CN101955966A - High starting intensity and wide host range constitutive expression plasmid pBSPPc and application thereof - Google Patents
High starting intensity and wide host range constitutive expression plasmid pBSPPc and application thereof Download PDFInfo
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Abstract
The invention discloses a high starting intensity and wide host range constitutive expression plasmid pBSPPc and the application of expressing catechol dioxygenase in gram negative bacteria. The plasmid not only can express constitutive startup exogenous genes without adding any inducers or carrying out any additional induction operation, but also has high starting intensity and high induced velocity. Therefore, the plasmid has large application potential in the fields of biodegradation and bioremediation.
Description
Technical field
What the present invention relates to is genetically engineered field expression plasmid and application, especially starts intensity wide host range constitutive expression plasmid pBSPPc about height and in the application in biological degradation and biological restoration field.
Background technology
Gene expression system has vital role in biological degradation and biological restoration field, is the important tool of carrying out exogenous gene expression.Gene expression system commonly used now, such as tac promoter expression system, Tn7 promoter expression system and some light induction types or temperature-induced type promoter expression system etc., a common defective is all arranged, be that the promotor abduction delivering needs the extra inducible factor that adds, as IPTG, temperature variation, illumination variation etc.This has brought extra operation for biological degradation and biological restoration, has increased cost, and, when environmental area is used, increased the difficulty that expression system is implemented, and may bring negative influence (Di Gennaro et al., 2008 to environment; Choi et al., 2005).Thereby, be necessary to develop the new constitutive expression plasmid that is not subjected to above-mentioned restriction.
Arene compounds is the important carcinogenic substance of a class, extensively exists in environment, not only environment has been caused severe contamination, and the serious threat human health.Utilize microorganism, eliminating environmental pollution by biological degradation and biological restoration has become present important research project.Catechol claims pyrocatechol again, is the metabolic intermediate products of many arene compounds.Catechol dioxygenase can act on the metabolic intermediate product pyrocatechol of arene compounds, the ortho position cracking of its catalysis phenyl ring.This characteristic makes this enzyme be in consequence in the arene compounds metabolism, and the environmental pollution of eliminating arene compounds is had great importance.
Reference
Di?Gennaro?P,Ferrara?S,Bestetti?G,et?al.Novel?auto-inducing?expression?systems?for?the?development?of?whole-cell?biocatalysts.Appl?Microbiol?Biotechnol,2008,79:617-625.
Choi?K?H,Gaynor?J?B,White?K?G,et?al.(2005)A?Tn7-based?broad-range?bacterial?cloning?and?expression?system.Nat?Methods,2005,2:443-448.
Summary of the invention
At the defective of existing plasmid and the present situation of present stage environmental pollution, the purpose of this invention is to provide the constitutive expression plasmid of the wide host range of a kind of high startup intensity, and make it be applied in Gram-negative bacteria, express catechol dioxygenase.
The high wide host range constitutive expression of the intensity plasmid that starts of the present invention, called after pBSPPc is by replicon ori
1600, selection markers gene bla (ammonia benzyl resistant gene), multiple clone site, intergenic sequence and the high intensity constitutive promoter Pc that starts form; It is characterized in that the nucleotide sequence of described plasmid pBSPPc is shown in SEQ ID No.4; Wherein, the described high nucleotide sequence of intensity constitutive promoter Pc that starts is shown in SEQ ID No.1.
The construction process of plasmid pBSPPc of the present invention is:
Synthetic promoter Pc, 225bp altogether.
With above-mentioned synthetic Pc fragment is template, and the design primer is as follows:
Pc.f (the line part is the KpnI restriction enzyme site) GATC
GGTACCTGCCGATACAAGAACAA
Pc.r (the line part is the XhoI restriction enzyme site) GTCA
CTCGAGACGGGTTCGCTACCTGC
Prepare reaction system: ddH then
2O 34 μ l, dNTP Mixture (2.5mM each) 4 μ l, 10 * Easy Taq Buffer, 5 μ l, Sense Primer (20 μ M) 0.5 μ l, Anti Primer (20 μ M) 0.5 μ l, template DNA (synthetic promotor Pc fragment) 0.5 μ l, Easy Taq 1 μ l.
Carry out polymerase chain reaction (PCR) after system prepares, it is as follows to circulate:
94 ℃ of 4min; 94 ℃ of 30s afterwards, 55 ℃ of 30s, 72 ℃ of 30s, totally 29 circulations; Last 72 ℃ of 10min.
Use the corresponding nucleic acids restriction endonuclease that amplified production and wide host range expression plasmid pBSPIISK (nucleotide sequence is shown in SEQ ID No.3) are carried out enzyme respectively and cut, dna ligase connects, and identifies, obtains positive recombinant plasmid, called after pBSPPc; The nucleotide sequence of described plasmid pBSPPc is shown in SEQ ID No.4.
Plasmid pBSPPc of the present invention expresses the application of catechol dioxygenase in Gram-negative bacteria.Wherein, the expression of described catechol dioxygenase is to realize with the gene bphC (nucleotide sequence is shown in SEQ IDNo.2) that inserts catechol dioxygenase behind plasmid pBSPPc promotor Pc.
The enzyme that catechol dioxygenase gene bphC expresses is catechol dioxygenase BphC, the characteristic of this enzyme is, the sticking furancarboxylic acid semialdehyde of its 2-of catalytic cpd catechol generation rapidly hydroxyl, the sticking furancarboxylic acid semialdehyde of 2-hydroxyl is a kind of compound that yellow color is arranged, and catechol is colourless.In the experiment by the endonuclease enzyme cut, DNA connects, transform the equimolecular operation, and catechol dioxygenase gene bphC is inserted into promotor Pc back, then plasmid is transformed different bacterial strains.By spraying the catechol aqueous solution to the transformant flat board, the colour-change of observing transformant detects transformant easily and whether has the ability that the catalytic cpd catechol generates the sticking furancarboxylic acid semialdehyde of 2-hydroxyl, whether can constitutive expression and host's scope of application of plasmid thereby detect plasmid of the present invention, and detect other characteristic of plasmid by the active mensuration of catechol dioxygenase.
The applying step of above-mentioned plasmid pBSPPc is: extract genome from pseudomonas P.putida B6-2 (CGMCCNo.3758), as template, pcr amplification obtains gene bphC.Design has the primer of endonuclease restriction enzyme site then, is template with the bphC gene, once more amplification.With endonuclease amplified production and plasmid pBSPPc are carried out enzyme afterwards and cut, dna ligase connects.Through identifying, obtain positive recombinant plasmid pBSPPcbphC.Based on this, recombinant plasmid is transformed different hosts with natural conversion method with the electroporation conversion method, as Gram-negative bacteria.Detect by the spraying catechol aqueous solution and bacterium colony PCR, result of study shows that pBSPPc can be applicable to preferably that ((Escherichia coli Mach T1) buys from the Beijing Quanshijin Biotechnology Co., Ltd (TransGen Biotech to intestinal bacteria Mach T1, Co.)), Pseudomonas fluorescens CICC23254 ((Pseudomonas fluorescens CICC 23254) is available from Chinese industrial microbial strains preservation administrative center), Pseudomonas stutzeri SDM (Pseudomonas stutzeri SDM, the contriver is preserved in Chinese typical culture collection center, CCTCC No.M206010) and pseudomonas putida KT2440 ((Pseudomonas putida KT2440, ATCC 47054) is available from the biological product preservation of USS center) etc.
The present invention further detects the enzyme work of plasmid pBSPPcbphC expression catechol dioxygenase.At first, will be through verifying the correct transformant P.putida KT2440/pBSPPcbphC incubated overnight that makes up plasmid that contains.Then, by 1% inoculum size switching 50ml/500ml LB triangular flask, the shaking table concussion is cultivated.Cell concentration (OD is measured in sampling in per two hours
600) and the enzyme of catechol dioxygenase BphC live.
Described enzyme activity determination method is: the ultrasonic disruption bacterium, centrifugal removal cell debris is got a certain amount of crude enzyme liquid and is diluted to 3ml with APbuffer (the PB damping fluid that contains the pH 7.5 of 10% acetone), adds the catechol solution of the 0.2M of 50 μ l, the vibration mixing.Measure the concrete data (see figure 3) that enzyme is lived with the UV-2500 ultraviolet spectrophotometer then.
The present invention utilizes promotor Pc and the wide host range expression plasmid pBSPIISK (Schweizer in the bacterium III type integron, 2001) made up new expression plasmid pBSPPc, because promotor Pc has and does not need to induce (being constitutive expression), start the intensity height, fast and advantage of wide range of application (the Xu et al. of toggle speed, 2007), this promotor is inserted on the plasmid, make the plasmid that makes up have the advantage of this promotor, promptly do not need to induce, start the intensity height, toggle speed is fast and applied widely, just the good alternative system of present gene expression system.In order to detect the application potential of plasmid of the present invention in biological degradation and biological restoration field, the present invention is a template with pseudomonas (P.putida) B6-2 genome, the gene bphC of polymerase chain reaction (PCR) clone's catechol dioxygenase, by the endonuclease enzyme cut, serial molecule manipulations such as DNA connection, conversion are inserted into the back of promotor Pc among the expression plasmid pBSPPc, and the enzyme that has detected catechol dioxygenase is lived.To make up plasmid by natural conversion method and electroporation conversion method and transform different gram negative bacteriums, find that plasmid of the present invention is preferably applied to intestinal bacteria, Pseudomonas stutzeri, pseudomonas putida or Pseudomonas fluorescens.Above characteristics show that plasmid pBSPPc of the present invention has that host range is wide, constitutive expression, characteristics that starting efficiency is high, do not need to add any inductor or carry out any extra operation of inducing.The characteristics of plasmid provided by the present invention make the good alternative system of its gene induced expression system that becomes present use, have great application prospect in biological degradation and biological restoration field.
Described host is meant the bacterial cell that plasmid transforms.
Described transformant is meant that plasmid is transformed in the bacterium, and the bacterial cell of accepting plasmid is called transformant.
Described active the detection is meant to connect foreign gene in promotor Pc back, transformed host cell then, and the activity of the enzyme by detecting exogenous gene expression, whether the promotor Pc fragment that characterizes acquisition has functionally active.
Described constitutive expression is and the corresponding notion of inducible expression to be meant that gene need not to induce can realize a kind of gene expression ways of expressing.
Described inducible expression be meant that gene is not expressed under normal conditions or the expression degree is very low, but under the effect of inductor, this gene transcription and expression is activated or a kind of gene expression ways of enhanced.
Reference
Schweizer?H?P.Vectors?to?express?foreign?genes?and?techniques?to?monitor?gene?expression?in?Pseudomonas.Curr?Opin?Biotechnol,2001,12:439-445.
Xu?H,Davies?J,Miao?V.(2007)Molecular?characterization?of?class?3integrons?from?Delftia?spp.J?Bacteriol,2007,189:6276-6283.
Description of drawings
Fig. 1 plasmid pBSPPc collection of illustrative plates, Pc is high intensity constitutive expression promoter gene, the P of starting among the figure
LacBe the inducible promoter gene, bla is an ampicillin resistance gene, and rep is a replication region, ori
1600Be the replicon gene.
Fig. 2 plasmid pBSPPcbphC collection of illustrative plates, Pc is high intensity constitutive expression promoter gene, the P of starting among the figure
LacBe the inducible promoter gene, bla is an ampicillin resistance gene, and rep is a replication region, ori
1600Be the replicon gene, bphC is the catechol dioxygenase gene.
The structure of plasmid pBSPPcbphC is in order to detect the characteristic of plasmid pMMPc by gene bphC.
Fig. 3 is transformant P.putida KT2440/pBSPPcbphC cell concentration and enzyme live data curve.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to illustrate the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted actual conditions among the following embodiment, according to ordinary method, clone as the Sambrook equimolecular: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment 1
PBSPIISK (Schweizer, 2001) plasmid extracts
A small amount of extraction of plasmid uses TIANGEN TIANperp Mini Plasmid Kit plasmid corpusculum test kit (centrifugal column type) to finish.
1. column equilibration step: to adsorption column CB3 (adsorption column is put into collection tube), add the balance liquid BL of 500 μ l, 12, (13400*g) centrifugal 1min outwells the waste liquid in the collection tube to 000rpm, and adsorption column is reentered in the collection tube.
2. get the bacterium of 1-5ml incubated overnight, add in the centrifuge tube, 12, (13400*g) centrifugal 1min absorbs supernatant (bacterium liquid can be collected bacterial sediment in the centrifuge tube by repeatedly centrifugal more for a long time) to 000rpm as far as possible.
3. in the centrifuge tube that leaves bacterial sediment, add 250 μ l solution P1 (please check earlier and added RNaseA whether), use pipettor or the vortex vibrator bacterial precipitation that thoroughly suspends.
Attention: if the not bacterium piece of thorough mixing is arranged, can influence cracking, cause extracted amount and purity on the low side.
4. in centrifuge tube, add 250 μ l solution P2, leniently spin upside down and make the abundant cracking of thalline for 4-6 time.
Attention: gentle mixing, concuss not in order to avoid interrupt genomic dna, causes in the plasmid of extraction and is mixed with genomic DNA fragment.This moment the bacterium liquid limpid thickness that becomes, the used time is no more than 5min, in order to avoid plasmid is damaged.
5. add 350 μ l solution P3 in centrifuge tube, gentle immediately spins upside down 6-8 time, and fully mixing white flocks will occur at this moment.12, (13400*g) centrifugal 10min, form precipitation in the centrifuge tube bottom this moment to 000rpm.
Attention: P3 should mix after adding immediately, avoids producing localized precipitation.If also have the minute white precipitation in the supernatant, but get supernatant behind the recentrifuge.
6. carefully supernatant is poured or is moved into into (adsorption column is put into collection tube) among the adsorption column CB3, the sucking-off precipitation of noting trying not.Room temperature is placed 1-2min, and 12,000rpm (13400*g) centrifugal 30-60 second, outwell the waste liquid in the collection tube, adsorption column is relay reclaim in the collector.
7. in adsorption column CB3, add 700 μ l rinsing liquid PW (please check earlier and added dehydrated alcohol whether), 12,000rpm (13400*g) centrifugal 30-60 second, outwell the waste liquid in the collection tube, adsorption column is relay reclaim in the collector.
8. in adsorption column CB3, add 500 μ l rinsing liquid PW, 12,000rpm (13400*g) centrifugal 30-60 second, outwells the waste liquid in the collection tube.
9. adsorption column CB3 is relay and reclaim in the collector, 12, (13400*g) centrifugal 2min, purpose is that rinsing liquid remaining on the adsorption column is removed to 000rpm.
Attention: experiment that alcoholic acid is residual in the rinsing liquid can the follow-up enzyme reaction of influence (enzyme is cut, PCR etc.).For guaranteeing that the downstream experiment is not subjected to the influence of residual ethanol, adsorption column CB3 is uncapped in suggestion, places room temperature or 50 ℃ of incubators to place several minutes, thoroughly to dry rinsing liquid remaining in the sorbing material.
10. adsorption column CB3 is placed a clean centrifuge tube, to adsorption film middle part Dropwise 5 0-100 μ l elution buffer EB, room temperature is placed 1min, and 12, (13400*g) centrifugal 2min collects plasmid solution in the centrifuge tube 000rpm.
The dna gel electrophoresis detection has been extracted plasmid pBSPIISK by test kit.
Pseudomonas (Pseudomonas putida) B6-2 (contriver is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, CGMCC No:3758) genome extracts.
Wizard Genomic DNA Purification Kit is used in genomic a small amount of extraction, and (WI USA) finishes for Promega Corporation, Madison.
1. get the bacterium of 1.5ml incubated overnight, add in the centrifuge tube, 12, (13400*g) centrifugal 1min absorbs supernatant (bacterium liquid can be collected bacterial sediment in the centrifuge tube by repeatedly centrifugal more for a long time) to 000rpm as far as possible.
2. add 600 μ l nucleic acid lysate, mixings gently.
3. 80 ℃, incubation 5min is cooled to room temperature then.
4. add 3 μ l RNA enzymes, mixing, 37 ℃, incubation 15-60min is cooled to room temperature then.
5. add 200 μ l albumen extracts, the vortex mixing.Ice bath 5min.Then 13,000rpm, centrifugal 3min.
6. supernatant is transferred in the new eppendorf pipe, adds 600 μ l Virahols, mixing.13,000rpm, centrifugal 3min discards supernatant liquor.
7. 70% ethanol that in the eppendorf pipe, adds 600 μ l room temperatures, the light shaking mixing.Then 13,000rpm, centrifugal 3min.
8. abandon supernatant, room temperature is placed 10-15min, and the eppendorf pipe is dried.
9. add 100 μ l lysates and place 1h, perhaps 4 ℃ of dissolving genomic dnas that spend the night for 65 ℃.
The dna gel electrophoresis detection has been extracted the genome of pseudomonas B6-2 by test kit.
Embodiment 3
1. polymerase chain reaction (PCR) clone gene bphC
Design of primers is as follows:
BphC.f (the line part is the SmaI restriction enzyme site)
CGTA
CCCGGGTCGACGAAGGAGACAGTAATGAGCATCA
BphC.r (the line part is the XbaI enzyme cutting site)
GATCAAGCT
TCTAGATCATGCTTTGTTGCGCGCAG
1. the preparation of reaction system: ddH
2O 34 μ l, dNTP Mixture (2.5mM each) 4 μ l, 10 * Easy Taq Buffer, 5 μ l, Sense Primer (20 μ M) 0.5 μ l, Anti Primer (20 μ M) 0.5 μ l, template DNA (P.putida B6-2 genome) 0.5 μ l, EasyTaq 1 μ l.
2. polymerase chain reaction (PCR) circulation
Concrete steps are: 94 ℃ of 4min; 94 ℃ of 30s afterwards, 55 ℃ of 30s, 72 ℃ of 1min, totally 29 circulations; Last 72 ℃ of 10min.
The dna gel electrophoresis detection has amplified gene bphC.
2. polymerase chain reaction (PCR) clone gene Pc (Pc gene fragment by Beijing Liuhe Huada Genomics Technology Co., Ltd synthetic).
Design of primers is as follows:
Pc.f (the line part is the KpnI restriction enzyme site) GATC
GGTACCTGCCGATACAAGAACAA
Pc.r (the line part is the XhoI restriction enzyme site) GTCA
CTCGAGACGGGTTCGCTACCTGC
1. the preparation of reaction system: ddH
2O 34 μ l, dNTP Mixture (2.5mM each) 4 μ l, 10 * Easy Taq Buffer, 5 μ l, Sense Primer (20 μ M) 0.5 μ l, Anti Primer (20 μ M) 0.5 μ l, template DNA (the promotor Pc fragment of synthetic) 0.5 μ l, EasyTaq 1 μ l.
2. polymerase chain reaction (PCR) circulation
Concrete steps are: 94 ℃ of 4min; 94 ℃ of 30s afterwards, 55 ℃ of 30s, 72 ℃ of 30s, totally 29 circulations; Last 72 ℃ of 10min.
The dna gel electrophoresis detection has amplified gene Pc.
Embodiment 4
PBSPPc, the structure of pBSPPcbphC plasmid
1.pBSPPc the structure of plasmid
Pcr amplification Pc promotor (Pc.f (KpnI) Pc.r (XhoI)); Cut Pc promotor (the endonuclease KpnI 0.5 μ l that handles pBSPIISK (-) and pcr amplification with KpnI and XhoI enzyme then, endonuclease XhoI 0.5 μ l, 10 * Buffer, 1 μ l, DNA 8 μ l, 37 ℃, 2 hours), connect (10 * T4DNALigase Buffer, 1 μ l, T4DNALigase 1 μ l, plasmid fragment 1.5 μ l, gene bphC fragment 6.5 μ l, 16 ℃, the connection of spending the night).Transformed into escherichia coli.Transformant by the ordinary method screening has gene activity has obtained plasmid pBSPPc, and its nucleotide sequence is shown in SEQ ID No.4.
2.pBSPPcbphC the structure of plasmid
Amplification gene bphC (is template with P.putida B6-2 genome) (bphC.f (SmaI) bphC.r (XbaI)); Use SmaI and XbaI enzyme cutting PCR product and pBSPPc plasmid (endonuclease SmaI 0.5 μ l, endonuclease XbaI 0.5 μ l, 10 * Buffer, 1 μ l then, DNA 8 μ l, 37 ℃, 2 hours), connect (10 * T4DNALigase Buffer, 1 μ l, T4DNALigase 1 μ l, plasmid fragment 1.5 μ l, gene bphC fragment 6.5 μ l, 16 ℃, the connection of spending the night), transformed into escherichia coli.Transformant by the ordinary method screening has gene activity has obtained plasmid pBSPPcbphC, and its nucleotide sequence is shown in SEQ IDNo.5.
Embodiment 5
Making up the plasmid host scope of application detects
1. plasmid pBSPPc, pBSPPcbphC is by nature conversion method transformed into escherichia coli (intestinal bacteria Mach T1 ((E.coli Mach T1) buys from the Beijing Quanshijin Biotechnology Co., Ltd (TransGen Biotech, Co.))).
1. prepare competent escherichia coli cell.
2. 10 μ l plasmids are added 100 μ l competence Bacillus coli cells, mix ice bath 30min.
3. 37 ℃ of water bath heat preservation 5min.
4. ice bath 2min adds 400 μ l LB liquid nutrient mediums, cultivates 1h for 37 ℃.
5. get 100 μ l cultures, coating resistant panel, overnight incubation.
Through the ordinary method checking, obtained E.coli Mach T1/pBSPPc, E.coli Mach T1/pBSPPcbphC transformant.
2. plasmid pBSPPcbphC transforms pseudomonas (pseudomonas putida KT2440 ((P.putidaKT2440 by the electroporation conversion method, ATCC 47054) available from the biological product preservation of USS center), Pseudomonas fluorescens CICC 23254 ((P.fluorescens CICC 23254) is available from Chinese industrial microbial strains preservation administrative center), Pseudomonas stutzeri SDM (P.stutzeri SDM, contriver are preserved in Chinese typical culture collection center C CTCC No.M206010).
1. prepare the pseudomonas competence.
2. in 150 μ l competent cells, add 10 μ l (50ng/ μ l) DNA, transfer in the electric revolving cup of ice bath precooling, place 1~1.5min on ice.
3. shock by electricity: 25 μ F, 200 Ω, time constant is 4.5~5.0ms, is lower than 4.2ms, then efficient obviously descends.
4. take out cup and add the 1ml recovery media immediately after electric shock finishes, hatch 1h for 30 ℃.
5. be coated with resistant panel, 30 ℃ of incubated overnight.
Through the ordinary method checking, obtained P.putida KT2440/pBSPPcbphC, P.florescens CICC23254/pBSPPcbphC, P.stutzeri SDM/pBSPPcbphC transformant.
3. to the dull and stereotyped spraying of the transformant catechol aqueous solution, change the detection plasmid whether can the constitutive expression and its scope of application by the transformant colony colour.
Found that plasmid pBSPPc can preferably be applicable to bacterial strain intestinal bacteria, pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens.
4. bacterium colony PCR detects transformant
Picking transformant bacterium colony shakes pipe to LB, the concussion of spending the night is cultivated, and gets 1ml bacterium liquid, and is centrifugal, abandon supernatant, bacterium mud suspends with 100 μ l sterilized waters, and boiling water bath 10min is centrifugal then, with supernatant as template, carry out embodiment 3 described polymerase chain reactions, amplified the band about 800bp, prove and contain correct structure plasmid in the transformant.
Make up plasmid and induce intensity detection
Will be through verifying the correct transformant P.putida KT2440/pBSPPcbphC incubated overnight that makes up plasmid that contains.
By 1% inoculum size switching 50ml/500ml LB triangular flask, 30 ℃ of shaking table concussions are cultivated.Transformant P.putidaKT2440/pBSPPcbphC does not add any inductor and does not carry out any other the operation of inducing yet.Cell concentration (OD is measured in sampling in per two hours
600) and the enzyme of catechol dioxygenase (BphC) live.
The enzyme activity determination method: the ultrasonic disruption bacterium, centrifugal removal cell debris is got a certain amount of crude enzyme liquid and is diluted to 3ml with AP buffer (the pH 7.5PB damping fluid that contains 10% acetone), adds the catechol solution of the 0.2M of 50 μ l, the vibration mixing.Measure the concrete data (concrete data are seen Fig. 3) that enzyme is lived with the UV-2500 ultraviolet spectrophotometer then.
Claims (4)
1. the one kind high wide host's constitutive expression of startup intensity plasmid, called after pBSPPc is by replicon ori
1600, selection markers gene bla (ammonia benzyl resistant gene), multiple clone site, intergenic sequence and the high intensity constitutive promoter Pc that starts form; It is characterized in that the nucleotide sequence of described plasmid pBSPPc is shown in SEQ ID No.4; Wherein, the described high nucleotide sequence of intensity constitutive promoter Pc that starts is shown in SEQ ID No.1.
2. the pBSPPc of plasmid described in the claim 1 expresses the application of catechol dioxygenase in Gram-negative bacteria.
3. application as claimed in claim 2 is characterized in that, the expression of described catechol dioxygenase is to realize with the gene bphC that inserts catechol dioxygenase behind plasmid pBSPPc promotor Pc.
4. application as claimed in claim 2 is characterized in that, described Gram-negative bacteria is intestinal bacteria, Pseudomonas stutzeri, pseudomonas putida or Pseudomonas fluorescens.
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《Curr Opin Biotechnol》 20111231 Schweizer H P Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonas 439-445 1-9 , 第12期 2 * |
《J Bacteriol》 20071231 Xu H et al Molecular characterization of class 3 integrons from Delftia spp. 6276-6283 1-9 第189卷, 2 * |
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