CN103409446A - Corynebacterium gene continuous knockout system, as well as construction method and application thereof - Google Patents
Corynebacterium gene continuous knockout system, as well as construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a construction method and an application of a corynebacterium gene continuous knockout system, belonging to the technical field of genetic engineering. The corynebacterium gene continuous knockout system comprises a template plasmid and a temperature-sensitive expression plasmid, wherein the template plasmid provides a kan fragment (also known as a kan box) with loxp or variant loxL/R sites at two ends; and the temperature-sensitive expression plasmid carries a Cm resistance gene cat and a temperature-sensitive C. glutamicum replicon, expresses a Cre recombinant enzyme and is used for removing a Km resistance gene kan. The corynebacterium continuous knockout system disclosed by the invention can be generally used for continuous gene transformation of corynebacterium and has the advantages of simplicity and convenience in operation and high efficiency. By utilizing the knockout system, gene (continuous) knockout of three major typical subspecies genomes of the corynebacterium can be successfully completed, and the system is proved to be applicable to research and production of corynebacterium metabolites.
Description
Technical field
The present invention relates to a kind of genes of corynebacteria and knock out continuously system and construction process and application, particularly a kind of based on homologous recombination and the system that knocks out that the specific site restructuring combines, belong to the microbiological genetic engineering field.
Background technology
Coryneform bacteria is a class gram-positive microorganism, belongs to and has the actinomycetes of moderate to high GC content.(Kinoshita et al. since people separate corynebacterium glutamicum to produce Pidolidone first, 1985), coryneform three main representatives: corynebacterium glutamicum, brevibacterium flavum, and brevibacterium lactofermentum, be widely used in producing amino acid.
A large amount of about corynebacterium glutamicum physiology owing to having added up, biological chemistry and genetics knowledge (Eggeling and Bott, 2005; Burkovski, 2008), metabolic engineering has replaced classical random mutation, becomes the main policies of coryneform bacteria strain improvement.Along with the complete genomic order-checking of corynebacterium glutamicum, genetic engineering means status in the metabolic engineering breeding is more and more important.Utilizing molecular biology method to carry out the metabolic engineering breeding, is at first that suitable carrier need to be arranged.At present, along with the endogenous plasmid of corynebacterium glutamicum comprises pCG1(Ozaki, 1984), pBL1(Santamaria, 1984) etc. the discovery of plasmid of family, the investigator has constructed a large amount of plasmids (Xu, 2010) be used to carrying out genetic expression.But, an other class plasmid, the carrier of modifying for chromogene but seems very deficient.Current widely used coryneform bacteria knockout carrier is pK18mobsacB/pK19mobsacB.But its inactivation of insertion due to the sacB gene is taken turns the exchange screening to second sometimes and is brought very burden.When the sacB gene is on corynebacterium glutamicum subspecies C.glutamicum ATCC14067 karyomit(e), do not have sucrose susceptibility, so need build a set of suitable system that knocks out for C.glutamicum ATCC14067.
In the present invention, at first we built a kind of system that knocks out combined with the specific site restructuring based on homologous recombination.Homologous recombination is mainly to depend on fragment or carrier to have certain identical homologous DNA sequence with the target gene group, through pairing and the splitting of chain of homologous region, practice midwifery and give birth to exchange again, completes the DNA restructuring.Homologous recombination is the most basic recombination form, can occur in any one bacterial strain, and wherein, homologous fragment is more long more to be beneficial to and to recombinate.In genetically engineered, as homology arm, by homologous recombination, goal gene is substituted by resistant gene due to the restructuring exchange of both sides homology arm at adding purpose gene both sides, resistant gene both sides DNA fragmentation.The specific site restructuring utilizes the special recombination system of Cre/lox.The Cre/lox recombination system is found in the P1 phage, comprises recombinase and recombination site two portions, and wherein, Cre is the recombinase that is under the jurisdiction of λ Int enzyme supergene family, can receive the deleted or restructuring of gene order between recombination site; The loxp site is can be for the recombination site of Cre recombinase recognition reaction, and when two loxp sites are positioned on a DNA chain and direction when identical, the Cre recombinase can excise two sequences between the loxp site.LoxL/loxR is sudden change loxp site, and two recombination sites, after the restructuring of Cre enzyme, produce a site loxLR that can not be identified by the Cre recombinase again, and this site no longer participates in the site restructuring.
The system that knocks out that the present invention builds is namely utilized homologous recombination and variation loxLR site, contains and overcomes the existing defect that knocks out system be used to the two ends of increasing, and can be used for the gene knockout of the main subspecies of coryneform bacteria; At first the process that knocks out replaces goal gene by homologous recombination with Km resistant gene kan, then by site-specific, recombinates and removes resistant gene on genome, easy and simple to handle efficient; Can be used for a plurality of genes in same bacterial strain knocks out continuously; Knocked out rear by the transformation bacterial strain do not carry any resistance.
Summary of the invention
The invention provides a kind of genes of corynebacteria and knock out continuously system and construction process thereof, by this, knock out system 1) successfully knock out the coryneform bacteria Three Represents: corynebacterium glutamicum ATCC13032, brevibacterium flavum ATCC14067, and aceE in brevibacterium lactofermentum ATCC13869; 2) complete knocking out continuously of aceE and ilvA in brevibacterium flavum ATCC14067.
Genes of corynebacteria provided by the invention knocks out system continuously, comprises template plasmid and temperature sensitive expression plasmid; Described template plasmid provides the kan fragment of two ends with loxp or variation loxL/R site, claims again the kan box; Described temperature sensitive expression plasmid carries Cm resistant gene cat and temperature sensitive C.glutamicum replicon, expresses the Cre recombinase, be used to removing Km resistant gene kan.Described template plasmid is pDTW201/202; Described temperature sensitive expression plasmid is pDTW109; Its nucleotide sequence is as shown in SEQ IDNO.1-NO.3.
Genes of corynebacteria knocks out system structure continuously:
The first step: with pK18mobsacB(Schafer et al., 1994) be template, using the kan-F/kan-R primer pair as template, pcr amplification obtains the kan gene, and introduces respectively XhoI and XbaI enzyme cutting site at its two ends, after enzyme is cut purifying, standby; Loxp site sequence loxp-F-F/loxp-F-R and the loxp-R-F/loxp-R-R of synthetic are annealed respectively, form double-stranded, standby; LoxL/R site sequence loxL-F/loxL-R and the loxR-F/loxR-R of synthetic are annealed respectively, form double-stranded, standby;
Second step: the kan fragment, loxp fragment and the process ApaI that have handled well are connected with pBluescript II SK (+) plasmid that the SacII enzyme is cut purifying, transform JM109, novel plasmid called after pDTW201.With loxpLE and loxpRE fragment, replace two sections loxp fragments, according to the construction process of pDTW201, the kan box template plasmid called after pDTW202 of structure.
Kan box template amplification primer is positioned at the recombination site outside, and the amplified production two ends, with the Km resistance fragment in loxp site or variation loxL/R site, are called the kan box, is convenient to follow-up site-specific restructuring.
Another technical problem to be solved by this invention is the construction process of temperature sensitive type Cre recombinase expression vector pDTW-109.
Carrier detection of the present invention comprises selection markers, replicon fragment and multiple clone site, it is characterized in that containing the Cm resistant gene cat(PtacM+cat that is useful on the plasmid screening); E.coli replicon oriE; The temperature sensitive replicon rep of C.glutamicum
TSCre recombinase expressing gene (PtacM+cre) under strong promoter tacM regulation and control.
Expression vector pDTW109 contains the temperature sensitive replicon rep of C.glutamicum
TSFragment, from 4764 to 5969.But pDTW109 is at 25 ° of C stable existences and copy, unstable and lose under 37 ° of C.
Expression vector pDTW109 contains the resistance marker paraxin mark resistant gene cat that is useful on the selection of coryneform bacteria transformant, from 166 to 825.
Expression vector pDTW109 contains E.coli replicon oriE, from 1315 to 2242.
Expression vector pDTW109 contains the Cre recombinase expressing gene (PtacM+cre) under strong promoter tacM regulation and control, and wherein PtacM is from 3301 to 3368, and cre is from 2242 to 3273.
For addressing the above problem, the concrete technical scheme adopted is
The first step: with pACYC184 (Chang A.C.Y.and Cohen S.N., 1978), be template, can move reading frame with corresponding primer amplification paraxin acyltransferase, and 5 of amplified fragments ' and 3 ' end introduce NheI and HindIII restriction enzyme site.Fragment is connected to form pDXW-10-cat after NheI and HindIII enzyme are cut purifying with through the same pDXW-10 processed, transform intestinal bacteria.The E.coli plasmid pKK223-3 of take is template, with corresponding primer amplification E.coli replicon oriE, and fragment 5 ' all introduce SmaI restriction enzyme point of contact with 3 ' end; The pDXW-10-cat of take is template, with corresponding primer amplification chloramphenicol resistance gene, and fragment 5 ' and 3 ' end introducing SmaI point of contact; Two fragments all connect after the SmaI enzyme is cut purifying, transform JM109, and novel plasmid is named as pDTW-101.The MCS of plasmid pDTW-101 and synthetic is cut to rear purifying with HindIII and NheI enzyme, connect, transform JM109.New plasmid is named as pDTW102;
Second step: with pC2 (the Goyal et al. that contains C.glutamicum pBL1 replicon, 1996) plasmid is as template, with corresponding primer amplification C.glutamicum replicon rep, and 5 of amplified fragments ' and 3 ' end introduce HindIII and SacII restriction enzyme site.Fragment through HindIII with after the SacII enzyme is cut purifying with through the same pDTW102 processed, is connected, conversion JM109, new plasmid is named as pDTW104;
The 3rd step: our reference (Nakamura, 2006), carry out point mutation to the contained pBL1 replicon of pDTW104.Use corresponding primer pair plasmid pDTW104 to carry out point mutation, make C.glutamicum/pDTW104 have temperature-sensing property.The new plasmid with temperature-sensing property is named as pDTW106;
The 4th step:, on pDTW106, introduce again a MCS sequence, the restriction enzyme site consumption that before making up, operation causes.Use oligonucleotide chain to anneal, SacI is connected with the pDTW106 that the SacII enzyme is cut purification process with process, transforms JM109, and novel plasmid is named as pDTW107;
The 5th step: introduce the tac promotor.Adopt oligonucleotide chain to anneal and form two strands, BglII is connected with the pDTW107 that the SacII enzyme is cut purification process with process, transforms JM109, and novel plasmid is named as pDTW108;
The 6th step: the pSH47 of take is template plasmid, with corresponding primer amplification recombinase fragment, and fragment 5 ' and 3 ' end introducing XhoI and PstI restriction enzyme site.The recombinase fragment is cut purifying and is connected through the same pDTW108 plasmid of processing with the PstI enzyme through XhoI, transforms JM109.Novel plasmid is named as pDTW109.
Another technical problem to be solved by this invention is the constructed system that knocks out continuously 1 of application) knock out ATCC13032, ATCC14067, aceE in ATCC13869; 2) the knocking out continuously of aceE and ilvA in ATCC14067.Utilize pDTW201/202 to build respectively aceE and ilvA knockout carrier pDTW301/302, knocked out the aceE in ATCC13032/14067/13869, obtain strains A TCC13032/14067/13869 Δ aceE; Complete knocking out continuously of aceE and ilvA in ATCC14067 and obtain strains A TCC14067 Δ aceE Δ ilvA.
But described knock out the system simple and effective complete knocking out continuously of a plurality of genes on the genes of corynebacteria group, be applicable to various representative genes of corynebacteria and knock out, knocked out rear bacterial strain and do not carried any antibiotics resistance.
The accompanying drawing explanation
The physical map of Fig. 1 template plasmid pDTW201/202.
The structure of Fig. 2 expression vector pDTW109.
Fig. 3 aceE& IlvA knocks out the plasmid physical map.
Fig. 4 gene (aceE& IlvA) knock out the process schematic diagram.
Phenotype and genotype proof diagram after Fig. 5 ATCC14067 Δ aceE Δ ilvA gene knockout
M：1kb?Marker；1：ATCC14067-aceE；2：ATCC14067ΔaceE-kan-aceE；3：ATCC14067ΔaceE–aceE；4：ATCC14067-ilvA；5：ATCC14067ΔaceEΔilvA-kan-ilvA；l6：ATCC14067ΔaceEΔilvA-ilvA。
Embodiment
Genes of corynebacteria provided by the invention knocks out system continuously, comprises template plasmid and temperature sensitive expression plasmid; Described template plasmid provides the kan fragment of two ends with loxp or variation loxL/R site, claims again the kan box; Described temperature sensitive expression plasmid carries Cm resistant gene cat and temperature sensitive C.glutamicum replicon, expresses the Cre recombinase, be used to removing Km resistant gene kan.Described template plasmid is pDTW201/202; Described temperature sensitive expression plasmid is pDTW109; Its nucleotide sequence is as shown in SEQ IDNO.1-NO.3.
Genes of corynebacteria knocks out system structure continuously:
The first step: with pK18mobsacB(Schafer et al., 1994) be template, using the kan-F/kan-R primer pair as template, pcr amplification obtains the kan gene, and introduces respectively XhoI and XbaI enzyme cutting site at its two ends, after enzyme is cut purifying, standby; Loxp site sequence loxp-F-F/loxp-F-R and the loxp-R-F/loxp-R-R of synthetic are annealed respectively, form double-stranded, standby; LoxL/R site sequence loxL-F/loxL-R and the loxR-F/loxR-R of synthetic are annealed respectively, form double-stranded, standby;
Second step: the kan fragment, loxp fragment and the process ApaI that have handled well are connected with pBluescript II SK (+) plasmid that the SacII enzyme is cut purifying, transform JM109, novel plasmid called after pDTW201.With loxpLE and loxpRE fragment, replace two sections loxp fragments, according to the construction process of pDTW201, the kan box template plasmid called after pDTW202 of structure.
Kan box template amplification primer is positioned at the recombination site outside, and the amplified production two ends, with the Km resistance fragment in loxp site or variation loxL/R site, are called the kan box, is convenient to follow-up site-specific restructuring.
Another technical problem to be solved by this invention is the construction process of temperature sensitive type Cre recombinase expression vector pDTW-109.
Carrier detection of the present invention comprises selection markers, replicon fragment and multiple clone site, it is characterized in that containing the Cm resistant gene cat(PtacM+cat that is useful on the plasmid screening); E.coli replicon oriE; The temperature sensitive replicon rep of C.glutamicum
TSCre recombinase expressing gene (PtacM+cre) under strong promoter tacM regulation and control.
Expression vector pDTW109 contains the temperature sensitive replicon rep of C.glutamicum
TSFragment, from 4764 to 5969.But pDTW109 is at 25 ° of C stable existences and copy, unstable and lose under 37 ° of C.
Expression vector pDTW109 contains the resistance marker paraxin mark resistant gene cat that is useful on the selection of coryneform bacteria transformant, from 166 to 825.
Expression vector pDTW109 contains E.coli replicon oriE, from 1315 to 2242.
Expression vector pDTW109 contains the Cre recombinase expressing gene (PtacM+cre) under strong promoter tacM regulation and control, and wherein PtacM is from 3301 to 3368, and cre is from 2242 to 3273.
For addressing the above problem, the concrete technical scheme adopted is
The first step: with pACYC184 (Chang A.C.Y.and Cohen S.N., 1978), be template, can move reading frame with corresponding primer amplification paraxin acyltransferase, and 5 of amplified fragments ' and 3 ' end introduce NheI and HindIII restriction enzyme site.Fragment is connected to form pDXW-10-cat after NheI and HindIII enzyme are cut purifying with through the same pDXW-10 processed, transform intestinal bacteria.The E.coli plasmid pKK223-3 of take is template, with corresponding primer amplification E.coli replicon oriE, and fragment 5 ' all introduce SmaI restriction enzyme point of contact with 3 ' end; The pDXW-10-cat of take is template, with corresponding primer amplification chloramphenicol resistance gene, and fragment 5 ' and 3 ' end introducing SmaI point of contact; Two fragments all connect after the SmaI enzyme is cut purifying, transform JM109, and novel plasmid is named as pDTW-101.The MCS of plasmid pDTW-101 and synthetic is cut to rear purifying with HindIII and NheI enzyme, connect, transform JM109.New plasmid is named as pDTW102;
Second step: with pC2 (the Goyal et al. that contains C.glutamicum pBL1 replicon, 1996) plasmid is as template, with corresponding primer amplification C.glutamicum replicon rep, and 5 of amplified fragments ' and 3 ' end introduce HindIII and SacII restriction enzyme site.Fragment through HindIII with after the SacII enzyme is cut purifying with through the same pDTW102 processed, is connected, conversion JM109, new plasmid is named as pDTW104;
The 3rd step: our reference (Nakamura, 2006), carry out point mutation to the contained pBL1 replicon of pDTW104.Use corresponding primer pair plasmid pDTW104 to carry out point mutation, make C.glutamicum/pDTW104 have temperature-sensing property.The new plasmid with temperature-sensing property is named as pDTW106;
The 4th step:, on pDTW106, introduce again a MCS sequence, the restriction enzyme site consumption that before making up, operation causes.Use oligonucleotide chain to anneal, SacI is connected with the pDTW106 that the SacII enzyme is cut purification process with process, transforms JM109, and novel plasmid is named as pDTW107;
The 5th step: introduce the tac promotor.Adopt oligonucleotide chain to anneal and form two strands, BglII is connected with the pDTW107 that the SacII enzyme is cut purification process with process, transforms JM109, and novel plasmid is named as pDTW108;
The 6th step: the pSH47 of take is template plasmid, with corresponding primer amplification recombinase fragment, and fragment 5 ' and 3 ' end introducing XhoI and PstI restriction enzyme site.The recombinase fragment is cut purifying and is connected through the same pDTW108 plasmid of processing with the PstI enzyme through XhoI, transforms JM109.Novel plasmid is named as pDTW109.
Another technical problem to be solved by this invention is the constructed system that knocks out continuously 1 of application) knock out ATCC13032, ATCC14067, aceE in ATCC13869; 2) the knocking out continuously of aceE and ilvA in ATCC14067.Utilize pDTW201/202 to build respectively aceE and ilvA knockout carrier pDTW301/302, knocked out the aceE in ATCC13032/14067/13869, obtain strains A TCC13032/14067/13869 Δ aceE; Complete knocking out continuously of aceE and ilvA in ATCC14067 and obtain strains A TCC14067 Δ aceE Δ ilvA.
In embodiment 2ATCC13032/14067/13869, aceE's knocks out
1. knock out the acquisition of plasmid pDTW301
The C.glutamicum ATCC14067 genome of take is template, with corresponding primer amplification, obtains respectively upstream fragment and the downstream fragment of aceE gene.5 of upstream fragment ' and 3 ' end introduce respectively PstI and BamHI, in 5 of downstream fragment ' and 3 ' end introduces respectively XbaI and SalI restriction enzyme site.Using pDTW201 as template, with corresponding primer amplification two ends, contain the kan box in loxp site, 5 ' and 3 ' end introduce respectively BamHI and XbaI enzyme cutting site.AceE upstream fragment is cut to purifying through PstI and BamHI enzyme, the downstream fragment is cut purifying through XbaI and SalI enzyme, kan box fragment is through BamHI and XbaI enzyme cutting purifying, pBluescript II SK (+) cuts purifying through PstI and SalI enzyme, four fragments connect, transform JM109, new plasmid is aceE and knocks out plasmid, called after pDTW301;
2. knocking out competence preparation and electricity turns
C.glutamicum ATCC13032/1406713869 adopts electrotransformation, and competence preparation and method for transformation are with reference to corresponding document (Van der, 1999; Xu, 2010; Tan, Y., 2012);
3. knock out flow process
(1) by plasmid pDTW301 from E.coli JM109, extracting, electricity transforms the C.glutamicum bacterial strain, coats the solid recovery media that contains potassium acetate and kantlex.30 ° of C cultivated approximately 36 hours;
(2) on picking A flat board, single bacterium colony carries out the PCR checking, chooses single bacterium colony that checking is correct, and namely C.glutamicum ATCC13032/14067/13869 Δ aceE-kan, carry out liquid culture, the preparation competence;
(3) by plasmid pDTW109 from E.coli JM109, extracting, electricity transforms the competence prepared in previous step, coats the solid recovery media that contains potassium acetate and paraxin.25 ° of C cultivated approximately 36 hours;
(4) on picking B flat board, single bacterium colony carries out the plasmid checking, chooses single bacterium colony that checking is correct, i.e. C.glutamicum ATCC13032/14067/13869 Δ aceE-kan/pDTW109;
4.pDTW109 removal
(1) C.glutamicum ATCC13032/14067/13869 Δ aceE-kan/pDTW109 is inoculated in liquid nutrient medium, 37 ° of C incubated overnight;
(2) overnight culture in previous step is lined to the LBG solid medium that contains potassium acetate, 300C cultivated approximately 24 hours;
(3) on the picking flat board, single bacterium colony carries out respectively resistance checking and PCR checking.The resistance checking comprises that resistance checking of card and chlorampenicol resistant checking.The kalamycin resistance checking: single bacterium colony is lined to the solid plate that contains potassium acetate and kantlex, 30 ° of C cultivated approximately 24 hours.Chlorampenicol resistant checking: by single bacterium colony line
The solid plate of paraxin, 25 ° of C cultivated approximately 24 hours.PCR verifies that bacterium colony correct and that can not grow on kantlex or paraxin flat board is namely to knock out successful purpose bacterial strain, i.e. C.glutamicum ATCC13032/14067/13869 Δ aceE.
PCR verifies primer:
Genotype proof diagram result is as shown in Figure 5.
In embodiment 3ATCC14067 Δ aceE, ilvA's knocks out
1. knock out the acquisition of plasmid pDTW302
The C.glutamicum ATCC14067 genome of take is template, with corresponding primer amplification, obtains respectively upstream fragment and the downstream fragment of ilvA gene.5 of upstream fragment ' and 3 ' end introduce respectively XhoI and XbaI, in 5 of downstream fragment ' and 3 ' end introduces respectively PstI and BamHI restriction enzyme site.Using pDTW202 as template, with corresponding primer amplification two ends, contain the kan box in loxL/R site, 5 ' and 3 ' end introduce respectively BamHI and XbaI enzyme cutting site.By ilvA upstream fragment process XhoI and XbaI enzyme cutting purifying, the downstream fragment is cut purifying through PstI and BamHI enzyme, kan box fragment is through BamHI and XbaI enzyme cutting purifying, pBluescript II SK (+) cuts purifying through PstI and XhoI enzyme, four fragments connect, transform JM109, new plasmid is ilvA and knocks out plasmid, called after pDTW302;
2. knocking out competence preparation and electricity turns
With embodiment 2, only in substratum, all need to add potassium acetate and ILE;
3. knock out flow process
With embodiment 2, only in substratum, all need to add potassium acetate and ILE; ATCC14067 Δ aceE ilvA::
LoxL-kan-loxR called after ATCC14067 Δ aceE Δ ilvA-kan.
4.pDTW109 removal
With embodiment 2, only in substratum, all need to add potassium acetate and ILE; ATCC14067 Δ aceE ilvA::loxLR called after ATCC14067 Δ aceE Δ ilvA.
PCR verifies primer:
Genotype proof diagram result is as shown in Figure 5.
References
Albert,H.,Dale,E.C.,Lee,E.,Ow,D.W.,1995.Site-specific?integration?of?DNA?into?wild-type?and?mutant?lox?sites?placed?in?the?plant?genome.The?Plant?Journal7,649–659.
Burkovski,A.,2008.Corynebacteria:Genomics?and?Molecular?Biology.Caister?Academic?Press,Norfolk.
Chang,A.C.Y.and?Cohen,S.N.,1978.Construction?and?characterization?of?amplifiable?multicopy?DNA?cloning?vehicles?derived?from?the?P15A?cryptic?miniplasmid.J?Bacteriol134,1141-1156.
Goyal,D.,Wachi,M.,Kijima,N.,et?al.,1996.A?cryptic?plasmid?pBL1from?Brevibacterium?lactofermentum?causes?growth?inhibition?and?filamentation?in?Escherichia?coli.Plasmid36,62–66.
Jaffe,R.,Santamaria,M.,Yunis,E.J.,et?al.,1984.The?neuroectodermal?tumor?of?bone.The?American?journal?of?surgical?pathology8,885-898.
Kinoshita,S.,1985.Glutamic?acid?bacteria,in:Demain,A.L.,Solomon,N.A.(Eds.),Biology?of?industrial?microorganisms?cummings.The?Benjamin/Cummings?Publishing?Company,London,pp.115–146
Nakamura,J.,Kanno,S.,Kimura,E.,2006.Temperature-sensitive?cloning?vector?for?Corynebacterium?glutamicum.Plasmid56,179-186.
Ozaki,M.,Kratohvil,S.,Matijevi
,E.,1984.Formation?of?monodispersed?spindle-type?hematite?particles.Journal?of?Colloid?and?Interface?Science102,146-151.
Schafer,A.,Tauch,A.,Jager,W.,et?al.,1994.Small?mobilizable?multi-purpose?cloning?vectors?derived?from?the?Escherichia?coli?plasmids?pK18and?pK19:selection?of?defined?deletions?in?the?chromosome?of?Corynebacterium?glutamicum.Gene145,69-73.
Wendisch,V.F.,Bott,M.,2005.Phosphorus?metabolism?of?Corynebacterium?glutamicum.
Xu,D.,Tan,Y.,Shi,F.,Wang,X.,2010.An?improved?shuttle?vector?constructed?for?metabolic?engineering?research?in?Corynebacterium?glutamicum.Plasmid64,85–91.
Claims (10)
1. a genes of corynebacteria knocks out system continuously, it is characterized in that comprising template plasmid and temperature sensitive expression plasmid; Described template plasmid provides the kan fragment of two ends with loxp or variation loxL/R site, claims again the kan box; Described temperature sensitive expression plasmid carries Cm resistant gene cat and temperature sensitive C.glutamicum replicon, expresses the Cre recombinase, be used to removing Km resistant gene kan.
2. the system that knocks out claimed in claim 1, is characterized in that described template plasmid is pDTW201/202.
3. the system that knocks out claimed in claim 1, is characterized in that described temperature sensitive expression plasmid is pDTW109.
4. the arbitrary described construction process that knocks out system of claim 1-3, is characterized in that comprising the steps:
The first step: the pK18mobsacB of take is template, and pcr amplification obtains the kan gene, and introduces respectively XhoI and XbaI enzyme cutting site at its two ends, after enzyme is cut purifying, standby; Loxp fragment loxp-F-F/loxp-F-R and the loxp-R-F/loxp-R-R of synthetic are annealed respectively, form double-stranded, standby; LoxL/R fragment loxL-F/loxL-R and the loxR-F/loxR-R of synthetic are annealed respectively, form double-stranded, standby;
Second step: kan fragment, loxp fragment and process ApaI that the first step is handled well are connected with pBluescript II SK (+) plasmid that the SacII enzyme is cut purifying, transform JM109, novel plasmid called after pDTW201; With loxL and loxR fragment, replace two sections loxp fragments, according to the construction process of pDTW201, the kan box template plasmid called after pDTW202 of structure;
The 3rd step: the pACYC184 of take is template, can move reading frame with corresponding primer amplification paraxin acyltransferase Cat, and 5 of amplified fragments ' and 3 ' end introduce NheI and HindIII restriction enzyme site; Fragment is connected to form pDXW-10-cat after NheI and HindIII enzyme are cut purifying with through the same pDXW-10 processed, transform intestinal bacteria; The E.coli plasmid pKK223-3 of take is template, with corresponding primer amplification E.coli replicon oriE, and fragment 5 ' all introduce SmaI restriction enzyme point of contact with 3 ' end; The pDXW-10-cat of take is template, with corresponding primer amplification chloramphenicol resistance gene, and fragment 5 ' and 3 ' end introducing SmaI point of contact; Two fragments all connect after the SmaI enzyme is cut purifying, transform JM109, and novel plasmid is named as pDTW-101; The MCS of plasmid pDTW-101 and synthetic is cut to rear purifying with HindIII and NheI enzyme, connect, transform JM109; New plasmid is named as pDTW102;
The 4th step: using contain C.glutamicum pBL1 replicon the pC2 plasmid as template, with corresponding primer amplification C.glutamicum replicon rep, and 5 of amplified fragments ' and 3 ' end introduce HindIII and SacII restriction enzyme site; Fragment through HindIII with after the SacII enzyme is cut purifying with through the same pDTW102 processed, is connected, conversion JM109, new plasmid is named as pDTW104;
The 5th step: use corresponding primer pair plasmid pDTW104 to carry out point mutation, make C.glutamicum/pDTW104 have temperature-sensing property; The new plasmid with temperature-sensing property is named as pDTW106;
The 6th step: introduce again a MCS sequence on pDTW106, the restriction enzyme site consumption that before making up, operation causes; Use oligonucleotide chain to anneal, SacI is connected with the pDTW106 that the SacII enzyme is cut purification process with process, transforms JM109, and novel plasmid is named as pDTW107;
The 7th step: introduce the tac promotor; Adopt oligonucleotide chain to anneal and form two strands, BglII is connected with the pDTW107 that the SacII enzyme is cut purification process with process, transforms JM109, and novel plasmid is named as pDTW108;
The 8th step: the pSH47 of take is template plasmid, with corresponding primer amplification recombinase fragment, and fragment 5 ' and 3 ' end introducing XhoI and PstI restriction enzyme site; The recombinase fragment is cut purifying and is connected through the same pDTW108 plasmid of processing with the PstI enzyme through XhoI, transforms JM109, and novel plasmid is named as pDTW109.
5. method claimed in claim 4, is characterized in that the contained kan box template nucleotide sequence of described pDTW201 is as shown in SEQ IDNO.1.
6. method claimed in claim 4, is characterized in that the contained kan box of described pDTW202 template nucleotide sequence is as shown in SEQ IDNO.2.
7. method claimed in claim 4, is characterized in that described temperature sensitive Cre recombinase expression plasmid pDTW109 nucleotide sequence is as shown in SEQ ID NO.3.
8. the arbitrary described application of system aceE in ATCC13869/14067/13032 in knocking out that knock out of claim 1-3.
9. arbitrary described system aceE and ilvA in the ATCC14067 application in knocking out continuously that knocks out of claim 1-3.
10. the arbitrary described application of claim 8 or 9 is characterized in that knocking out flow process and is:
(1) will knock out with plasmid from E.coli JM109, extracting, electricity transforms the C.glutamicum bacterial strain, coats the solid recovery media that contains potassium acetate and kantlex, and 30 ° of C cultivated approximately 36 hours;
(2) on the picking flat board, single bacterium colony carries out the PCR checking, chooses single bacterium colony that checking is correct, carries out liquid culture, the preparation competence;
(3) by plasmid pDTW109 from E.coli JM109, extracting, electricity transforms the competence prepared in previous step, coats the solid recovery media that contains potassium acetate and paraxin, 25 ° of C cultivated approximately 36 hours;
(4) on the picking flat board, single bacterium colony carries out the plasmid checking, chooses single bacterium colony that checking is correct;
(5) inoculation previous step obtained is in liquid nutrient medium, 37 ° of C incubated overnight;
(6) overnight culture in previous step is lined to the LBG solid medium that contains potassium acetate, 30 ° of C cultivated approximately 24 hours;
(7) on the picking flat board, single bacterium colony carries out respectively resistance checking and PCR checking acquisition knocks out successful purpose bacterial strain.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710375A (en) * | 2013-12-30 | 2014-04-09 | 江南大学 | Novel plasmid for gene modification of Corynebacterium glutamicum and application thereof |
| CN103849640A (en) * | 2014-03-12 | 2014-06-11 | 南京师范大学 | Method for carrying out point mutations on essential genes on escherichia coli by using co-transformation of oligonucleotide and eliminable plasmids |
| CN104004678A (en) * | 2014-05-05 | 2014-08-27 | 江南大学 | Construction of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and method for fermentation production of L-valine |
| CN107119067A (en) * | 2017-04-25 | 2017-09-01 | 华南理工大学 | A kind of method of the continuous traceless knockout of gene in Corynebacterium glutamicum |
| CN107384951A (en) * | 2017-07-14 | 2017-11-24 | 江南大学 | Gene editing carrier, preparation method, system and its application of a kind of corynebacterium glutamicum |
| CN112251457A (en) * | 2019-07-22 | 2021-01-22 | 江南大学 | Adaptive evolution method and application of 4-hydroxyisoleucine producing strain |
| WO2021103498A1 (en) * | 2019-11-25 | 2021-06-03 | 华农（肇庆）生物产业技术研究院有限公司 | Corynebacterium atcc 13032 improved strain capable of effectively expressing foreign protein and construction method therefor |
| CN113355346A (en) * | 2020-08-21 | 2021-09-07 | 深圳蓝晶生物科技有限公司 | Method for deleting redundant segments on a stable vector, stable vector obtained by said method and use thereof |
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-
2013
- 2013-05-31 CN CN2013102155089A patent/CN103409446A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| YANZHEN TAN,ET AL: "Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum", 《PLASMID》 * |
| 徐大庆 等: "一株黄色短杆菌基因工程菌株的构建及其L-缬氨酸积累", 《食品科学》 * |
| 谭延振: "谷氨酸棒状杆菌基因敲除系统的构建", 《中国优秀硕士学位论文全文数据库》 * |
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| CN103710375A (en) * | 2013-12-30 | 2014-04-09 | 江南大学 | Novel plasmid for gene modification of Corynebacterium glutamicum and application thereof |
| CN103849640A (en) * | 2014-03-12 | 2014-06-11 | 南京师范大学 | Method for carrying out point mutations on essential genes on escherichia coli by using co-transformation of oligonucleotide and eliminable plasmids |
| CN103849640B (en) * | 2014-03-12 | 2016-01-20 | 南京师范大学 | A kind of oligonucleotide and the method for plasmid cotransformation for the point mutation of intestinal bacteria indispensable gene can be eliminated |
| CN104004678A (en) * | 2014-05-05 | 2014-08-27 | 江南大学 | Construction of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and method for fermentation production of L-valine |
| CN107119067A (en) * | 2017-04-25 | 2017-09-01 | 华南理工大学 | A kind of method of the continuous traceless knockout of gene in Corynebacterium glutamicum |
| CN107119067B (en) * | 2017-04-25 | 2019-10-18 | 华南理工大学 | A kind of method of the continuous seamless knockout of gene in Corynebacterium glutamicum |
| CN107384951A (en) * | 2017-07-14 | 2017-11-24 | 江南大学 | Gene editing carrier, preparation method, system and its application of a kind of corynebacterium glutamicum |
| CN112251457A (en) * | 2019-07-22 | 2021-01-22 | 江南大学 | Adaptive evolution method and application of 4-hydroxyisoleucine producing strain |
| WO2021103498A1 (en) * | 2019-11-25 | 2021-06-03 | 华农（肇庆）生物产业技术研究院有限公司 | Corynebacterium atcc 13032 improved strain capable of effectively expressing foreign protein and construction method therefor |
| CN113355346A (en) * | 2020-08-21 | 2021-09-07 | 深圳蓝晶生物科技有限公司 | Method for deleting redundant segments on a stable vector, stable vector obtained by said method and use thereof |
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