CN101955965A

CN101955965A - Constitutive transposition expression plasmid pUCTn7Pc with high start strength and wide host range and application thereof

Info

Publication number: CN101955965A
Application number: CN 201010277553
Authority: CN
Inventors: 许平; 徐友强; 陶飞; 马翠卿
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2010-09-09
Filing date: 2010-09-09
Publication date: 2011-01-26
Anticipated expiration: 2030-09-09
Also published as: CN101955965B

Abstract

The invention discloses a constitutive transposition expression plasmid pUCTn7Pc with high start strength, high start speed and wide host range and application of the constitutive transposition expression plasmid pUCTn7Pc for expressing catechol dioxygenase in Gram negative bacteria. The plasmid of the invention can constitutively start the expression of an exogenous gene without adding any inducer or carrying out any additional induction operation and has the advantages of high start strength and high inducing speed and the characteristics of transposition; and proved by the tests, the plasmid has great application potentials in the fields of biodegradation and bioremediation.

Description

High startup intensity wide host range composing type swivel base expression plasmid pUCTn7Pc and application thereof

Technical field

What the present invention relates to is genetically engineered field expression plasmid and application, especially about the swivel base plasmid pUCTn7Pc of constitutive expression 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.(2008)Novel?auto-inducing?expression?systems?for?the?development?of?whole-cell?biocatalysts.Appl?Microbiol?Biotechnol?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?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 composing type of the intensity swivel base expression plasmid that starts of the present invention, called after pUCTn7Pc is made up of oriT, selection markers gene bla (ampicillin resistance gene), selection markers gene Gm (gentamicin resistant gene), multiple clone site, intergenic sequence and the high intensity constitutive promoter Pc that starts; It is characterized in that the nucleotide sequence of described plasmid pUCTn7Pc 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 pUCTn7Pc 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.

Using the corresponding nucleic acids restriction endonuclease that amplified production and wide host range swivel base type expression plasmid pUC18miniTn7T Gm (nucleotide sequence is shown in SEQ ID No.3) are carried out enzyme respectively cuts, dna ligase connects, identify, obtain positive recombinant plasmid, called after pUCTn7Pc; The nucleotide sequence of described plasmid pUCTn7Pc is shown in SEQ ID No.4.

Plasmid pUCTn7Pc 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 ID No.2) that inserts catechol dioxygenase behind plasmid pUCTn7Pc 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, whether 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, can constitutive expression and host's scope of application of plasmid thereby detect plasmid of the present invention.

The applying step of above-mentioned plasmid pUCTn7Pc is: extract genome from pseudomonas P.putida B6-2 (CGMCC No.3758), as template, pcr amplification obtains gene bphC, design the primer that has different nucleic acid endonuclease digestions site then respectively, with the amplified production is that template increases again, with the corresponding nucleic acids restriction endonuclease amplified production and plasmid pUCTn7Pc are carried out enzyme respectively then and cut, dna ligase connects.Through identifying, obtain positive recombinant plasmid pUCTn7PcbphC.Based on this, recombinant plasmid is transformed different hosts by the nature conversion method, as Gram-negative bacteria.Detect by the spraying catechol aqueous solution (seeing Fig. 4 and Fig. 6) and bacterium colony PCR (seeing Fig. 3 and Fig. 5).Result of study shows that pUCTn7Pc preferably is applicable to Gram-negative bacteria Pseudomonas fluorescens CICC 23254 ((Pseudomonas fluorescens CICC 23254), available from Chinese industrial microbial strains preservation administrative center) or Klebsiella pneumonia (Klebsiella pneumoniae, CCTCC M208097) etc.

The present invention utilizes promotor Pc and the wide host range swivel base type expression plasmid pUC18miniTn7T Gm in the bacterium III type integron to make up new expression plasmid pUCTn7Pc, 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 back that the endonuclease enzyme is cut, serial molecule manipulation such as DNA connection, conversion is inserted into promotor Pc among the expression plasmid pUCTn7Pc, and, detected the enzyme of catechol dioxygenase and lived by the spraying catechol aqueous solution.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 can be preferably applied to intestinal bacteria, Pseudomonas fluorescens or Klebsiella pneumonia.Above characteristics show that plasmid pUCTn7Pc 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.Because plasmid pUC18 miniTn7TGm is a swivel base type plasmid, thereby can be incorporated on host's the karyomit(e) makes the plasmid pUCTn7Pc of structure have the characteristic of swivel base, the gene of plasmid swivel base is not easy to lose, and avoided the adding of artificial selective pressure, from the environmental pollution treatment angle, reasonable more economically.The characteristics of the constructed plasmid that obtains of 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

Choi?KH，Gaynor?JB，White?KG，et?al.A?Tn7-based?broad-host-range?bacterial?cloning?and?expression?system.Nature?Mathods，2005，2(6)：443-448.

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 pUCTn7Pc collection of illustrative plates, oriT is a replicon among the figure, bla is an ampicillin resistance gene, Gm is the gentamicin resistant gene, Tn7L and Tn7R are transposon gene, and Terminator 1 and 2 is that two genes stop the zone, and Pc is the high intensity groups moulding expression promoter gene that starts.

Fig. 2 plasmid pUCTn7PcbphC collection of illustrative plates, oriT is a replicon among the figure, bla is an ampicillin resistance gene, Gm is the gentamicin resistant gene, Tn7L and Tn7R are transposon gene, Terminator 1 and 2 is that two genes stop the zone, and Pc is the high intensity groups moulding expression promoter gene that starts, and bphC is the gene of catechol dioxygenase.

The structure of plasmid pUCTn7PcbphC is in order to detect the characteristic of plasmid pMMPc by gene bphC.

Fig. 3 K.pneumoniae/pUCTn7PcbphC bacterium colony PCR qualification result

Fig. 3 transforms Klebsiella pneumonia transformant bacterium colony PCR result for the pUCTn7PcbphC plasmid.1-9 is nine transformants ,+,-be respectively positive and negative contrast, as can be seen from the figure, 7,8,9 bands (band of bphC gene) that amplified about 800bp may be correct transformant, 1-6 does not amplify the band of 800bp, should be the false positive transformant.On this basis, by the transformant line, spraying catechol solution (0.2M) sees whether bacterium colony produces color reaction and further transformant is judged.

Fig. 4 K.pneumoniae/pUCTn7PcbphC transformant spraying catechol aqueous solution result

The left side flat board is a blank among the figure, and the right side flat board is the K.pneumoniae/pUCTn7PcbphC transformant.Result from figure contains the transformant that transforms plasmid and show glassy yellow, and the blank of bacterial strain does not develop the color, and illustrates that this plasmid can be applied to host's Klebsiella pneumonia as can be seen.

Fig. 5 P.fluorescens/pUCTn7PcbphC bacterium colony PCR qualification result

Be the result of P.fluorescens/pUCTn7PcbphC bacterium colony PCR among the figure, the band (band of bphC gene) that three transformants have all amplified about 800bp may be correct transformant.On this basis, by the transformant line, spraying catechol solution (0.2M) sees whether bacterium colony produces color reaction and further transformant is judged.

Fig. 6 P.fluorescens/pUCTn7PcbphC transformant spraying catechol aqueous solution result

The left side flat board is a blank among the figure, and the right side flat board is the P.fluorescens/pUCTn7PcbphC transformant.Result from figure contains the transformant that transforms plasmid and show glassy yellow, and the blank of bacterial strain does not develop the color, and illustrates that this plasmid can be applied to the host Pseudomonas fluorescens as can be seen.

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

(come from GenBank, numbering: AY737004) plasmid extracts pUC18miniTn7T Gm

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 pUC18 miniTn7T Gm by test kit.

Embodiment 2

Pseudomonas (Pseudomonas putida) B6-2 (contriver is preserved in the common micro-organisms center C GMCC No:3758 of China Committee for Culture Collection of Microorganisms) 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 pseudomonas (P.putida) B6-2 genome 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

Design of primers is as follows:

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 (synthetic promotor Pc fragment) 0.5 μ l, Easy Taq 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

PUCTn7Pc, the structure of pUCTn7PcbphC plasmid

1.pUCTn7Pc 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 pUC18minTn7T Gm and pcr amplification with KpnI and XhoI enzyme, 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 screens transformant with colony polymerase chain reaction (PCR) method.Obtained plasmid pUCTn7Pc by screening, its nucleotide sequence is shown in SEQ ID No.4.

2.pUCTn7PcbphC the structure of plasmid

Amplification gene bphC (P.putida B6-2 genome is a template) (bphC.f (SmaI) bphC.r (XbaI)); SmaI and XbaI enzyme cutting PCR product and pUCTn7Pc plasmid (endonuclease SmaI 0.5 μ l, endonuclease XbaI 0.5 μ l, 10 * Buffer, 1 μ l, DNA 8 μ l, 37 ℃, 2 hours), the T4DNA ligase enzyme connects 10 * T4DNA Ligase Buffer1 μ l then, T4DNALigase 1 μ l, plasmid fragment 1.5 μ l, gene bphC fragment 6.5 μ l, 16 ℃, the connection of spending the night), the transformant of transformed into escherichia coli screening with gene activity.Transformant by the ordinary method screening has gene activity has obtained plasmid pUCTn7PcbphC, and its nucleotide sequence is shown in SEQ ID No.5.

Embodiment 5

Making up the plasmid host scope of application detects

1. plasmid pUCTn7Pc, (((E.coli Mach T1) buys from the Beijing Quanshijin Biotechnology Co., Ltd (TransGen Biotech, Co.)) intestinal bacteria Mach T1 pUCTn7PcbphC by nature conversion method transformed into escherichia coli.

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/pUCTn7Pc, E.coli Mach T1/pUCTn7PcbphC transformant.

2. plasmid pUCTn7PcbphC transforms pseudomonas (Pseudomonas fluorescens CICC 23254 ((P.fluorescens CICC 23254) is available from Chinese industrial microbial strains preservation administrative center)) by electrotransformation.

1. the competent preparation of pseudomonas.

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.fluorescens CICC 23254/pUCTn7PcbphC transformant.

3. plasmid pUCTn7PcbphC transforms Klebsiella pneumonia (contriver is preserved in Chinese typical culture collection center C CTCC M 208097) by electrotransformation.

1. the competent preparation of Klebsiella pneumonia.

2. in 50 μ l competent cells, add 1-2 μ l (50ng/ μ l) DNA, transfer in the electric revolving cup of ice bath precooling.

3. shock by electricity: 25 μ F, 200 Ω, 1.8kV.

4. take out cup and add 1ml LB substratum immediately after electric shock finishes, hatch 2h for 37 ℃.

5. be coated with resistant panel, 37 ℃ of incubated overnight.

Through the ordinary method checking, obtained K.pneumonia CCTCC M 208097/pUCTn7PcbphC transformant.

4. 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 pUCTn7Pc can preferably be applicable to bacterial strain Pseudomonas fluorescens or Klebsiella pneumonia (seeing Fig. 4 and 6).

5. 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 (seeing Fig. 3 and 5) in the transformant.

Claims

1. the one kind high wide host's composing type of startup intensity swivel base expression plasmid, called after pUCTn7Pc is made up of replicon oriT, selection markers gene bla (ampicillin resistance gene), selection markers gene Gm (gentamicin resistant gene), multiple clone site, intergenic sequence and the high intensity constitutive promoter Pc that starts; It is characterized in that the nucleotide sequence of described plasmid pUCTn7Pc is shown in SEQ ID No.4; Wherein, the described high nucleotide sequence of intensity constitutive promoter Pc that starts is shown in SEQID No.1.

2. the pUCTn7Pc 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 pUCTn7Pc promotor Pc.

4. application as claimed in claim 2 is characterized in that, described Gram-negative bacteria is Pseudomonas fluorescens or Klebsiella pneumonia.