CN101955966B - High starting intensity and wide host range constitutive expression plasmid pBSPPc and application thereof - Google Patents

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

High startup intensity wide host range constitutive expression plasmid pBSPPc and application thereof

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 prosthetic field.

Background technology

Gene expression system has vital role in biological degradation and biological prosthetic 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 extra interpolation inducible factor, like IPTG, temperature variation, illumination variation etc.This has brought extra operation for biological degradation and biological prosthetic, 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 does not receive above-mentioned restriction.

Arene compounds is one type of important carcinogenic substance, in environment, extensively exists, and not only environment has been caused severe contamination, and the serious threat human health.Utilize mikrobe, eliminating environmental pollution through biological degradation and biological prosthetic has become present important research project.Catechol is claimed 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 in the arene compounds metabolism, be in consequence, 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

To 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 according to the invention, called after pBSPPc is by replicon ori ₁₆₀₀, selection markers gene bla (ammonia benzyl resistant gene), MCS, intergenic sequence and the high intensity constitutive promoter Pc that starts form; It is characterized in that the nucleotide sequence of said plasmid pBSPPc is shown in SEQ ID No.4; Wherein, the said high nucleotide sequence that starts intensity constitutive promoter Pc 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 following:

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, circulate as follows:

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 said plasmid pBSPPc is shown in SEQ ID No.4.

Plasmid pBSPPc according to the invention expresses the application of catechol dioxygenase in Gram-negative bacteria.Wherein, the expression of said catechol dioxygenase is to realize with the gene bphC (nucleotide sequence is shown in SEQ IDNo.2) that behind plasmid pBSPPc promotor Pc, inserts catechol dioxygenase.

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 through 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.Through spraying the catechol aqueous solution to the transformant flat board; Observe the colour-change of transformant and come to detect easily the ability whether transformant has the sticking furancarboxylic acid semialdehyde of catalytic cpd catechol generation 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 through the active mensuration of catechol dioxygenase.

The applying step of above-mentioned plasmid pBSPPc is: from pseudomonas P.putida B6-2 (CGMCCNo.3758), extract genome, 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, like Gram-negative bacteria.Detect through 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 article preservation of USS center) etc.

The present invention further detects the enzyme work of plasmid pBSPPcbphC expression catechol dioxygenase.At first, will pass through the transformant P.putida KT2440/pBSPPcbphC incubated overnight that correct the containing of checking makes up plasmid.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.

Said 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 does not have need induce (being constitutive expression), start the intensity height, toggle speed is fast and advantage of wide range of application (Xu et al.; 2007), this promotor is inserted on the plasmid, makes the plasmid that makes up have the advantage of this promotor; Promptly need not induce, start the intensity height, toggle speed is fast and applied widely, the good alternative system of gene expression system at present just.In order to detect the application potential of plasmid of the present invention in biological degradation and biological prosthetic 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; Through 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 through 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, need not 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 prosthetic 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 at promotor Pc to connect foreign gene at the back, transformed host cell then, and the activity of the enzyme through 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 through gene bphC.

Fig. 3 is transformant P.putida KT2440/pBSPPcbphC cell concentration and enzyme live data curve.

Embodiment

Below in conjunction with concrete embodiment, further set forth the present invention.These embodiment only are used to explain the present invention, and are not used in restriction scope of the present invention.The experimental technique of unreceipted actual conditions among the following embodiment; According to ordinary method; Clone like 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 accomplish.

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 through 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. in centrifuge tube, add 350 μ l solution P3, gentle immediately spins upside down 6-8 time, and fully mixing white flocks will occur at this moment.12, (13400*g) centrifugal 10min, form deposition 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 deposition 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 deposition 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 absolute ethyl 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 does not receive 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 through test kit.

Embodiment 2

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) accomplishes 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 through 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.

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 through test kit.

Embodiment 3

1. polymerase chain reaction (PCR) clone gene bphC

Design of primers is following:

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 following:

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 (endonuclease KpnI 0.5 μ l, endonuclease XhoI 0.5 μ l, 10 * Buffer, the 1 μ l that handles pBSPIISK (-) and pcr amplification with KpnI and XhoI enzyme 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 through 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, DNA 8 μ l then; 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 through 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 through 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 cultures.

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 through the electroporation conversion method; ATCC 47054) available from the biological article 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 through the transformant colony colour.

The result finds 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, and the concussion of spending the night is cultivated, and gets 1ml bacterium liquid; Centrifugal, abandon supernatant, bacterium mud suspends with 100 μ l sterilized waters; Boiling water bath 10min, 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.

Embodiment 6

Make up plasmid and induce intensity detection

To pass through the transformant P.putida KT2440/pBSPPcbphC incubated overnight that correct the containing of checking makes up plasmid.

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 (3)

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 ammonia benzyl resistant gene, MCS, intergenic sequence and the high intensity constitutive promoter Pc that starts form; It is characterized in that the nucleotide sequence of said plasmid pBSPPc is shown in SEQ ID No.4; Wherein, the said high nucleotide sequence that starts intensity constitutive promoter Pc is shown in SEQ ID No.1.

2. the pBSPPc of plasmid described in the claim 1 expresses the application of catechol dioxygenase in intestinal bacteria, Pseudomonas stutzeri, pseudomonas putida or Pseudomonas fluorescens.

3. application as claimed in claim 2; It is characterized in that; The expression of said catechol dioxygenase is to realize that with the gene bphC that behind plasmid pBSPPc promotor Pc, inserts catechol dioxygenase the gene bphC nucleotide sequence of wherein said catechol dioxygenase is shown in SEQ ID No.2.

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