasmid pLP12-ascS) transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on ascS-MF1/ascS – MR2 primer pair flat board, correct clone can produce the amplified band of 703bp.PCR fragment carries out sequence verification again, jointly determines correct clone, and this is correctly cloned containing connecting product-plasmid pLP12-ascS.Enlarged culturing is correctly cloned, and extracts plasmid pLP12-ascS.
(3) pLP12-ascS electricity transformation of E. coli β 2163 cell.Electricity conversion condition and the rear cell culture condition of electric shock are with embodiment 2.Picking is cloned arbitrarily, and purifying is also preserved, and namely obtains the donor bacterium-intestinal bacteria β 2163 (pLP12-ascS) comprising suicide plasmid pLP12-ascS.
(4) incubated overnight intestinal bacteria β 2163 (pLP12-ascS) and recipient bacterium Vibrio parahaemolyticus E06135.Get 100 μ L donor bacterium to mix with 100 μ L recipient bacteriums, 8000g is centrifugal, and 2min abandons supernatant, and it is resuspended to add 400 μ L fresh LB, the careful supernatant discarded of the centrifugal 2min of 8000g again.Add 10 μ L fresh LB resuspended, re-suspension liquid is all sucked and drops on LB agar plate.After super clean bench is air-dry, is inverted for 37 DEG C and cultivates 8hr.
(5) fully blow outstanding above-mentioned culture with 1ml fresh LB, get 100 μ L and be coated with LB agar plate (containing 0.3%D-glucose, 10 μ g/ml paraxin), 37 DEG C of incubated overnight.On this flat board, only have and first time homologous recombination occurs, chromosomal integration has the insertion mutation Vibrio parahaemolyticus cell of suicide plasmid pLP12-ascS just can grow.Random picked clones carries out PCR qualification, PCR primer ascS-MF1/ascS – MR2, and correct clone produces 952bp and 703bp bis-bands (swimming lane 14 in Fig. 3), obtains the strain of ascS insertion mutation.
(6) strain of ascS insertion mutation cultivates 4 hours, gradient dilution in LB (0.3%D-glucose), respectively gets 100 μ L and is coated with LB agar plate (0.2%L-pectinose), 37 DEG C of incubated overnight.On this flat board, only have and second time homologous recombination occurs, the cell that chromosome elimination integrates state suicide plasmid pLP12-degS can be survived.After second time restructuring occurs bacterial cell, the cell that the cell of an ascS genetically deficient and comprise wild-type ascS gene can be produced.
(7) PCR detection is carried out with the clone on ascS-MF1/ascS-MR2 primer pair LB agar plate (0.2%L-pectinose), correct ascS genetically deficient clone produces the band (swimming lane 15 in Fig. 3) of 703bp, and the Wild type clone that second time restructuring is formed produces 952bp band (swimming lane 13 in Fig. 3).Correct ascS genetically deficient clone, again through PCR primer sequence verification, namely obtains ascS gene-deleted strain, thus completes Vibrio parahaemolyticus ascS gene knockout.In the present embodiment, Negative selection flat board (10 -1dilution) upper random picking 16 clones in, have 3 for correct ascS genetically deficient clone.
Embodiment 7 is applied pLP12T and is knocked out Vibrio vulnificus VI type excretory system gene (impB)
(1) the impB gene in-frame deletion fusion fragment of Vibrio vulnificus ATCC27562 is obtained with overlapping pcr amplification.First round PCR primer pair is respectively impB-MF1/impB-MR1, impB-MF2/impB-MR2, and template is Vibrio vulnificus ATCC27562 genomic dna.After two PCR primer purifying, the template of PCR is taken turns in equal-volume mixing as second.Second takes turns PCR adopts primer impB-MF1/impB – MR2, adopts regular Taq DNA polymerase, obtains second and take turns PCR primer.
(2) second take turns PCR primer purified after, the general gene knockout suicide vector pLP12T of the vibrios of directly preparing with embodiment 1 is connected.Connect product (plasmid pLP12-impB) directly transformation of E. coli DH5 α λ pir cell, and be coated with LB flat board (containing 20 μ g/ml paraxin, 0.3%D-glucose).Carry out PCR screening with the clone on impB-MF1/impB-MR2 primer pair flat board, correct clone can produce long 909bp amplified band.PCR fragment carries out sequence verification again, jointly determines correct clone, and this is correctly cloned containing connecting product-plasmid pLP12-impB.Enlarged culturing is correctly cloned, and extracts plasmid pLP12-impB.
(3) pLP12-impB electricity transformation of E. coli β 2163 cell.Electricity conversion condition and the rear cell culture condition of electric shock are with embodiment 2.Incubated overnight, picking is cloned arbitrarily, and purifying is also preserved, and namely obtains the donor bacterium-intestinal bacteria β 2163 (pLP12-impB) comprising suicide plasmid pLP12-impB.
(4) incubated overnight intestinal bacteria β 2163 (pLP12-impB) and recipient bacterium Vibrio vulnificus ATCC27562.Get 100 μ L donor bacterium to mix with 100 μ L recipient bacteriums, 8000g is centrifugal, and 3min abandons supernatant, and it is resuspended to add 600 μ L fresh LB, the centrifugal 3min of 8000g again, careful supernatant discarded.Add 10 μ L fresh LB resuspended, re-suspension liquid is all sucked and drops on LB agar plate.After super clean bench is air-dry, is inverted for 37 DEG C and cultivates 7hr.
(5) fully blow outstanding above-mentioned culture with 1ml fresh LB, get 100 μ L and be coated with LB agar plate (containing 0.3%D-glucose, 10 μ g/ml paraxin), 37 DEG C of incubated overnight.On this flat board, the insertion mutation Vibrio vulnificus cell only having generation first time homologous recombination after stain colour solid to be integrated with suicide plasmid pLP12-impB just can grow.Random picked clones carries out PCR qualification, PCR primer impB-MF1/impB-MR2, and correct clone produces 1290bp and 909bp bis-bands (swimming lane 17 in Fig. 3), obtains the strain of impB insertion mutation.
(6) strain of impB insertion mutation cultivates 3 hours, gradient dilution in LB (0.3%D-glucose), respectively gets 100 μ L and is coated with LB agar plate (0.2%L-pectinose), 37 DEG C of incubated overnight.On this flat board, only have and second time homologous recombination occurs, the cell that chromosome elimination integrates state suicide plasmid pLP12-impB can be survived.After second time restructuring occurs bacterial cell, the cell that the cell of an impB genetically deficient and comprise wild-type impB gene can be produced.
(7) PCR detection is carried out with the clone on impB-MF1/impB-MR2 primer pair LB agar plate (0.2%L-pectinose), correct impB genetically deficient clone produces the band (swimming lane 18 in Fig. 3) of 909bp, and the Wild type clone that second time restructuring is formed then produces 1290bp band (swimming lane 16 in Fig. 3).Correct impB genetically deficient clone, again through PCR primer sequence verification, namely obtains impB gene-deleted strain, thus completes Vibrio parahaemolyticus impB gene knockout.In the present embodiment, Negative selection flat board (10 -1dilution) upper random picking 20 clones in, have 6 for correct impB genetically deficient clone.
Negative selection flat board in above-described embodiment 2-7 all has the existence of NaCl, but deletion mutantion strain still has very high appearance ratio, illustrates that lethal gene has played effect, and not by the impact that NaCl exists.

Claims (8)

1. a gene knockout suicide vector pLP12, is circular vectors, it is characterized in that, comprise P bADpromotor, repressor protein gene araC, RP4 shift initiation site, chloramphenicol resistance gene, R6K replication origin, multiple clone site district and lethal gene vmi480, described multiple clone site district at least comprises two AhdI restriction enzyme sites, the nucleotide sequence of described lethal gene vmi480 is as shown in the 21-617 bit base sequence of SEQIDNO.1, and described lethal gene vmi480 is positioned at P bADpromotor downstream is also in P bADunder promotor controls.
2. gene knockout suicide vector pLP12 according to claim 1, is characterized in that, described gene knockout suicide vector pLP12, its nucleotide sequence is as shown in SEQIDNO.1.
3. a gene knockout suicide vector pLP12T, for linear carrier, it is characterized in that, be the linear carrier will obtained after two AhdI restriction enzyme sites in gene knockout suicide vector pLP12 AhdI Mei Qie multiple clone site district according to claim 1, be gene knockout suicide vector pLP12T.
4. gene knockout suicide vector pLP12T according to claim 3, is characterized in that, described gene knockout suicide vector pLP12T, its nucleotide sequence is as shown in SEQIDNO.2.
5. the application of gene knockout suicide vector pLP12T according to claim 3 in gene knockout.
6. application according to claim 5, is characterized in that, the described application of gene knockout suicide vector pLP12T in vibrios gene knockout.
7. application according to claim 5, it is characterized in that, the in-frame deletion of goal gene is merged fragment be connected with gene knockout suicide vector pLP12T, the in-frame deletion obtaining carrying goal gene merges the pLP12T of fragment, called after pLP12T-X, pLP12T-X enters in donor bacterium through transforming, then this donor bacterium is mixed with recipient bacterium, through conjugation, pLP12T-X transfer is entered recipient bacterium, select through paraxin, obtain the goal gene insertion mutation type bacterial strain that pLP12T-X is incorporated into recipient bacterium genome target site, then this goal gene insertion mutation type bacterial strain is under pectinose induction vmi480 gene produces the Negative selection pressure of toxin Vmi480, the clone of goal gene disappearance is obtained through screening, thus reach goal gene is knocked out, obtain the bacterial strain of goal gene disappearance.
8. application according to claim 7, is characterized in that, described recipient bacterium is vibrios.
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Application publication date: 20151118