CN101148679A

CN101148679A - Extraneous sources IPTG and O2 concentration simultaneously regulating and controlling expression carrier and construction method thereof

Info

Publication number: CN101148679A
Application number: CNA2007100926787A
Authority: CN
Inventors: 胡宗利; 陈绪清; 陈国平; 赵志平; 潘宇; 胡廷章
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2007-09-10
Filing date: 2007-09-10
Publication date: 2008-03-26
Anticipated expiration: 2027-09-10
Also published as: CN101148679B

Abstract

The present invention discloses one kind of expression vector controlled by both exogenous IPTG and O2 concentration and its construction process. The controlling gene LacIq and operator gene Lac0 of lac operon and the promoter of Rhodobacter sphaeroides puc operon are constructed into one powerful hetero promoter controlled by both exogenous IPTG and O2; and into the structural gene pucB, are inserted an Xa factor, a restriction endonuclease site and 10 histidine coding sequences for easy access of exogenous gene to construct pucB fusion protein. The expression vector may be induction expressed in the deletion mutant of Rhodobacter sphaeroides LH2 in a great amount, and exogenous protein may be separated and purified by means of histidine affinity chromatography. The present invention may be applied in the research on the expression of various types of photosynthetic bacteria genes, etc.

Description

A kind of while extraneous sources IPTG and O 2The expression vector and the construction process thereof of concentration regulation and control

Technical field

The present invention relates to a kind of biological expression vector, relate in particular to a kind of while extraneous sources IPTG and O ₂The expression vector and the structure thereof of concentration regulation and control.

Background technology

Photosynthetic bacterium is distributed widely in fresh water, seawater, and polar region or hot spring (comprising high hot water) and high salt in the different ecotope such as high organic content, are that a class is carried out non-oxygen-production photosynthesis, have the microorganism of complicated metabolic function.The red bacterium Rhodobactersphaeroides of class ball that the red bacterium of purple nonsulfur bacteria section belongs to is a kind of Gram-negative photosynthetic bacterium, is the important model biology that photosystem is caught in research.

When utilizing luminous energy to obtain energy, the red bacterium of class ball carries out in the process of growth metabolism, catch photosystem and brought into play important effect, at first utilize and catch photosystem 2 (Light-harvesting2 or LH2) absorption photon, be delivered to then and catch photosystem 1 (Light-harvesting1 or LH1), LH1 is encircling photosynthetic reaction center (Reaction Center or RC), LH1 directly arrives RC with the photon transfer that obtains, and energy exchange finally takes place in RC.LH2, LH1 and RC are that the red bacterium of class ball carries out photosynthetic instrument, are typical transmembrane protein.At present, people utilize technology such as X-ray diffraction, transmission electron microscope, atomic force microscope that the 26S Proteasome Structure and Function of LH2, LH1 and RC has been carried out comparatively deep research, and are especially more deep to the understanding of LH2.The red bacterium LH2 of class ball is made up of nine alpha subunits and nine beta subunits; alpha subunit and beta subunit are respectively in conjunction with the bacteriochlorophyll and the carotenoid of different numbers; the effect of bacteriochlorophyll mainly is to absorb photon, and the effect of carotenoid mainly is the destruction that the protection bacterium avoids photosynthetic oxygenation.Because the existence of bacteriochlorophyll and carotenoid, LH2 has charateristic avsorption band at 800nm and 850nm place.Wherein alpha subunit size is 5.457KDa, and the size of beta subunit is 6.809KDa, respectively by structure gene pucA in the puc operon (puc operon) and pucB coding.In addition, the structure gene pucC in the puc operon plays an important role to the LH2 that alpha subunit and beta subunit correctly are assembled into function.The expression of puc operon mainly is subjected to the influence of oxygen density and intensity of illumination.Oxygen density is high more, and expression level is low more, otherwise, then high more; Intensity of illumination is strong more, and expression level is low more, otherwise, high more; Oxygen density is better than intensity of illumination to the influence of puc operon expression level.When under suitable oxygen concentration or intensity of illumination condition, the inner membrance of photosynthetic bacterium is depression automatically, increased the endomembrane system of photosynthetic bacterium greatly, for the expression of membranin provides a large amount of spaces, in order to fill these spaces, structure gene pucB, pucA in the photosynthetic bacterium puc operon and pucC great expression, and on inner membrance, be assembled into rapidly and catch photosystem 2.Because natural puc promotor has the part background to express, and oxygen concentration is difficult to real-time monitoring in the nutrient solution, make that utilizing the puc operon to express foreign protein in the red bacterium of class ball is difficult to artificial adjustment under aerobic conditions.

At present, intestinal bacteria lactose operon regulatory gene LacIq and operator gene Lac0 have been utilized, the strong hybrid promoter and the expression vector of a series of extraneous sources IPTG regulation and control have been made up, and be widely used in escherichia expression system, produce the pharmaceutical protein or the polypeptide that in a large number human health are played an important role, for great contribution has been made in the mankind's development.The red bacterium of photosynthetic bacterium class ball is that a class extensively is present in natural important microbe, and growth is easy to cultivate rapidly, and is simple to operate.But, up to the present, also do not produced useful pharmaceutical protein or polypeptide by the human use.Its basic reason just be also not set up one can be in the red bacterium of class ball the strong hybrid promoter and the expression vector of artificial adjustment exogenous protein expression.Therefore, making up one can artificially regulate and control, and starts the strong promoter and the expression vector thereof of exogenous protein expression in the red bacterium of class ball, for foundation and the Application and Development of the red bacterial expression system of class ball provides indispensable instrument and precondition.

Summary of the invention

The present invention can utilize lactose operon regulatory gene LacIq and operator gene Lac0 in the characteristics of great expression under the conditions suitable according to the red bacterium puc of class ball operon, is built into an extraneous sources IPTG and an O simultaneously with the promotor of the red bacterium puc of class ball operon ₂The strong hybrid promoter of regulation and control, behind structure gene pucB, insert simultaneously Xa factor, restriction endonuclease sites and 10 Histidine encoding sequences, be convenient to insert foreign gene and be built into the pucB fusion rotein, can be in the red bacterium LH2 of the class ball deletion mutantion strain a large amount of abduction deliverings of this expression vector, and can be through Histidine affinity chromatography separation and purification foreign protein.This invention can be used for utilizing various photosynthetical system expression of gene in the red bacterial studies photosynthetic bacterium of class ball and the expression and the purifying of artificial adjustment foreign protein in the red bacterial expression system of class ball.

Therefore, the purpose of this invention is to provide a kind of while extraneous sources IPTG and O ₂The construction process of the expression vector of concentration regulation and control, this method mainly may further comprise the steps:

(1) pcr amplification of intestinal bacteria lactose operon regulatory gene LacIq and being connected on the expression vector pRK415;

(2) pcr amplification of the promotor of the red bacterium puc of class ball operon and the operator gene Lac0 of intestinal bacteria lactose operon reaches with the product that is connected of the middle gained of step (1) and connects;

(3) the SD sequence of the red bacterium puc of class ball operon, the pcr amplification of structure gene pucB and Xa factor reach with the product that is connected of the middle gained of step (2) and connect;

The structure gene pucA of (4) 10 Histidine encoding sequences and the red bacterium operon of class ball, the pcr amplification of pucC and terminator and with step (3) in gained be connected the product connection.

In the further scheme of the present invention, the PCR primer that adopts in the above-mentioned steps (1) is the nucleotide sequence of SEQ ID:2 and SEQ ID:3, and the nucleotides sequence of gained PCR product is classified SEQ ID NO:4 as.

The PCR primer that adopts in the step (2) is the nucleotide sequence of SEQ ID:5 and SEQ ID:6, and the nucleotides sequence of gained PCR product is classified SEQ ID NO:7 as.

The PCR primer that adopts in the step (3) is the nucleotide sequence of SEQ ID:8 and SEQ ID:9, and the nucleotides sequence of gained PCR product is classified SEQ ID NO:10 as.

The PCR primer that adopts in the step (4) is the nucleotide sequence of SEQ ID:11 and SEQ ID:12, and the nucleotides sequence of gained PCR product is classified SEQ ID NO:13 as.

The nucleotides sequence that makes up the expression vector of finishing in the step (4) is classified SEQ ID NO:14 as.

Make up the expression vector of finishing in the step (4) and have an extraneous sources IPTG and an O simultaneously ₂The strong hybrid promoter of concentration regulation and control.

In order to prove constructed while extraneous sources IPTG of the present invention and O ₂The validity of the expression vector of concentration regulation and control the invention provides this expression vector and transforms the method that host bacterium, abduction delivering and expression conditions detect, and mainly may further comprise the steps:

(a) expression vector transformed into escherichia coli S _17-1

(b) expression vector arrives the red bacterium of class ball through conjugal transfer;

(c) abduction delivering of the red bacterium pucB of class ball, pucA and pucC;

(d) detection of the red bacterium pucB of class ball, pucA and pucC expression conditions.

In the preferred technique scheme of the present invention, the recipient bacterium of adopting in the above-mentioned steps (a) is intestinal bacteria S _17-1

Adopt CaCl in the step (a) ₂Conversion method is with above-mentioned while extraneous sources IPTG and O ₂The expression vector of concentration regulation and control is transformed into above-mentioned intestinal bacteria S _17-1

The recipient bacterium of adopting in the step (b) is the red bacterium DD13 of class ball.

Adopt the conjugal transfer method with above-mentioned while extraneous sources IPTG and O in the step (b) ₂The expression vector of concentration regulation and control is from above-mentioned intestinal bacteria S _17-1Transfer to the red bacterium DD13 of above-mentioned class ball.

Adopt external source IPTG and O in the step (c) ₂Concentration is induced above-mentioned while extraneous sources IPTG and O ₂The red bacterium pucB of class ball, pucA and pucC expression of gene on the expression vector of concentration regulation and control.

Adopt spectrophotometer scanning to detect described pucB, pucA and pucC expression of gene situation in the step (d).

Advantage of the present invention is: the expression vector that makes up through above-mentioned steps has extraneous sources IPTG and O simultaneously ₂The strong hybrid promoter of concentration regulation and control, make this expression vector in the red bacterium of photosynthetic bacterium class ball, can express by artificial adjustment, foreign protein for the first time can be in the red bacterium of class ball, express by external source IPTG artificial adjustment, provide indispensable instrument for setting up the red bacterium exogenous protein expression of photosynthetic bacterium class ball system.In addition, before adding external source IPTG, the constructed expression vector of the present invention can reach no background in the red bacterium of class ball expresses, and has avoided poisonous foreign protein to the host bacterium---the mushroom influence of the red bacterium of class ball, and significant to the great expression of poisonous foreign protein.

For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.

Description of drawings

Fig. 1 is while extraneous sources IPTG of the present invention and O ₂The building process synoptic diagram of the expression vector of concentration regulation and control;

Fig. 2 is that recombinant plasmid pRK-Iq of the present invention identifies electrophorogram through the double digestion of EcoRI and XhoI;

Fig. 3 is the detection collection of illustrative plates of pucB of the present invention, pucA and pucC expression conditions;

Fig. 4 is a step synoptic diagram of the present invention.

Embodiment

Material:

1.PrimeSTAR ^TMHS archaeal dna polymerase (Takara company product, DaLian, China)

2. various restriction enzymes (Takara company product, DaLian, China)

3. connect test kit DNA Ligation Kit Ver.2.0 (Takara company product, DaLian, China)

4.DNA purification kit, centrifugal column type plain agar sugar gel DNA reclaim test kit (sky is Time Inc.'s product, the BeiJing, China)

5.DNA extract test kit Universal Genomic DNA Extraction Kit Ver.3.0 (Takara company product, DaLian, China)

6. escherichia coli jm109 competent cell (Takara company product, DaLian, China)

7. intestinal bacteria S _17-1(U.S. representative microbial DSMZ, preservation registration number is: ATCC No.47055, preservation time: 1960)

8. (DSMZ of Britain country, preservation registration number is the red bacterium RS-1 of class ball: UKNCC No.8253, preservation time: 1956)

Annotate: following reagent is the commercially available prod.

9.LB substratum

Yeast extract 5g

Tryptones 10g

NaCl 10g

Solid medium: add 2% (w/v) agar powder

Be dissolved in the 1000ml deionized water, and regulate pH value to 7.0, high pressure steam sterilization with the NaOH of 1mol/L.

10.M22+ substratum

Solid M22+: in 100ml 10 * M22 storing solution, add 900ml deionized water and 15g agar powder, mixing, high pressure steam sterilization.Make the solid medium dissolving before using, add 1000 * vitamin mixture of 1ml filtration sterilization when cooling to 60 ℃.

Wherein: the preparation of 10 * M22 storing solution

KH ₂PO ₄ 30.6g

K ₂HPO ₄.3H ₂O 30g

Sodium.alpha.-hydroxypropionate 25g

(NH4) ₂SO ₄ 5g

NaCl 5g

Sodium succinate 43.425g

Sodium Glutamate 2.7g

Aspartic acid 0.4g

Solution?C 200ml

The deionized water constant volume is regulated pH value to 6.8, high pressure steam sterilization to 1000ml with NaOH.

Wherein: the preparation of Solution C

MgCl ₂.6H ₂O 24g

CaCl ₂ 3.34g

EDTA.Na ₂.2H ₂O 0.125g

ZnCl ₂ 0.261g

FeCl ₂.4H ₂O 0.25g

MnCl ₂.4H ₂O 0.09g

(NH4) ₆Mo ₇O ₂₄·4H ₂O?0.009g

CuCl ₂.2H ₂O 0.008g

Co(NO ₃) ₂.6H ₂O 0.0124g

Boric acid 0.0057g

Nitrilotriacetic acid 10g

The deionized water constant volume is to 1000ml, and-20 ℃ of preservations need not sterilization.

Wherein: the preparation of 1000 * vitamin mixture

Nicotinic acid 1g

VitB1 0.5g

Para-amino benzoic acid 0.1g

Vitamin H 0.01g

The deionized water constant volume is to 1000ml, filtration sterilization ,-20 ℃ of preservations.

Liquid M22+: on the Bechtop to the 10 * M22 storing solution that in the 1L culturing bottle of crossing high pressure steam sterilization, adds the 100ml high pressure steam sterilization respectively, 1000 * vitamin mixture of 1ml filtration sterilization, the acid hydrolysis casein of 20ml 5% high pressure steam sterilization and 879ml sterilization deionized water, standby behind the mixing in 4 ℃ of preservations.

Wherein: the preparation of 5% acid hydrolysis casein

Take by weighing 5g acid hydrolysis casein, use the deionized water dissolving constant volume to 100ml, high pressure steam sterilization ,-20 ℃ of preservations.

Embodiment oneThe pcr amplification of intestinal bacteria lactose operon regulatory gene LacIq be connected

Operation instructions according to the DNA extraction test kit, from e. coli jm109, extract genomic dna, go up according to GenBank the sudden change promotor of intestinal bacteria lactose operon regulatory gene LacIq of report and protein coding sequence (GenBank accession number: EF189157) design one couple of PCR primers A1 and A2 (SEQID NO:2 and SEQ ID NO:3); 5 of upstream primer A1 ' end contains protection base and restriction enzyme site SacI, XhoI; 5 of downstream primer A2 ' end contains protection base and restriction enzyme site EcoRI, and primer sequence is as follows:

A1：5’-CTCTGAGCTCGAGGACACCATCG?AATGGTGC-3’(SEQ?ID?NO：2)

A2：5’-CTCTGAATTCTAATTGCGTTGCGCTCACTG-3’(SEQ?ID?NO：3)

The reaction system of PCR is 100 μ l, wherein contains each 20pmol of primer A1 and A2, the dNTP of 200 μ mol, 1 μ l genomic dna, 5 * PrimeSTAR ^TMReaction buffer 20 μ l, the PrimeSTAR of high-fidelity ^TMHSDNA polysaccharase 5U.The reaction conditions of PCR is: fs 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change of subordinate phase 30 seconds were annealed 30 seconds for 60 ℃, and 72 ℃ were extended 1 minute, and circulated 30 times; Phase III 72 ℃ of extensions 5 minutes.Gained PCR product (SEQ ID NO:4) detects visible about 1200bp size electrophoresis band through 1% agarose electrophoresis, with the recovery of PCR product, purifying.

With reference to Fig. 1, getting an amount of purified pcr product carries out the EcoRI enzyme and cuts, the enzyme system of cutting is: 2 μ l, 10 * H damping fluid, 1 μ l EcoRI enzyme liquid, an amount of PCR product, add the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification EcoRI enzyme and cut product, got an amount of purifying EcoRI enzyme then and cut product and carry out the SacI enzyme and cut, the enzyme system of cutting is: 2 μ l, 10 * L damping fluid, 1 μ l SacI enzyme liquid, an amount of EcoRI enzyme is cut product, adds the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification SacI enzyme and cut product.Simultaneously, cut system with wide host's property expression vector pRK415 plasmid DNA (SEQ ID NO:1) (pRK415 plasmid source: Keen according to above-mentioned enzyme, N.T., S.Tamaki, D.Kobayashi, and D.Trollinger.1988.Improved broad-host-range plasmids for DNAcloning in Gram-negative bacteria.Gene 70:191 197.) carrying out EcoRI and SacI substep enzyme cuts and purifying.

To be connected with LacIq PCR product with the pRK415 plasmid DNA that the SacI enzyme is cut through EcoRI in right amount according to the operation instructions that connects test kit.Connect product and pass through CaCl ₂Conversion method transforms the JM109 competent escherichia coli cell, after the screening of 10 μ g/ml tetracyclin resistances, white colony on the picking LB flat board in the LB liquid nutrient medium that contains 10 μ g/ml tsiklomitsins, 37 ℃, 250rpm, shaking culture is spent the night, extract plasmid DNA next day, carry out double digestion with EcoRI and XhoI and identify that the enzyme system of cutting is: 2 μ l, 10 * H damping fluid, 0.5 μ l EcoRI enzyme liquid, 0.5 μ l XhoI enzyme liquid, an amount of plasmid DNA adds the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, restriction enzyme mapping as shown in Figure 2, wherein the molecular weight marker thing is that (molecular weight is DL2000plus from big to small: 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp).Because the pRK415 plasmid DNA does not have the XhoI restriction enzyme site, 5 ' the end of above-mentioned PCR primer A1 has been introduced an XhoI restriction enzyme site, if above-mentioned PCR product correctly inserts the pRK415 plasmid DNA, recombinant plasmid just has an XhoI single endonuclease digestion site, can cut out the above-mentioned PCR product fragment of about 1200bp size through EcoRI and XhoI double digestion, cutting correct recombinant plasmid for enzyme checks order, sequencing result shows that the dna fragmentation that inserts in the positive recombinant plasmid multiple clone site promptly is the promotor of intestinal bacteria lactose operon regulatory gene LacIq and complete encoding sequence, this recombinant plasmid called after pRK-Iq.

Embodiment twoThe pcr amplification of the red bacterium puc of class ball operon promotor pucP and intestinal bacteria lactose operon operator gene Lac0 be connected

Utilize the DNA extraction test kit to extract the red bacterium RS-1 of class ball genomic dna, according to GenBank go up report the puc operon sequence (GenBank accession number: X68796) design one couple of PCR primers B1 and B2 (SEQ ID NO:5 and (SEQ ID NO:6); 5 ' the end of upstream primer B1 contains protection base and restriction enzyme site XhoI; 5 ' the end of downstream primer B2 contains protection base and restriction enzyme site KpnI and intestinal bacteria lactose operon operator gene Lac0, and primer sequence is as follows:

B1：5’-ATATCTCGAGGCCTCGGA?CACCCTCGTT?TTTGC-3’(SEQ?ID?NO：5)

B2：5’

-CTCTGGTACCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATGGGTGTCAC-3’(SEQID?NO：6)

The reaction system of PCR is 100 μ l, wherein contains each 20pmol of primer B1 and B2, the dNTP of 200 μ mol, 1 μ l RS-1 genomic dna, 5 * PrimeSTAR ^TMReaction buffer 20 μ l, the PrimeSTAR of high-fidelity ^TMHS archaeal dna polymerase 5U.The reaction conditions of PCR is: fs 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change of subordinate phase 30 seconds were annealed 30 seconds for 60 ℃, and 72 ℃ were extended 30 seconds, and circulated 30 times; Phase III 72 ℃ of extensions 5 minutes.Gained PCR product (SEQ ID NO:7) detects visible about 250bp size electrophoresis band through 1.5% agarose electrophoresis, with the recovery of PCR product, purifying.

With reference to Fig. 1, getting an amount of purified pcr product carries out the XhoI enzyme and cuts, the enzyme system of cutting is: 2 μ l, 10 * H damping fluid, 1 μ l XhoI enzyme liquid, an amount of PCR product, add the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification XhoI enzyme and cut product, got an amount of purifying XhoI enzyme then and cut product and carry out the KpnI enzyme and cut, the enzyme system of cutting is: 2 μ l, 10 * L damping fluid, 1 μ l KpnI enzyme liquid, an amount of XhoI enzyme is cut product, adds the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification KpnI enzyme and cut product.Simultaneously, according to the above-mentioned enzyme system of cutting the pRK-Iq plasmid DNA being carried out XhoI and KpnI substep enzyme cuts and purifying.

To be connected with the PCR product with the pRK-Iq plasmid DNA that the KpnI enzyme is cut through XhoI in right amount according to the operation instructions that connects test kit.Connecting product transforms, screens and identify positive recombinant plasmid and carry out sequencing according to method described in the embodiment 1, sequencing result shows that the dna fragmentation that inserts in the positive recombinant plasmid multiple clone site promptly is the red bacterium puc of class ball operon promotor pucP and intestinal bacteria lactose operon operator gene Lac0, this recombinant plasmid called after pRK-PO.

Embodiment threeThe red bacterium puc of class ball operon SD sequence, the pcr amplification of structure gene pucB and Xa factor be connected

According to GenBank go up report the puc operon sequence (GenBank accession number: X68796) design one couple of PCR primers C1 and C2 (SEQ ID NO:8 and SEQ ID NO:9); 5 of upstream primer C1 ' end contains protection base and restriction enzyme site KpnI; 5 of downstream primer C2 ' end contains the protection base; restriction enzyme site XbaI and SacI; and the encoding sequence of 3 ' end insertion Xa factor, primer sequence is as follows:

C1：5’-ATATGGTACCAGTTGGGAGACGACACAGTGACTG-3’(SEQ?ID?NO：8)

C2：5’-ATATTCTAGAGAGCTCCCTTCCCTCGATGCCGAGCCACGGGGTC-3’(SEQ?IDNO：9)

The reaction system of PCR is 100 μ l, wherein contains each 20pmol of primer C1 and C2, the dNTP of 200 μ mol, 1 μ l RS-1 genomic dna, 5 * PrimeSTAR ^TMReaction buffer 20 μ l, the PrimeSTAR of high-fidelity ^TMHS archaeal dna polymerase 5U.The reaction conditions of PCR is: fs 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change of subordinate phase 30 seconds were annealed 30 seconds for 60 ℃, and 72 ℃ were extended 30 seconds, and circulated 30 times; Phase III 72 ℃ of extensions 5 minutes.Gained PCR product (SEQ ID NO:10) detects visible about 200bp size electrophoresis band through 1.5% agarose electrophoresis, with the recovery of PCR product, purifying.

With reference to Fig. 1, getting an amount of purified pcr product carries out the KpnI enzyme and cuts, the enzyme system of cutting is: 2 μ l, 10 * L damping fluid, 1 μ l KpnI enzyme liquid, an amount of PCR product, add the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification KpnI enzyme and cut product, got an amount of purifying KpnI enzyme then and cut product and carry out XbaI enzyme cutting, the enzyme system of cutting is: 2 μ l, 10 * M+BSA damping fluid, 1 μ l XbaI enzyme liquid, an amount of KpnI enzyme is cut product, adds the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification XbaI enzyme cutting product.Simultaneously, according to the above-mentioned enzyme system of cutting the pRK-PO plasmid DNA being carried out KpnI and XbaI substep enzyme cuts and purifying.

According to the operation instructions that connects test kit an amount of pRK-PO plasmid DNA through KpnI and XbaI enzyme cutting is connected with the PCR product.Connecting product transforms, screens and identify positive recombinant plasmid and carry out sequencing according to method described in the embodiment one, sequencing result shows that the dna fragmentation that inserts in the positive recombinant plasmid multiple clone site promptly is the red bacterium puc of a class ball operon SD sequence, structure gene pucB and Xa factor encoding sequence, this recombinant plasmid called after pRK-BXa.

Embodiment four10 Histidine encoding sequences and the red bacterium operon of class ball structure gene pucA, the pcr amplification of pucC and terminator be connected

According to GenBank go up report the puc operon sequence (GenBank accession number: X68796) design one couple of PCR primers D1 and D2 (SEQ ID NO:11 and SEQ ID NO:12); 5 of upstream primer D1 ' end contains protection base and restriction enzyme site XbaI, and 10 Histidine encoding sequences, and 5 of downstream primer D2 ' end contains the protection base; restriction enzyme site PstI, primer sequence is as follows:

D1：5’-ATATTCTAGACACCACCACCACCACCACCACCACCACCACTAAGTCGACGGATCTACTAGTCAGGAGAAGACTGACATGAC-3’(SEQ?ID?NO：11)

D2：5’-ATATCTGCAGCAGTGGCAGGCAGGTCGCTGCCTAG-3’(SEQ?ID?NO：12)

The reaction system of PCR is 100ul, wherein contains each 20pmol of primer D1 and D2, the dNTP of 200 μ mol, 1 μ l RS-1 genomic dna, 5 * PrimeSTAR ^TMReaction buffer 20 μ l, the PrimeSTARTMHS archaeal dna polymerase 5U of high-fidelity.The reaction conditions of PCR is: fs 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change of subordinate phase 30 seconds were annealed 30 seconds for 60 ℃, and 72 ℃ were extended 2 minutes, and circulated 30 times; Phase III 72 ℃ of extensions 10 minutes.Gained PCR product (SEQ ID NO:13) detects visible about 2000bp size electrophoresis band through 1.0% agarose electrophoresis, with the recovery of PCR product, purifying.

With reference to Fig. 1, get an amount of purified pcr product and carry out XbaI enzyme cutting, the enzyme system of cutting is: 2 μ l, 10 * M+BSA damping fluid, 1 μ l XbaI enzyme liquid, an amount of PCR product, add the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification XbaI enzyme cutting product, got an amount of purifying XbaI enzyme cutting product then and carried out the PstI enzyme and cut, the enzyme system of cutting is: 2 μ l, 10 * H damping fluid, 1 μ l PstI enzyme liquid, an amount of XbaI enzyme cutting product adds the sterilization distilled water to 20 μ l, 37 ℃ of enzymes were cut 1 hour, and the DNA purification kit is crossed column purification PstI enzyme and cut product.Simultaneously, according to the above-mentioned enzyme system of cutting the pRK-BXa plasmid DNA being carried out XbaI and PstI substep enzyme cuts and purifying.

To be connected with the PCR product with the pRK-BXa plasmid DNA that the PstI enzyme is cut through XbaI in right amount according to the operation instructions that connects test kit.Connecting product transforms, screens and identify positive recombinant plasmid and carry out sequencing according to method described in the embodiment one, sequencing result shows that the dna fragmentation that inserts in the positive recombinant plasmid multiple clone site promptly is 10 Histidine encoding sequences, terminator codon, the red bacterium puc of class ball operon structure gene pucA, the terminator of pucC and puc operon, this recombinant plasmid called after pRK-CH.

Through the vector construction process of the foregoing description 1-4, be built into one and had extraneous sources IPTG and O simultaneously ₂The expression vector of the strong hybrid promoter of concentration regulation and control, this expression vector is above-mentioned pRK-CH (SEQID NO:14) just.

Embodiment fiveExpression vector transformed into escherichia coli S _17-1

(1) S _17-1The preparation of competent cell

Get the intestinal bacteria S of-80 ℃ of preservations _17-1Line on the LB flat board 37 ℃ of incubated overnight; Choose single colony inoculation in 10ml LB nutrient solution, 37 ℃, 250rpm, shaking culture is spent the night; Get the 1ml overnight culture and add in the 100ml LB nutrient solution, 37 ℃, 250rpm, shaking culture 2 hours; Place frozen water to make bacterium be cooled to 0 ℃ in 10 minutes the 100ml culture; Then in 4 ℃, 2500rpm, centrifugal 7 minutes; Abandon supernatant, with the 0.1M CaCl of 10ml ice bath precooling ₂Resuspended; 4 ℃, 2000rpm, centrifugal 5 minutes; Abandon supernatant, with the 0.1M CaCl of 2ml ice bath precooling ₂Resuspended, leave standstill in the frozen water 2 hours standby.

(2) transform

Get the S of 100 μ l ice baths _17-1Competent cell is added in the 1.5ml EP pipe of precooling, adds the expression vector pRK-CH plasmid DNA that 1 μ l builds again, mixes lightly, places 30 minutes in the frozen water; In 42 ℃ of heat shocks 90 seconds, place frozen water rapidly then, placed 2 minutes; Add 500 μ l LB nutrient solutions, 37 ℃, 150rpm, shaking culture 1 hour; Get 50 μ l bacterium liquid and evenly coat on the LB flat board that contains 10 μ g/ml tsiklomitsins, 37 ℃, be inverted overnight incubation.Next day, that normal growth is S on the tetracyclin resistance flat board _17-1Transformed bacteria.

Embodiment sixExpression vector arrives the red bacterium of class ball through conjugal transfer

According to (Jones MR such as Jones, Fowler GJ, Gibson LC, Grief GG, Olsen JD, Crielaard W, Hunter CN.Mutants of Rhodobacter sphaeroides lacking oneor more pigment-protein complexes and complementation withreaction-centre, LH1, and LH2 genes.Mol Microbiol.1992; 6 (9): 1173-1184.) reported method, will catch photosystem 1 among the red bacterium RS-1 of class ball, catch photosystem 2 and photosynthetic reaction center and knock out, obtain Rhodobacter sphaeroides mutant strain DD ₁₃When expression vector pRK-CH is transformed into Rhodobacter sphaeroides mutant strain DD ₁₃After, under external source IPTG and suitable oxygen concn condition, give expression to the alpha subunit and the beta subunit of catching photosystem 2, what correctly be assembled into function catches photosystem 2, can detect charateristic avsorption band at 800nm and 850nm place, complementary Rhodobacter sphaeroides mutant strain DD ₁₃Catch the function of photosystem 2 disappearances.

With Rhodobacter sphaeroides mutant strain DD ₁₃Line on the M22+ substratum that contains 20 μ g/ml Xin Meisus, 34 ℃, dark is inverted and was cultivated 3 days; Picking DD ₁₃Single colony inoculation contains in the M22+ nutrient solution of 20 μ g/ml Xin Meisus in 10ml, and 34 ℃, 250rpm, dark shaking culture is spent the night; Next day, 4 ℃, 6000rpm, centrifugal 10 minutes collection thalline, the resuspended thalline of 1ml LB nutrient solution; Scrape and get S _17-1Transformed bacteria list bacterium colony adds in the resuspended liquid of 100 μ l LB bacteriums, abundant mixing, drip to then on the LB substratum of thorough drying, 34 ℃, cultivated 6h-8 hour, scrape from the LB substratum and to get bacterium, the resuspended thalline of 1ml M22+ is got the resuspended bacterium liquid of 100 μ l and is coated on the M22+ substratum and (to contain tsiklomitsin: 1 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, can see red bacterium colony about dark culturing 3-4 days; Owing to contain tetracycline resistance gene, Rhodobacter sphaeroides mutant strain DD on the expression vector pRK-CH ₁₃Contain neomycin resistance gene in the genome, only contain the red bacterium of class ball of expression vector pRK-CH plasmid DNA could be in having these two kinds of antibiotic substratum normal growth, so these red bacterium colonies are the red bacterium of class ball that contains expression vector pRK-CH plasmid DNA; Picking list colony inoculation to 10ml M22+ nutrient solution (tsiklomitsin: 1 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, 250rpm, dark shaking culture is spent the night; Next day ,-80 ℃ of culture presevation, preservation bacterium called after RS-CH.

Embodiment sevenThe abduction delivering of the red bacterium pucB of class ball, pucA and pucC

Get-80 ℃ of preservation bacterium RS-CH streak culture in the M22+ substratum (tsiklomitsin: 1 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, the dark inversion cultivated 3 days; Picking list colony inoculation to 10ml M22+ nutrient solution (tsiklomitsin: 1 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, 250rpm, shaking culture is spent the night under the dark condition; Get the 1ml overnight culture be inoculated in the 200ml M22+ nutrient solution (tsiklomitsin: 1 μ g/ml, Xin Meisu: 20 μ g/ml, nutrient solution volume be container 50%), 34 ℃, 250rpm, shaking culture is 20 hours under the dark condition; Add IPTG to final concentration 1mM, 34 ℃, induced under the 150rpm, dark condition 5 hours.Simultaneously, under the same conditions, do not have IPTG inductive RS-CH bacterium, IPTG induces DD ₁₃Bacterium and do not reach under half anaerobic condition (shaking culture under the dark condition＜20 hour) and promptly add IPTG inductive RS-CH bacterium as negative control.

Embodiment eightThe detection of the red bacterium pucB of class ball, pucA and pucC expression conditions

Adopt spectrophotometer Lambda 900UV/VIS/NIR spectrometer (U.S.), with the M22+ nutrient solution is reference, inducing culture liquid at 700-900nm wavelength region interscan transformed bacteria RS-CH, charateristic avsorption band appears respectively at 800nm and 850nm, and the no charateristic avsorption band in the respective wavelength scope of three negative control nutrient solutions described in the foregoing description seven occurs, scanning result as shown in Figure 3, A does not promptly add IPTG inductive RS-CH bacterium liquid scanning result for reaching under half anaerobic condition (shaking culture under the dark condition＜20 hour) among the figure, B is not for adding IPTG inductive RS-CH bacterium liquid scanning result among the figure, (shaking culture is 20 hours under the dark condition) just add IPTG inductive RS-CH bacterium liquid scanning result to C under half anaerobic condition in order to reach among the figure, and (shaking culture is 20 hours under the dark condition) just add IPTG inductive DD to D under half anaerobic condition in order to reach among the figure ₁₃Bacterium liquid scanning result.This shows, the expression vector pRK-CH that the present invention makes up successfully changes among the red bacteria variants DD13 of class ball, and only under external source IPTG and anaerobic condition, could go out alpha and the beta subunit that photosynthetic bacterium catches photosystem 2 by abduction delivering, and correctly be assembled into function catch photosystem 2, thereby in conjunction with bacteriochlorophyll and carotenoid, occur charateristic avsorption band respectively in 800nm and 850nm place, the complementary red bacteria variants DD13 of class ball catches the function of photosystem 2 disappearances.This shows while extraneous sources IPTG and O in the expression vector that the present invention makes up ₂The hybrid promoter of concentration regulation and control, can be in the red bacterium of class ball effective, artificial startup pucB, pucA and pucC expression of gene.

The construction process of above-mentioned expression vector provided by the invention and the step synoptic diagram that proves its validity are as shown in Figure 4, experimental result of the present invention shows, the promotor of the puc operon by the red bacterium of class ball that the red bacterium of lactose operon operator gene Lac0 and purple nonsulfur bacteria section is belonged to is carried out heterozygosis and is connected into a strong hybrid promoter, and introducing lactose operon regulatory gene LacIq, the dna sequence dna of Xa factor and 10 Histidines, be built into the expression vector of regulating and control by external source IPTG and oxygen concentration simultaneously, this expression vector can be in the red bacterium of class ball the abduction delivering photosynthetic bacterium catch the alpha and the beta subunit of photosystem 2 and other foreign proteins.

Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.

＜110〉University Of Chongqing

＜120〉expression vector and the construction process thereof of the regulation and control of a kind of while extraneous sources IPTG and O2 concentration

<130>

<160>14

<170>PatentIn?version?3.2

<210>1

<211>10690

<212>DNA

＜213〉carrier pRK415 plasmid dna sequence

<400>1

ctgccatttt?tggggtgagg?ccgttcgcgg?ccgaggggcg?cagcccctgg?ggggatggga 60

ggcccgcgtt?agcgggccgg?gagggttcga?gaaggggggg?cacccccctt?cggcgtgcgc 120

ggtcacgcgc?acagggcgca?gccctggtta?aaaacaaggt?ttataaatat?tggtttaaaa 180

gcaggttaaa?agacaggtta?gcggtggccg?aaaaacgggc?ggaaaccctt?gcaaatgctg 240

gattttctgc?ctgtggacag?cccctcaaat?gtcaataggt?gcgcccctca?tctgtcagca 300

ctctgcccct?caagtgtcaa?ggatcgcgcc?cctcatctgt?cagtagtcgc?gcccctcaag 360

tgtcaatacc?gcagggcact?tatccccagg?cttgtccaca?tcatctgtgg?gaaactcgcg 420

taaaatcagg?cgttttcgcc?gatttgcgag?gctggccagc?tccacgtcgc?cggccgaaat 480

cgagcctgcc?cctcatctgt?caacgccgcg?ccgggtgagt?cggcccctca?agtgtcaacg 540

tccgcccctc?atctgtcagt?gagggccaag?ttttccgcga?ggtatccaca?acgccggcgg 600

ccgcggtgtc?tcgcacacgg?cttcgacggc?gtttctggcg?cgtttgcagg?gccatagacg 660

gccgccagcc?cagcggcgag?ggcaaccagc?ccggtgagcg?tcggaaaggc?gctcttccgc 720

ttcctcgctc?actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca 780

ctcaaaggcg?gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg 840

agcaaaaggc?cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca 900

taggctccgc?ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa 960

cccgacagga?ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc 1020

tgttccgacc?ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc 1080

gccattcgcc?attcaggctg?cgcaactgtt?gggaagggcg?atcggtgcgg?gcctcttcgc 1140

tattacgcca?gctggcgaaa?gggggatgtg?ctgcaaggcg?attaagttgg?gtaacgccag 1200

ggttttccca?gtcacgacgt?tgtaaaacga?cggccagtga?attcgagctc?ggtacccggg 1260

gatcctctag?agtcgacctg?caggcatgca?agcttggcgt?aatcatggtc?atagctgttt 1320

cctgtgtgaa?attgttatcc?gctcacaatt?ccacacaaca?tacgagccgg?aagcataaag 1380

tgtaaagcct?ggggtgccta?atgagtgagc?taactcacat?taattgcgtt?gcgctcactg 1440

cccgctttcc?agtcgggaaa?cctgtcgtgc?cagctgcatt?aatgaatcgg?ccaacgcgcg 1500

gggagaggcg?gtttgcgtat?tgggcgctcg?gtcttgcctt?gctcgtcggt?gatgtacttc 1560

accagctccg?cgaagtcgct?cttcttgatg?gagcgcatgg?ggacgtgctt?ggcaatcacg 1620

cgcacccccc?ggccgtttta?gcggctaaaa?aagtcatggc?tctgccctcg?ggcggaccac 1680

gcccatcatg?accttgccaa?gctcgtcctg?cttctcttcg?atcttcgcca?gcagggcgag 1740

gatcgtggca?tcaccgaacc?gcgccgtgcg?cgggtcgtcg?gtgagccaga?gtttcagcag 1800

gccgcccagg?cggcccaggt?cgccattgat?gcgggccagc?tcgcggacgt?gctcatagtc 1860

cacgacgccc?gtgattttgt?agccctggcc?gacggccagc?aggtaggccg?acaggctcat 1920

gccggccgcc?gccgcctttt?cctcaatcgc?tcttcgttcg?tctggaaggc?agtacacctt 1980

gataggtggg?ctgcccttcc?tggttggctt?ggtttcatca?gccatccgct?tgccctcatc 2040

tgttacgccg?gcggtagccg?gccagcctcg?cagagcagga?ttcccgttga?gcaccgccag 2100

gtgcgaataa?gggacagtga?agaaggaaca?cccgctcgcg?ggtgggccta?cttcacctat 2160

cctgcccggc?tgacgccgtt?ggatacacca?aggaaagtct?acacgaaccc?tttggcaaaa 2220

tcctgtatat?cgtgcgaaaa?aggatggata?taccgaaaaa?atcgctataa?tgaccccgaa 2280

gcagggttat?gcagcggaaa?agcgccacgc?ttcccgaagg?gagaaaggcg?gacaggtatc 2340

cggtaagcgg?cagggtcgga?acaggagagc?gcacgaggga?gcttccaggg?ggaaacgcct 2400

ggtatcttta?tagtcctgtc?gggtttcgcc?acctctgact?tgagcgtcga?tttttgtgat 2460

gctcgtcagg?ggggcggagc?ctatggaaaa?acgccagcaa?cgcggccttt?ttacggttcc 2520

tggccttttg?ctggcctttt?gctcacatgt?tctttcctgc?gttatcccct?gattctgtgg 2580

ataaccgtat?taccgccttt?gagtgagctg?ataccgctcg?ccgcagccga?acgaccgagc 2640

gcagcgagtc?agtgagcgag?gaagcggaag?agcgccagaa?ggccgccaga?gaggccgagc 2700

gcggccgtga?ggcttggacg?ctagggcagg?gcatgaaaaa?gcccgtagcg?ggctgctacg 2760

ggcgtctgac?gcggtggaaa?gggggagggg?atgttgtcta?catggctctg?ctgtagtgag 2820

tgggttgcgc?tccggcagcg?gtcctgatca?atcgtcaccc?tttctcggtc?cttcaacgtt 2880

cctgacaacg?agcctccttt?tcgccaatcc?atcgacaatc?accgcgagtc?cctgctcgaa 2940

cgctgcgtcc?ggaccggctt?cgtcgaaggc?gtctatcgcg?gcccgcaaca?gcggcgagag 3000

cggagcctgt?tcaacggtgc?cgccgcgctc?gccggcatcg?ctgtcgccgg?cctgctcctc 3060

aagcacggcc?ccaacagtga?agtagctgat?tgtcatcagc?gcattgacgg?cgtccccggc 3120

cgaaaaaccc?gcctcgcaga?ggaagcgaag?ctgcgcgtcg?gccgtttcca?tctgcggtgc 3180

gcccggtcgc?gtgccggcat?ggatgcgcgc?gccatcgcgg?taggcgagca?gcgcctgcct 3240

gaagctgcgg?gcattcccga?tcagaaatga?gcgccagtcg?tcgtcggctc?tcggcaccga 3300

atgcgtatga?ttctccgcca?gcatggcttc?ggccagtgcg?tcgagcagcg?cccgcttgtt 3360

cctgaagtgc?cagtaaagcg?ccggctgctg?aacccccaac?cgttccgcca?gtttgcgtgt 3420

cgtcagaccg?tctacgccga?cctcgttcaa?caggtccagg?gcggcacgga?tcactgtatt 3480

cggctgcaac?tttgtcatgc?ttgacacttt?atcactgata?aacataatat?gtccaccaac 3540

ttatcagtga?taaagaatcc?gcgcgttcaa?tcggaccagc?ggaggctggt?ccggaggcca 3600

gacatgaaac?ccaacatacc?cctgatcgta?attctgagca?ctgtcgcgct?cgacgctgtc 3660

ggcatcggcc?tgattatgcc?ggtgctgccg?ggcctcctgc?gcgatctggt?tcactcgaac 3720

gacgtcaccg?cccactatgg?cattctgctg?gcgctgtatg?cgttggtgca?atttgcctgc 3780

gcacctgtgc?tgggcgcgct?gtcggatcgt?ttcgggcggc?ggccaatctt?gctcgtctcg 3840

ctggccggcg?ccactgtcga?ctacgccatc?atggcgacag?cgcctttcct?ttgggttctc 3900

tatatcgggc?ggatcgtggc?cggcatcacc?ggggcgactg?gggcggtagc?cggcgcttat 3960

attgccgata?tcactgatgg?cgatgagcgc?gcgcggcact?tcggcttcat?gagcgcctgt 4020

ttcgggttcg?ggatggtcgc?gggacctgtg?ctcggtgggc?tgatgggcgg?tttctccccc 4080

cacgctccgt?tcttcgccgc?ggcagccttg?aacggcctca?atttcctgac?gggctgtttc 4140

cttttgccgg?agtcgcacaa?aggcgaacgc?cggccgttac?gccgggaggc?tctcaacccg 4200

ctcgcttcgt?tccggtgggc?ccggggcatg?accgtcgtcg?ccgccctgat?ggcggtcttc 4260

ttcatcatgc?aacttgtcgg?acaggtgccg?gccgcgcttt?gggtcatttt?cggcgaggat 4320

cgctttcact?gggacgcgac?cacgatcggc?atttcgcttg?ccgcatttgg?cattctgcat 4380

tcactcgccc?aggcaatgat?caccggccct?gtagccgccc?ggctcggcga?aaggcgggca 4440

ctcatgctcg?gaatgattgc?cgacggcaca?ggctacatcc?tgcttgcctt?cgcgacacgg 4500

ggatggatgg?cgttcccgat?catggtcctg?cttgcttcgg?gtggcatcgg?aatgccggcg 4560

ctgcaagcaa?tgttgtccag?gcaggtggat?gaggaacgtc?aggggcagct?gcaaggctca 4620

ctggcggcgc?tcaccagcct?gacctcgatc?gtcggacccc?tcctcttcac?ggcgatctat 4680

gcggcttcta?taacaacgtg?gaacgggtgg?gcatggattg?caggcgctgc?cctctacttg 4740

ctctgcctgc?cggcgctgcg?tcgcgggctt?tggagcggcg?cagggcaacg?agccgatcgc 4800

tgatcgtgga?aacgataggc?ctatgccatg?cgggtcaagg?cgacttccgg?caagctatac 4860

gcgccctagg?agtgcggttg?gaacgttggc?ccagccagat?actcccgatc?acgagcagga 4920

cgccgatgat?ttgaagcgca?ctcagcgtct?gatccaagaa?caaccatcct?agcaacacgg 4980

cggtccccgg?gctgagaaag?cccagtaagg?aaacaactgt?aggttcgagt?cgcgagatcc 5040

cccggaacca?aaggaagtag?gttaaacccg?ctccgatcag?gccgagccac?gccaggccga 5100

gaacattggt?tcctgtaggc?atcgggattg?gcggatcaaa?cactaaagct?actggaacga 5160

gcagaagtcc?tccggccgcc?agttgccagg?cggtaaaggt?gagcagaggc?acgggaggtt 5220

gccacttgcg?ggtcagcacg?gttccgaacg?ccatggaaac?cgcccccgcc?aggcccgctg 5280

cgacgccgac?aggatctagc?gctgcgtttg?gtgtcaacac?caacagcgcc?acgcccgcag 5340

ttccgcaaat?agcccccagg?accgccatca?atcgtatcgg?gctacctagc?agagcggcag 5400

agatgaacac?gaccatcagc?ggctgcacag?cgcctaccgt?cgccgcgacc?cgcccggcag 5460

gcggtagacc?gaaataaaca?acaagctcca?gaatagcgaa?atattaagtg?cgccgaggat 5520

gaagatgcgc?atccaccaga?ttcccgttgg?aatctgtcgg?acgatcatca?cgagcaataa 5580

acccgccggc?aacgcccgca?gcagcatacc?ggcgacccct?cggcctcgct?gttcgggctc 5640

cacgaaaacg?ccggacagat?gcgccttgtg?agcgtccttg?gggccgtcct?cctgtttgaa 5700

gaccgacagc?ccaatgatct?cgccgtcgat?gtaggcgccg?aatgccacgg?catctcgcaa 5760

ccgttcagcg?aacgcctcca?tgggcttttt?ctcctcgtgc?tcgtaaacgg?acccgaacat 5820

ctctggagct?ttcttcaggg?ccgacaatcg?gatctcgcgg?aaatcctgca?cgtcggccgc 5880

tccaagccgt?cgaatctgag?ccttaatcac?aattgtcaat?tttaatcctc?tgtttatcgg 5940

cagttcgtag?agcgcgccgt?gcgcccgagc?gatactgagc?gaagcaagtg?cgtcgagcag 6000

tgcccgcttg?ttcctgaaat?gccagtaaag?cgctggctgc?tgaaccccca?gccggaactg 6060

accccacaag?gccctagcgt?ttgcaatgca?ccaggtcatc?attgacccag?gcgtgttcca 6120

ccaggccgct?gcctcgcaac?tcttcgcagg?cttcgccgac?ctgctcgcgc?cacttcttca 6180

cgcgggtgga?atccgatccg?cacatgaggc?ggaaggtttc?cagcttgagc?gggtacggct 6240

cccggtgcga?gctgaaatag?tcgaacatcc?gtcgggccgt?cggcgacagc?ttgcggtact 6300

tctcccatat?gaatttcgtg?tagtggtcgc?cagcaaacag?cacgacgatt?tcctcgtcga 6360

tcaggacctg?gcaacgggac?gttttcttgc?cacggtccag?gacgcggaag?cggtgcagca 6420

gcgacaccga?ttccaggtgc?ccaacgcggt?cggacgtgaa?gcccatcgcc?gtcgcctgta 6480

ggcgcgacag?gcattcctcg?gccttcgtgt?aataccggcc?attgatcgac?cagcccaggt 6540

cctggcaaag?ctcgtagaac?gtgaaggtga?tcggctcgcc?gataggggtg?cgcttcgcgt 6600

actccaacac?ctgctgccac?accagttcgt?catcgtcggc?ccgcagctcg?acgccggtgt 6660

aggtgatctt?cacgtccttg?ttgacgtgga?aaatgacctt?gttttgcagc?gcctcgcgcg 6720

ggattttctt?gttgcgcgtg?gtgaacaggg?cagagcgggc?cgtgtcgttt?ggcatcgctc 6780

gcatcgtgtc?cggccacggc?gcaatatcga?acaaggaaag?ctgcatttcc?ttgatctgct 6840

gcttcgtgtg?tttcagcaac?gcggcctgct?tggcctcgct?gacctgtttt?gccaggtcct 6900

cgccggcggt?ttttcgcttc?ttggtcgtca?tagttcctcg?cgtgtcgatg?gtcatcgact 6960

tcgccaaacc?tgccgcctcc?tgttcgagac?gacgcgaacg?ctccacggcg?gccgatggcg 7020

cgggcagggc?agggggagcc?agttgcacgc?tgtcgcgctc?gatcttggcc?gtagcttgct 7080

ggaccatcga?gccgacggac?tggaaggttt?cgcggggcgc?acgcatgacg?gtgcggcttg 7140

cgatggtttc?ggcatcctcg?gcggaaaacc?ccgcgtcgat?cagttcttgc?ctgtatgcct 7200

tccggtcaaa?cgtccgattc?attcaccctc?cttgcgggat?tgccccgact?cacgccgggg 7260

caatgtgccc?ttattcctga?tttgacccgc?ctggtgcctt?ggtgtccaga?taatccacct 7320

tatcggcaat?gaagtcggtc?ccgtagaccg?tctggccgtc?cttctcgtac?ttggtattcc 7380

gaatcttgcc?ctgcacgaat?accagcgacc?ccttgcccaa?atacttgccg?tgggcctcgg 7440

cctgagagcc?aaaacacttg?atgcggaaga?agtcggtgcg?ctcctgcttg?tcgccggcat 7500

cgttgcgcca?ctcttcatta?accgctatat?cgaaaattgc?ttgcggcttg?ttagaattgc 7560

catgacgtac?ctcggtgtca?cgggtaagat?taccgataaa?ctggaactga?ttatggctca 7620

tatcgaaagt?ctccttgaga?aaggagactc?tagtttagct?aaacattggt?tccgctgtca 7680

agaactttag?cggctaaaat?tttgcgggcc?gcgaccaaag?gtgcgagggg?cggcttccgc 7740

tgtgtacaac?cagatatttt?tcaccaacat?ccttcgtctg?ctcgatgagc?ggggcatgac 7800

gaaacatgag?ctgtcggaga?gggcaggggt?ttcaatttcg?tttttatcag?acttaaccaa 7860

cggtaaggcc?aacccctcgt?tgaaggtgat?ggaggccatt?gccgacgccc?tggaaactcc 7920

cctacctctt?ctcctggagt?ccaccgacct?tgaccgcgag?gcactcgcgg?agattgcggg 7980

tcatcctttc?aagagcagcg?tgccgcccgg?atacgaacgc?atcagtgtgg?ttttgccgtc 8040

acataaggcg?tttatcgtaa?agaaatgggg?cgacgacacc?cgaaaaaagc?tgcgtggaag 8100

gctctgacgc?caagggttag?ggcttgcact?tccttcttta?gccgctaaaa?cggccccttc 8160

tctgcgggcc?gtcggctcgc?gcatcatatc?gacatcctca?acggaagccg?tgccgcgaat 8220

ggcatcgggc?gggtgcgctt?tgacagttgt?tttctatcag?aacccctacg?tcgtgcggtt 8280

cgattagctg?tttgtcttgc?aggctaaaca?ctttcggtat?atcgtttgcc?tgtgcgataa 8340

tgttgctaat?gatttgttgc?gtaggggtta?ctgaaaagtg?agcgggaaag?aagagtttca 8400

gaccatcaag?gagcgggcca?agcgcaagct?ggaacgcgac?atgggtgcgg?acctgttggc 8460

cgcgctcaac?gacccgaaaa?ccgttgaagt?catgctcaac?gcggacggca?aggtgtggca 8520

cgaacgcctt?ggcgagccga?tgcggtacat?ctgcgacatg?cggcccagcc?agtcgcaggc 8580

gattatagaa?acggtggccg?gattccacgg?caaagaggtc?acgcggcatt?cgcccatcct 8640

ggaaggcgag?ttccccttgg?atggcagccg?ctttgccggc?caattgccgc?cggtcgtggc 8700

cgcgccaacc?tttgcgatcc?gcaagcgcgc?ggtcgccatc?ttcacgctgg?aacagtacgt 8760

cgaggcgggc?atcatgaccc?gcgagcaata?cgaggtcatt?aaaagcgccg?tgattgatga 8820

tatagcggcc?cggctgctcc?tggttctcgc?gcaccgaaat?gggtgacttc?accccgcgct 8880

ctttgatcgt?ggcaccgatt?tccgcgatgc?tctccgggga?aaagccgggg?ttgtcggccg 8940

tccgcggctg?atgcggatct?tcgtcgatca?ggtccaggtc?cagctcgata?gggccggaac 9000

cgccctgaga?cgccgcagga?gcgtccagga?ggctcgacag?gtcgccgatg?ctatccaacc 9060

ccaggccgga?cggctgcgcc?gcgcctgcgg?cttcctgagc?ggccgcagcg?gtgtttttct 9120

tggtggtctt?ggcttgagcc?gcagtcattg?ggaaatctcc?atcttcgtga?acacgtaatc 9180

agccagggcg?cgaacctctt?tcgatgcctt?gcgcgcggcc?gttttcttga?tcttccagac 9240

cggcacaccg?gatgcgaggg?catcggcgat?gctgctgcgc?aggccaacgg?tggccggaat 9300

catcatcttg?gggtacgcgg?ccagcagctc?ggcttggtgg?cgcgcgtggc?gcggattccg 9360

cgcatcgacc?ttgctgggca?ccatgccaag?gaattgcagc?ttggcgttct?tctggcgcac 9420

gttcgcaatg?gtcgtgacca?tcttcttgat?gccctggatg?ctgtacgcct?caagctcgat 9480

gggggacagc?acatagtcgg?ccgcgaagag?ggcggccgcc?aggccgacgc?caagggtcgg 9540

ggccgtgtcg?atcaggcaca?cgtcgaagcc?ttggttcgcc?agggccttga?tgttcgcccc 9600

gaacagctcg?cgggcgtcgt?ccagcgacag?ccgttcggcg?ttcgccagta?ccgggttgga 9660

ctcgatgagg?gcgaggcgcg?cggcctggcc?gtcgccggct?gcgggtgcgg?tttcggtcca 9720

gccgccggca?gggacagcgc?cgaacagctt?gcttgcatgc?aggccggtag?caaagtcctt 9780

gagcgtgtag?gacgcattgc?cctgggggtc?caggtcgatc?acggcaaccc?gcaagccgcg 9840

ctcgaaaaag?tcgaaggcaa?gatgcacaag?ggtcgaagtc?ttgccgacgc?cgcctttctg 9900

gttggccgtg?accaaagttt?tcatcgtttg?gtttcctgtt?ttttcttggc?gtccgcttcc 9960

cacttccgga?cgatgtacgc?ctgatgttcc?ggcagaaccg?ccgttacccg?cgcgtacccc 10020

tcgggcaagt?tcttgtcctc?gaacgcggcc?cacacgcgat?gcaccgcttg?cgacactgcg 10080

cccctggtca?gtcccagcga?cgttgcgaac?gtcgcctgtg?gcttcccatc?gactaagacg 10140

ccccgcgcta?tctcgatggt?ctgctgcccc?acttccagcc?cctggatcgc?ctcctggaac 10200

tggctttcgg?taagccgttt?cttcatggat?aacacccata?atttgctccg?cgccttggtt 10260

gaacatagcg?gtgacagccg?ccagcacatg?agagaagttt?agctaaacat?ttctcgcacg 10320

tcaacacctt?tagccgctaa?aactcgtcct?tggcgtaaca?aaacaaaagc?ccggaaaccg 10380

ggctttcgtc?tcttgccgct?tatggctctg?cacccggctc?catcaccaac?aggtcgcgca 10440

cgcgcttcac?tcggttgcgg?atcgacactg?ccagcccaac?aaagccggtt?gccgccgccg 10500

ccaggatcgc?gccgatgatg?ccggccacac?cggccatcgc?ccaccaggtc?gccgccctca 10560

ctgcccggca?cctggtcgct?gaatgtcgat?gccagcacct?gcggcacgtc?aatgcttccg 10620

ggcgtcgcgc?tcgggctgat?cgcccatccc?gttactgccc?cgatcccggc?aatggcaagg 10680

actgccagcg 10690

<210>2

<211>31

<212>DNA

＜213〉according to the sudden change promotor of intestinal bacteria lactose operon regulatory gene LacIq and the upstream primer of protein coding sequence design and synthetic

<400>2

ctctgagctc?gaggacacca?tcgaatggtg?c 31

<210>3

<211>30

<212>DNA

＜213〉according to the sudden change promotor of intestinal bacteria lactose operon regulatory gene LacIq and the downstream primer of protein coding sequence design and synthetic

<400>3

ctctgaattc?taattgcgtt?gcgctcactg 30

<210>4

<211>1198

<212>DNA

＜213〉promotor of intestinal bacteria lactose operon regulatory gene LacIq and the nucleotide sequence of complete genome

<400>4

Ctctgagctc?gaggacacca?tcgaatggtg?caaaaccttt?cgcggtatgg?catgatagcg 60

Cccggaagag?agtcaattca?gggtggtgaa?tgtgaaacca?gtaacgttat?acgatgtcgc 120

agagtatgcc?ggtgtctctt?atcagaccgt?ttcccgcgtg?gtgaaccagg?ccagccacgt 180

ttctgcgaaa?acgcgggaaa?aagtggaagc?ggcgatggcg?gagctgaatt?acattcccaa 240

ccgcgtggca?caacaactgg?cgggcaaaca?gtcgttgctg?attggcgttg?ccacctccag 300

tctggccctg?cacgcgccgt?cgcaaattgt?cgcggcgatt?aaatctcgcg?ccgatcaact 360

gggtgccagc?gtggtggtgt?cgatggtaga?acgaagcggc?gtcgaagcct?gtaaagcggc 420

ggtgcacaat?cttctcgcgc?aacgcgtcag?tgggctgatc?attaactatc?cgctggatga 480

ccaggatgcc?attgctgtgg?aagctgcctg?cactaatgtt?ccggcgttat?ttcttgatgt 540

ctctgaccag?acacccatca?acagtattat?tttctcccat?gaagacggta?cgcgactggg 600

cgtggagcat?ctggtcgcat?tgggtcacca?gcaaatcgcg?ctgttagcgg?gcccattaag 660

ttctgtctcg?gcgcgtctgc?gtctggctgg?ctggcataaa?tatctcactc?gcaatcaaat 720

tcagccgata?gcggaacggg?aaggcgactg?gagtgccatg?tccggttttc?aacaaaccat 780

gcaaatgctg?aatgagggca?tcgttcccac?tgcgatgctg?gttgccaacg?atcagatggc 840

gctgggcgca?atgcgcgcca?ttaccgagtc?cgggctgcgc?gttggtgcgg?atatctcggt 900

agtgggatac?gacgataccg?aagacagctc?atgttatatc?ccgccgtcaa?ccaccatcaa 960

acaggatttt?cgcctgctgg?ggcaaaccag?cgtggaccgc?ttgctgcaac?tctctcaggg 1020

ccaggcggtg?aagggcaatc?agctgttgcc?cgtctcactg?gtgaaaagaa?aaaccaccct 1080

ggcgcccaat?acgcaaaccg?cctctccccg?cgcgttggcc?gattcattaa?tgcagctggc 1140

acgacaggtt?tcccgactgg?aaagcgggca?gtgagcgcaa?cgcaattaga?attcagag 1198

<210>5

<211>33

<212>DNA

＜213〉design the also upstream primer of synthetic according to the puc operon sequence

<400>5

atatctcgag?gcctcggaca?ccctcgtttt?tgc 33

<210>6

<211>58

<212>DNA

＜213〉design the also downstream primer of synthetic according to the puc operon sequence

<400>6

ctctggtacc?tgtgtgaaat?tgttatccgc?tcacaattcc?acacattatg?ggtgtcac 58

<210>7

<211>276

<212>DNA

＜213〉nucleotide sequence of the red bacterium puc of class ball operon promotor pucP and intestinal bacteria lactose operon operator gene Lac0

<400>7

atatctcgag?gcctcggaca?ccctcgtttt?tgcagcagcg?agaggctgcg?ggacggccct 60

gtggggccgg?gacaggcagc?gtcaatttcc?cgcgcgcctg?cggcaaaatt?gtcccttttc 120

aagccgttac?gcaggattcc?cggccgatct?ggcggccaat?aagtcgcacc?caaaacggcc 180

ttgtcagcca?acactgacat?tgaatctgtc?agcgcaatgt?gacacccata?atgtgtggaa 240

ttgtgagcgg?ataacaattt?cacacaggta?ccagag 276

<210>8

<211>34

<212>DNA

<400>8

atatggtacc?agttgggaga?cgacacagtg?actg 34

<210>9

<211>44

<212>DNA

<400>9

atattctaga?gagctccctt?ccctcgatgc?cgagccacgg?ggtc 44

<210>10

<211>208

<212>DNA

＜213〉the red bacterium puc of class ball operon SD sequence, the nucleotide sequence of structure gene pucB and Xa factor

<400>10

atatggtacc?agttgggaga?cgacacagtg?actgacgatc?tgaacaaagt?ctggccgagc 60

ggcctgaccg?ttgccgaagc?cgaagaagtt?cataagcaac?tcatcctcgg?cacccgcgtc 120

ttcggtggca?tggcgctcat?cgcgcacttc?ctcgccgccg?ctgcgacccc?gtggctcggc 180

atcgagggaa?gggagctctc?tagaatat 208

<210>11

<211>81

<212>DNA

<400>11

atattctaga?caccaccacc?accaccacca?ccaccaccac?taagtcgacg?gatctactag 60

tcaggagaag?actgacatga?c 81

<210>12

<211>35

<212>DNA

<400>12

atatctgcag?cagtggcagg?caggtcgctg?cctag 35

<210>13

<211>2283

<212>DNA

＜213〉10 Histidine encoding sequences, terminator codon, the red bacterium puc of class ball operon structure gene pucA, the nucleotide sequence of pucC and terminator

<400>13

atattctaga?caccaccacc?accaccacca?ccaccaccac?taagtcgacg?gatctactag 60

tcaggagaag?actgacatga?ccaacggcaa?aatctggctc?gtggtgaaac?cgaccgtcgg 120

cgttccgctg?ttcctcagcg?ctgccgtcat?cgcctccgtc?gttatccacg?ctgctgtgct 180

gacgaccacc?acctggctgc?ccgcctacta?ccaaggctcg?gctgcggtcg?cggccgagta 240

atgctgcgca?aggcgcgggc?ctgcgggccc?acgccagcca?gtccgtgagt?tccgagcagg 300

ccgggatcct?tgggttccgg?cctgctcgtc?acaccagcga?cgtgttcctg?tcgccttgcc 360

cgttcgcggt?gtgtgcctga?agcccgacat?cctttccagt?cgcgcagtct?gcgctacccc 420

gggattcttc?acgcatgagc?cgaattgccg?aacatctggt?ccgcatcgga?ccgcggtttc 480

tgccatttgc?cgacgcggcc?tcggaccagc?tgcccctgcg?caagctgctg?cgcctgtcgc 540

tgtttcaggt?cgccgtgggc?atggccatcg?tcctgctcgt?cggcacgctg?aaccgggtga 600

tgatcgtcga?gctgaaggtg?cccgcctcgg?tggtggggat?catgatctcg?ctgccgctcc 660

tgttcgcgcc?cttccgcgcg?ctgatcgggt?tcaagtccga?cacccatgtc?tccgcactcg 720

gctggcggcg?cgtgccctgg?atctaccgcg?ggacgctggc?gctgtggggc?ggcttcgcca 780

tcatgccctt?cgcgctgatc?gtgctcggcg?ggcagggcta?tgccgagggc?cagcccttct 840

ggctcggcgt?ctcctcggcc?gcgctcgcct?tcctgatggt?gggcggcggc?gtgcacacga 900

tccagacggt?ggggcttgcg?ctggccaccg?acctcgcccc?gcgcgaggac?cagccgaagg 960

tcgtgggcct?gatgtatgtg?gtgctgctca?tcagcatgat?cttcgcctcg?atcggcttcg 1020

gctggctgct?cgatccctat?tatgacgcgc?agctcatcaa?ggtcatctcg?ggcgtcgccg 1080

tcgccgtgtt?cttcctgaac?atgatcgccc?tgtggaagat?ggagccgcgc?aaccgcgcct 1140

tcaccgtgaa?gcccgagaag?gagcccgagt?tcggcgacca?ctggcgcgag?ttcatcagcc 1200

gcgagaacgc?gctccacggg?ctgatcgtga?tcgggctcgg?cacgctgggc?ttcggcatgg 1260

ccgacgtgat?cctcgagccc?tacggcggcg?aggtgctctc?gatgacggtg?gccgagacga 1320

cccggctgac?cgcgaccttc?gcggggggcg?ggctcgtggg?cttctggctg?gcctcgtggg 1380

tgctcggccg?gggcttcgat?cccctgcgga?tggccttcct?cggcgccgcc?gcggggctgc 1440

cgggcttctt?cgccatcatg?ggcgcgaccg?agatgacgaa?cgtctgggtc?ttcctcctcg 1500

gcacgctggt?ggtggggttc?ggcggcgggc?tcttcagcca?cgggacgctc?acggccacga 1560

tgcggctggc?gccgaaggag?caggtgggcc?ttgcgctcgg?cgcctggggt?gcggtgcagg 1620

ccacggccgc?gggcgtggcc?atcgcgggcg?caggcgtgct?gcgcgacatc?ctgcaggcga 1680

tgccggatct?ctccggctac?ggtcccggcg?cgccctacgt?cgcggtcttc?gccctcgagg 1740

caggcttcct?cttcctgacc?atgatcgtga?tcctgcctct?cttgcgctcg?gctctcgccg 1800

cccgccgcct?gtgacgaatc?accacgccct?gcgtgcccgc?ttcccggagg?tgcaaaaggc 1860

tactcctttg?ggggtctttg?aaatctcagc?ctgcaggttc?gcgccctcgg?cgcgcttcgc 1920

cgcagggttg?aaccaggaca?accctgacgg?cgagattccg?cgcacgtccc?ccactcgccg 1980

ccgaggcgaa?acccttggtt?aacacaacgt?taacaagacc?ttccctgccc?cgacttgtcc 2040

acaacctgac?catttcggca?gggcacaggg?tagcctcccc?ctttttcctg?ctataacaga 2100

cctttgcggc?atcattggct?gcatgctaaa?gtatggaaat?cgacaaaact?ttgaagagta 2160

ggatttagcc?gggtttgcgt?cccaaacaca?aagtggacag?attgtgatga?atcattcgac 2220

cgactctttt?tcgaaatctt?tgaataccct?aggcagcgac?ctgcctgcca?ctgctgcaga 2280

tat 2283

<210>14

<211>14557

<212>DNA

＜213〉the carrier pRK-CH plasmid dna sequence of the present invention's structure

<400>14

ctgccatttt?tggggtgagg?ccgttcgcgg?ccgaggggcg?cagcccctgg?ggggatggga 60

ggcccgcgtt?agcgggccgg?gagggttcga?gaaggggggg?cacccccctt?cggcgtgcgc 120

ggtcacgcgc?acagggcgca?gccctggtta?aaaacaaggt?ttataaatat?tggtttaaaa 180

gcaggttaaa?agacaggtta?gcggtggccg?aaaaacgggc?ggaaaccctt?gcaaatgctg 240

gattttctgc?ctgtggacag?cccctcaaat?gtcaataggt?gcgcccctca?tctgtcagca 300

ctctgcccct?caagtgtcaa?ggatcgcgcc?cctcatctgt?cagtagtcgc?gcccctcaag 360

tgtcaatacc?gcagggcact?tatccccagg?cttgtccaca?tcatctgtgg?gaaactcgcg 420

taaaatcagg?cgttttcgcc?gatttgcgag?gctggccagc?tccacgtcgc?cggccgaaat 480

cgagcctgcc?cctcatctgt?caacgccgcg?ccgggtgagt?cggcccctca?agtgtcaacg 540

tccgcccctc?atctgtcagt?gagggccaag?ttttccgcga?ggtatccaca?acgccggcgg 600

ccgcggtgtc?tcgcacacgg?cttcgacggc?gtttctggcg?cgtttgcagg?gccatagacg 660

gccgccagcc?cagcggcgag?ggcaaccagc?ccggtgagcg?tcggaaaggc?gctcttccgc 720

ttcctcgctc?actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca 780

ctcaaaggcg?gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg 840

agcaaaaggc?cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca 900

taggctccgc?ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa 960

cccgacagga?ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc 1020

tgttccgacc?ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc 1080

gccattcgcc?attcaggctg?cgcaactgtt?gggaagggcg?atcggtgcgg?gcctcttcgc 1140

tattacgcca?gctggcgaaa?gggggatgtg?ctgcaaggcg?attaagttgg?gtaacgccag 1200

ggttttccca?gtcacgacgt?tgtaaaacga?cggccagtga?attctaattg?cgttgcgctc 1260

actgcccgct?ttccagtcgg?gaaacctgtc?gtgccagctg?cattaatgaa?tcggccaacg 1320

cgcggggaga?ggcggtttgc?gtattgggcg?ccagggtggt?ttttcttttc?accagtgaga 1380

cgggcaacag?ctgattgccc?ttcaccgcct?ggccctgaga?gagttgcagc?aagcggtcca 1440

cgctggtttg?ccccagcagg?cgaaaatcct?gtttgatggt?ggttaacggc?gggatataac 1500

atgagctgtc?ttcggtatcg?tcgtatccca?ctaccgagat?atccgcacca?acgcgcagcc 1560

cggactcggt?aatggcgcgc?attgcgccca?gcgccatctg?atcgttggca?accagcatcg 1620

cagtgggaac?gatgccctca?ttcagcattt?gcatggtttg?ttgaaaaccg?gacatggcac 1680

tccagtcgcc?ttcccgttcc?gctatcggct?gaatttgatt?gcgagtgaga?tatttatgcc 1740

agccagccag?acgcagacgc?gccgagacag?aacttaatgg?gcccgctaac?agcgcgattt 1800

gctggtgacc?caatgcgacc?agatgctcca?cgcccagtcg?cgtaccgtct?tcatgggaga 1860

aaataatact?gttgatgggt?gtctggtcag?agacatcaag?aaataacgcc?ggaacattag 1920

tgcaggcagc?ttccacagca?atggcatcct?ggtcatccag?cggatagtta?atgatcagcc 1980

cactgacgcg?ttgcgcgaga?agattgtgca?ccgccgcttt?acaggcttcg?acgccgcttc 2040

gttctaccat?cgacaccacc?acgctggcac?ccagttgatc?ggcgcgagat?ttaatcgccg 2100

cgacaatttg?cgacggcgcg?tgcagggcca?gactggaggt?ggcaacgcca?atcagcaacg 2160

actgtttgcc?cgccagttgt?tgtgccacgc?ggttgggaat?gtaattcagc?tccgccatcg 2220

ccgcttccac?tttttcccgc?gttttcgcag?aaacgtggct?ggcctggttc?accacgcggg 2280

aaacggtctg?ataagagaca?ccggcatact?ctgcgacatc?gtataacgtt?actggtttca 2340

cattcaccac?cctgaattga?ctctcttccg?ggcgctatca?tgccataccg?cgaaaggttt 2400

tgcaccattc?gatggtgtcc?tcgaggcctc?ggacaccctc?gtttttgcag?cagcgagagg 2460

ctgcgggacg?gccctgtggg?gccgggacag?gcagcgtcaa?tttcccgcgc?gcctgcggca 2520

aaattgtccc?ttttcaagcc?gttacgcagg?attcccggcc?gatctggcgg?ccaataagtc 2580

gcacccaaaa?cggccttgtc?agccaacact?gacattgaat?ctgtcagcgc?aatgtgacac 2640

ccataatgtg?tggaattgtg?agcggataac?aatttcacac?aggtaccagt?tgggagacga 2700

cacagtgact?gacgatctga?acaaagtctg?gccgagcggc?ctgaccgttg?ccgaagccga 2760

agaagttcat?aagcaactca?tcctcggcac?ccgcgtcttc?ggtggcatgg?cgctcatcgc 2820

gcacttcctc?gccgccgctg?cgaccccgtg?gctcggcatc?gagggaaggg?agctctctag 2880

acaccaccac?caccaccacc?accaccacca?ctaagtcgac?ggatctacta?gtcaggagaa 2940

gactgacatg?accaacggca?aaatctggct?cgtggtgaaa?ccgaccgtcg?gcgttccgct 3000

gttcctcagc?gctgccgtca?tcgcctccgt?cgttatccac?gctgctgtgc?tgacgaccac 3060

cacctggctg?cccgcctact?accaaggctc?ggctgcggtc?gcggccgagt?aatgctgcgc 3120

aaggcgcggg?cctgcgggcc?cacgccagcc?agtccgtgag?ttccgagcag?gccgggatcc 3180

ttgggttccg?gcctgctcgt?cacaccagcg?acgtgttcct?gtcgccttgc?ccgttcgcgg 3240

tgtgtgcctg?aagcccgaca?tcctttccag?tcgcgcagtc?tgcgctaccc?cgggattctt 3300

cacgcatgag?ccgaattgcc?gaacatctgg?tccgcatcgg?accgcggttt?ctgccatttg 3360

ccgacgcggc?ctcggaccag?ctgcccctgc?gcaagctgct?gcgcctgtcg?ctgtttcagg 3420

tcgccgtggg?catggccatc?gtcctgctcg?tcggcacgct?gaaccgggtg?atgatcgtcg 3480

agctgaaggt?gcccgcctcg?gtggtgggga?tcatgatctc?gctgccgctc?ctgttcgcgc 3540

ccttccgcgc?gctgatcggg?ttcaagtccg?acacccatgt?ctccgcactc?ggctggcggc 3600

gcgtgccctg?gatctaccgc?gggacgctgg?cgctgtgggg?cggcttcgcc?atcatgccct 3660

tcgcgctgat?cgtgctcggc?gggcagggct?atgccgaggg?ccagcccttc?tggctcggcg 3720

tctcctcggc?cgcgctcgcc?ttcctgatgg?tgggcggcgg?cgtgcacacg?atccagacgg 3780

tggggcttgc?gctggccacc?gacctcgccc?cgcgcgagga?ccagccgaag?gtcgtgggcc 3840

tgatgtatgt?ggtgctgctc?atcagcatga?tcttcgcctc?gatcggcttc?ggctggctgc 3900

tcgatcccta?ttatgacgcg?cagctcatca?aggtcatctc?gggcgtcgcc?gtcgccgtgt 3960

tcttcctgaa?catgatcgcc?ctgtggaaga?tggagccgcg?caaccgcgcc?ttcaccgtga 4020

agcccgagaa?ggagcccgag?ttcggcgacc?actggcgcga?gttcatcagc?cgcgagaacg 4080

cgctccacgg?gctgatcgtg?atcgggctcg?gcacgctggg?cttcggcatg?gccgacgtga 4140

tcctcgagcc?ctacggcggc?gaggtgctct?cgatgacggt?ggccgagacg?acccggctga 4200

ccgcgacctt?cgcggggggc?gggctcgtgg?gcttctggct?ggcctcgtgg?gtgctcggcc 4260

ggggcttcga?tcccctgcgg?atggccttcc?tcggcgccgc?cgcggggctg?ccgggcttct 4320

tcgccatcat?gggcgcgacc?gagatgacga?acgtctgggt?cttcctcctc?ggcacgctgg 4380

tggtggggtt?cggcggcggg?ctcttcagcc?acgggacgct?cacggccacg?atgcggctgg 4440

cgccgaagga?gcaggtgggc?cttgcgctcg?gcgcctgggg?tgcggtgcag?gccacggccg 4500

cgggcgtggc?catcgcgggc?gcaggcgtgc?tgcgcgacat?cctgcaggcg?atgccggatc 4560

tctccggcta?cggtcccggc?gcgccctacg?tcgcggtctt?cgccctcgag?gcaggcttcc 4620

tcttcctgac?catgatcgtg?atcctgcctc?tcttgcgctc?ggctctcgcc?gcccgccgcc 4680

tgtgacgaat?caccacgccc?tgcgtgcccg?cttcccggag?gtgcaaaagg?ctactccttt 4740

gggggtcttt?gaaatctcag?cctgcaggtt?cgcgccctcg?gcgcgcttcg?ccgcagggtt 4800

gaaccaggac?aaccctgacg?gcgagattcc?gcgcacgtcc?cccactcgcc?gccgaggcga 4860

aacccttggt?taacacaacg?ttaacaagac?cttccctgcc?ccgacttgtc?cacaacctga 4920

ccatttcggc?agggcacagg?gtagcctccc?cctttttcct?gctataacag?acctttgcgg 4980

catcattggc?tgcatgctaa?agtatggaaa?tcgacaaaac?tttgaagagt?aggatttagc 5040

cgggtttgcg?tcccaaacac?aaagtggaca?gattgtgatg?aatcattcga?ccgactcttt 5100

ttcgaaatct?ttgaataccc?taggcagcga?cctgcctgcc?actgctgcag?gcatgcaagc 5160

ttggcgtaat?catggtcata?gctgtttcct?gtgtgaaatt?gttatccgct?cacaattcca 5220

cacaacatac?gagccggaag?cataaagtgt?aaagcctggg?gtgcctaatg?agtgagctaa 5280

ctcacattaa?ttgcgttgcg?ctcactgccc?gctttccagt?cgggaaacct?gtcgtgccag 5340

ctgcattaat?gaatcggcca?acgcgcgggg?agaggcggtt?tgcgtattgg?gcgctcggtc 5400

ttgccttgct?cgtcggtgat?gtacttcacc?agctccgcga?agtcgctctt?cttgatggag 5460

cgcatgggga?cgtgcttggc?aatcacgcgc?accccccggc?cgttttagcg?gctaaaaaag 5520

tcatggctct?gccctcgggc?ggaccacgcc?catcatgacc?ttgccaagct?cgtcctgctt 5580

ctcttcgatc?ttcgccagca?gggcgaggat?cgtggcatca?ccgaaccgcg?ccgtgcgcgg 5640

gtcgtcggtg?agccagagtt?tcagcaggcc?gcccaggcgg?cccaggtcgc?cattgatgcg 5700

ggccagctcg?cggacgtgct?catagtccac?gacgcccgtg?attttgtagc?cctggccgac 5760

ggccagcagg?taggccgaca?ggctcatgcc?ggccgccgcc?gccttttcct?caatcgctct 5820

tcgttcgtct?ggaaggcagt?acaccttgat?aggtgggctg?cccttcctgg?ttggcttggt 5880

ttcatcagcc?atccgcttgc?cctcatctgt?tacgccggcg?gtagccggcc?agcctcgcag 5940

agcaggattc?ccgttgagca?ccgccaggtg?cgaataaggg?acagtgaaga?aggaacaccc 6000

gctcgcgggt?gggcctactt?cacctatcct?gcccggctga?cgccgttgga?tacaccaagg 6060

aaagtctaca?cgaacccttt?ggcaaaatcc?tgtatatcgt?gcgaaaaagg?atggatatac 6120

cgaaaaaatc?gctataatga?ccccgaagca?gggttatgca?gcggaaaagc?gccacgcttc 6180

ccgaagggag?aaaggcggac?aggtatccgg?taagcggcag?ggtcggaaca?ggagagcgca 6240

cgagggagct?tccaggggga?aacgcctggt?atctttatag?tcctgtcggg?tttcgccacc 6300

tctgacttga?gcgtcgattt?ttgtgatgct?cgtcaggggg?gcggagccta?tggaaaaacg 6360

ccagcaacgc?ggccttttta?cggttcctgg?ccttttgctg?gccttttgct?cacatgttct 6420

ttcctgcgtt?atcccctgat?tctgtggata?accgtattac?cgcctttgag?tgagctgata 6480

ccgctcgccg?cagccgaacg?accgagcgca?gcgagtcagt?gagcgaggaa?gcggaagagc 6540

gccagaaggc?cgccagagag?gccgagcgcg?gccgtgaggc?ttggacgcta?gggcagggca 6600

tgaaaaagcc?cgtagcgggc?tgctacgggc?gtctgacgcg?gtggaaaggg?ggaggggatg 6660

ttgtctacat?ggctctgctg?tagtgagtgg?gttgcgctcc?ggcagcggtc?ctgatcaatc 6720

gtcacccttt?ctcggtcctt?caacgttcct?gacaacgagc?ctccttttcg?ccaatccatc 6780

gacaatcacc?gcgagtccct?gctcgaacgc?tgcgtccgga?ccggcttcgt?cgaaggcgtc 6840

tatcgcggcc?cgcaacagcg?gcgagagcgg?agcctgttca?acggtgccgc?cgcgctcgcc 6900

ggcatcgctg?tcgccggcct?gctcctcaag?cacggcccca?acagtgaagt?agctgattgt 6960

catcagcgca?ttgacggcgt?ccccggccga?aaaacccgcc?tcgcagagga?agcgaagctg 7020

cgcgtcggcc?gtttccatct?gcggtgcgcc?cggtcgcgtg?ccggcatgga?tgcgcgcgcc 7080

atcgcggtag?gcgagcagcg?cctgcctgaa?gctgcgggca?ttcccgatca?gaaatgagcg 7140

ccagtcgtcg?tcggctctcg?gcaccgaatg?cgtatgattc?tccgccagca?tggcttcggc 7200

cagtgcgtcg?agcagcgccc?gcttgttcct?gaagtgccag?taaagcgccg?gctgctgaac 7260

ccccaaccgt?tccgccagtt?tgcgtgtcgt?cagaccgtct?acgccgacct?cgttcaacag 7320

gtccagggcg?gcacggatca?ctgtattcgg?ctgcaacttt?gtcatgcttg?acactttatc 7380

actgataaac?ataatatgtc?caccaactta?tcagtgataa?agaatccgcg?cgttcaatcg 7440

gaccagcgga?ggctggtccg?gaggccagac?atgaaaccca?acatacccct?gatcgtaatt 7500

ctgagcactg?tcgcgctcga?cgctgtcggc?atcggcctga?ttatgccggt?gctgccgggc 7560

ctcctgcgcg?atctggttca?ctcgaacgac?gtcaccgccc?actatggcat?tctgctggcg 7620

ctgtatgcgt?tggtgcaatt?tgcctgcgca?cctgtgctgg?gcgcgctgtc?ggatcgtttc 7680

gggcggcggc?caatcttgct?cgtctcgctg?gccggcgcca?ctgtcgacta?cgccatcatg 7740

gcgacagcgc?ctttcctttg?ggttctctat?atcgggcgga?tcgtggccgg?catcaccggg 7800

gcgactgggg?cggtagccgg?cgcttatatt?gccgatatca?ctgatggcga?tgagcgcgcg 7860

cggcacttcg?gcttcatgag?cgcctgtttc?gggttcggga?tggtcgcggg?acctgtgctc 7920

ggtgggctga?tgggcggttt?ctccccccac?gctccgttct?tcgccgcggc?agccttgaac 7980

ggcctcaatt?tcctgacggg?ctgtttcctt?ttgccggagt?cgcacaaagg?cgaacgccgg 8040

ccgttacgcc?gggaggctct?caacccgctc?gcttcgttcc?ggtgggcccg?gggcatgacc 8100

gtcgtcgccg?ccctgatggc?ggtcttcttc?atcatgcaac?ttgtcggaca?ggtgccggcc 8160

gcgctttggg?tcattttcgg?cgaggatcgc?tttcactggg?acgcgaccac?gatcggcatt 8220

tcgcttgccg?catttggcat?tctgcattca?ctcgcccagg?caatgatcac?cggccctgta 8280

gccgcccggc?tcggcgaaag?gcgggcactc?atgctcggaa?tgattgccga?cggcacaggc 8340

tacatcctgc?ttgccttcgc?gacacgggga?tggatggcgt?tcccgatcat?ggtcctgctt 8400

gcttcgggtg?gcatcggaat?gccggcgctg?caagcaatgt?tgtccaggca?ggtggatgag 8460

gaacgtcagg?ggcagctgca?aggctcactg?gcggcgctca?ccagcctgac?ctcgatcgtc 8520

ggacccctcc?tcttcacggc?gatctatgcg?gcttctataa?caacgtggaa?cgggtgggca 8580

tggattgcag?gcgctgccct?ctacttgctc?tgcctgccgg?cgctgcgtcg?cgggctttgg 8640

agcggcgcag?ggcaacgagc?cgatcgctga?tcgtggaaac?gataggccta?tgccatgcgg 8700

gtcaaggcga?cttccggcaa?gctatacgcg?ccctaggagt?gcggttggaa?cgttggccca 8760

gccagatact?cccgatcacg?agcaggacgc?cgatgatttg?aagcgcactc?agcgtctgat 8820

ccaagaacaa?ccatcctagc?aacacggcgg?tccccgggct?gagaaagccc?agtaaggaaa 8880

caactgtagg?ttcgagtcgc?gagatccccc?ggaaccaaag?gaagtaggtt?aaacccgctc 8940

cgatcaggcc?gagccacgcc?aggccgagaa?cattggttcc?tgtaggcatc?gggattggcg 9000

gatcaaacac?taaagctact?ggaacgagca?gaagtcctcc?ggccgccagt?tgccaggcgg 9060

taaaggtgag?cagaggcacg?ggaggttgcc?acttgcgggt?cagcacggtt?ccgaacgcca 9120

tggaaaccgc?ccccgccagg?cccgctgcga?cgccgacagg?atctagcgct?gcgtttggtg 9180

tcaacaccaa?cagcgccacg?cccgcagttc?cgcaaatagc?ccccaggacc?gccatcaatc 9240

gtatcgggct?acctagcaga?gcggcagaga?tgaacacgac?catcagcggc?tgcacagcgc 9300

ctaccgtcgc?cgcgacccgc?ccggcaggcg?gtagaccgaa?ataaacaaca?agctccagaa 9360

tagcgaaata?ttaagtgcgc?cgaggatgaa?gatgcgcatc?caccagattc?ccgttggaat 9420

ctgtcggacg?atcatcacga?gcaataaacc?cgccggcaac?gcccgcagca?gcataccggc 9480

gacccctcgg?cctcgctgtt?cgggctccac?gaaaacgccg?gacagatgcg?ccttgtgagc 9540

gtccttgggg?ccgtcctcct?gtttgaagac?cgacagccca?atgatctcgc?cgtcgatgta 9600

ggcgccgaat?gccacggcat?ctcgcaaccg?ttcagcgaac?gcctccatgg?gctttttctc 9660

ctcgtgctcg?taaacggacc?cgaacatctc?tggagctttc?ttcagggccg?acaatcggat 9720

ctcgcggaaa?tcctgcacgt?cggccgctcc?aagccgtcga?atctgagcct?taatcacaat 9780

tgtcaatttt?aatcctctgt?ttatcggcag?ttcgtagagc?gcgccgtgcg?cccgagcgat 9840

actgagcgaa?gcaagtgcgt?cgagcagtgc?ccgcttgttc?ctgaaatgcc?agtaaagcgc 9900

tggctgctga?acccccagcc?ggaactgacc?ccacaaggcc?ctagcgtttg?caatgcacca 9960

ggtcatcatt?gacccaggcg?tgttccacca?ggccgctgcc?tcgcaactct?tcgcaggctt 10020

cgccgacctg?ctcgcgccac?ttcttcacgc?gggtggaatc?cgatccgcac?atgaggcgga 10080

aggtttccag?cttgagcggg?tacggctccc?ggtgcgagct?gaaatagtcg?aacatccgtc 10140

gggccgtcgg?cgacagcttg?cggtacttct?cccatatgaa?tttcgtgtag?tggtcgccag 10200

caaacagcac?gacgatttcc?tcgtcgatca?ggacctggca?acgggacgtt?ttcttgccac 10260

ggtccaggac?gcggaagcgg?tgcagcagcg?acaccgattc?caggtgccca?acgcggtcgg 10320

acgtgaagcc?catcgccgtc?gcctgtaggc?gcgacaggca?ttcctcggcc?ttcgtgtaat 10380

accggccatt?gatcgaccag?cccaggtcct?ggcaaagctc?gtagaacgtg?aaggtgatcg 10440

gctcgccgat?aggggtgcgc?ttcgcgtact?ccaacacctg?ctgccacacc?agttcgtcat 10500

cgtcggcccg?cagctcgacg?ccggtgtagg?tgatcttcac?gtccttgttg?acgtggaaaa 10560

tgaccttgtt?ttgcagcgcc?tcgcgcggga?ttttcttgtt?gcgcgtggtg?aacagggcag 10620

agcgggccgt?gtcgtttggc?atcgctcgca?tcgtgtccgg?ccacggcgca?atatcgaaca 10680

aggaaagctg?catttccttg?atctgctgct?tcgtgtgttt?cagcaacgcg?gcctgcttgg 10740

cctcgctgac?ctgttttgcc?aggtcctcgc?cggcggtttt?tcgcttcttg?gtcgtcatag 10800

ttcctcgcgt?gtcgatggtc?atcgacttcg?ccaaacctgc?cgcctcctgt?tcgagacgac 10860

gcgaacgctc?cacggcggcc?gatggcgcgg?gcagggcagg?gggagccagt?tgcacgctgt 10920

cgcgctcgat?cttggccgta?gcttgctgga?ccatcgagcc?gacggactgg?aaggtttcgc 10980

ggggcgcacg?catgacggtg?cggcttgcga?tggtttcggc?atcctcggcg?gaaaaccccg 11040

cgtcgatcag?ttcttgcctg?tatgccttcc?ggtcaaacgt?ccgattcatt?caccctcctt 11100

gcgggattgc?cccgactcac?gccggggcaa?tgtgccctta?ttcctgattt?gacccgcctg 11160

gtgccttggt?gtccagataa?tccaccttat?cggcaatgaa?gtcggtcccg?tagaccgtct 11220

ggccgtcctt?ctcgtacttg?gtattccgaa?tcttgccctg?cacgaatacc?agcgacccct 11280

tgcccaaata?cttgccgtgg?gcctcggcct?gagagccaaa?acacttgatg?cggaagaagt 11340

cggtgcgctc?ctgcttgtcg?ccggcatcgt?tgcgccactc?ttcattaacc?gctatatcga 11400

aaattgcttg?cggcttgtta?gaattgccat?gacgtacctc?ggtgtcacgg?gtaagattac 11460

cgataaactg?gaactgatta?tggctcatat?cgaaagtctc?cttgagaaag?gagactctag 11520

tttagctaaa?cattggttcc?gctgtcaaga?actttagcgg?ctaaaatttt?gcgggccgcg 11580

accaaaggtg?cgaggggcgg?cttccgctgt?gtacaaccag?atatttttca?ccaacatcct 11640

tcgtctgctc?gatgagcggg?gcatgacgaa?acatgagctg?tcggagaggg?caggggtttc 11700

aatttcgttt?ttatcagact?taaccaacgg?taaggccaac?ccctcgttga?aggtgatgga 11760

ggccattgcc?gacgccctgg?aaactcccct?acctcttctc?ctggagtcca?ccgaccttga 11820

ccgcgaggca?ctcgcggaga?ttgcgggtca?tcctttcaag?agcagcgtgc?cgcccggata 11880

cgaacgcatc?agtgtggttt?tgccgtcaca?taaggcgttt?atcgtaaaga?aatggggcga 11940

cgacacccga?aaaaagctgc?gtggaaggct?ctgacgccaa?gggttagggc?ttgcacttcc 12000

ttctttagcc?gctaaaacgg?ccccttctct?gcgggccgtc?ggctcgcgca?tcatatcgac 12060

atcctcaacg?gaagccgtgc?cgcgaatggc?atcgggcggg?tgcgctttga?cagttgtttt 12120

ctatcagaac?ccctacgtcg?tgcggttcga?ttagctgttt?gtcttgcagg?ctaaacactt 12180

tcggtatatc?gtttgcctgt?gcgataatgt?tgctaatgat?ttgttgcgta?ggggttactg 12240

aaaagtgagc?gggaaagaag?agtttcagac?catcaaggag?cgggccaagc?gcaagctgga 12300

acgcgacatg?ggtgcggacc?tgttggccgc?gctcaacgac?ccgaaaaccg?ttgaagtcat 12360

gctcaacgcg?gacggcaagg?tgtggcacga?acgccttggc?gagccgatgc?ggtacatctg 12420

cgacatgcgg?cccagccagt?cgcaggcgat?tatagaaacg?gtggccggat?tccacggcaa 12480

agaggtcacg?cggcattcgc?ccatcctgga?aggcgagttc?cccttggatg?gcagccgctt 12540

tgccggccaa?ttgccgccgg?tcgtggccgc?gccaaccttt?gcgatccgca?agcgcgcggt 12600

cgccatcttc?acgctggaac?agtacgtcga?ggcgggcatc?atgacccgcg?agcaatacga 12660

ggtcattaaa?agcgccgtga?ttgatgatat?agcggcccgg?ctgctcctgg?ttctcgcgca 12720

ccgaaatggg?tgacttcacc?ccgcgctctt?tgatcgtggc?accgatttcc?gcgatgctct 12780

ccggggaaaa?gccggggttg?tcggccgtcc?gcggctgatg?cggatcttcg?tcgatcaggt 12840

ccaggtccag?ctcgataggg?ccggaaccgc?cctgagacgc?cgcaggagcg?tccaggaggc 12900

tcgacaggtc?gccgatgcta?tccaacccca?ggccggacgg?ctgcgccgcg?cctgcggctt 12960

cctgagcggc?cgcagcggtg?tttttcttgg?tggtcttggc?ttgagccgca?gtcattggga 13020

aatctccatc?ttcgtgaaca?cgtaatcagc?cagggcgcga?acctctttcg?atgccttgcg 13080

cgcggccgtt?ttcttgatct?tccagaccgg?cacaccggat?gcgagggcat?cggcgatgct 13140

gctgcgcagg?ccaacggtgg?ccggaatcat?catcttgggg?tacgcggcca?gcagctcggc 13200

ttggtggcgc?gcgtggcgcg?gattccgcgc?atcgaccttg?ctgggcacca?tgccaaggaa 13260

ttgcagcttg?gcgttcttct?ggcgcacgtt?cgcaatggtc?gtgaccatct tcttgatgcc 13320

ctggatgctg?tacgcctcaa?gctcgatggg?ggacagcaca?tagtcggccg?cgaagagggc 13380

ggccgccagg?ccgacgccaa?gggtcggggc?cgtgtcgatc?aggcacacgt?cgaagccttg 13440

gttcgccagg?gccttgatgt?tcgccccgaa?cagctcgcgg?gcgtcgtcca?gcgacagccg 13500

ttcggcgttc?gccagtaccg?ggttggactc?gatgagggcg?aggcgcgcgg?cctggccgtc 13560

gccggctgcg?ggtgcggttt?cggtccagcc?gccggcaggg?acagcgccga?acagcttgct 13620

tgcatgcagg?ccggtagcaa?agtccttgag?cgtgtaggac?gcattgccct?gggggtccag 13680

gtcgatcacg?gcaacccgca?agccgcgctc?gaaaaagtcg?aaggcaagat?gcacaagggt 13740

cgaagtcttg?ccgacgccgc?ctttctggtt?ggccgtgacc?aaagttttca?tcgtttggtt 13800

tcctgttttt?tcttggcgtc?cgcttcccac?ttccggacga?tgtacgcctg?atgttccggc 13860

agaaccgccg?ttacccgcgc?gtacccctcg?ggcaagttct?tgtcctcgaa?cgcggcccac 13920

acgcgatgca?ccgcttgcga?cactgcgccc?ctggtcagtc?ccagcgacgt?tgcgaacgtc 13980

gcctgtggct?tcccatcgac?taagacgccc?cgcgctatct?cgatggtctg?ctgccccact 14040

tccagcccct?ggatcgcctc?ctggaactgg?ctttcggtaa?gccgtttctt?catggataac 14100

acccataatt?tgctccgcgc?cttggttgaa?catagcggtg?acagccgcca?gcacatgaga 14160

gaagtttagc?taaacatttc?tcgcacgtca?acacctttag?ccgctaaaac?tcgtccttgg 14220

cgtaacaaaa?caaaagcccg?gaaaccgggc?tttcgtctct?tgccgcttat?ggctctgcac 14280

ccggctccat?caccaacagg?tcgcgcacgc?gcttcactcg?gttgcggatc?gacactgcca 14340

gcccaacaaa?gccggttgcc?gccgccgcca?ggatcgcgcc?gatgatgccg?gccacaccgg 14400

ccatcgccca?ccaggtcgcc?gccctcactg?cccggcacct?ggtcgctgaa?tgtcgatgcc 14460

agcacctgcg?gcacgtcaat?gcttccgggc?gtcgcgctcg?ggctgatcgc?ccatcccgtt 14520

actgccccga?tcccggcaat?ggcaaggact?gccagcg 14557

Claims

1. simultaneously extraneous sources IPTG and O ₂The construction process of the expression vector of concentration regulation and control is characterized in that the method comprising the steps of:

2. method according to claim 1 is characterized in that the PCR primer that adopts in the step (1) is the nucleotide sequence of SEQ ID:2 and SEQ ID:3, and the nucleotides sequence of gained PCR product is classified SEQ IDN0:4 as.

3. method according to claim 1 is characterized in that the PCR primer that adopts in the step (2) is the nucleotide sequence of SEQ ID:5 and SEQ ID:6, and the nucleotides sequence of gained PCR product is classified SEQ IDNO:7 as.

4. method according to claim 1 is characterized in that the PCR primer that adopts in the step (3) is the nucleotide sequence of SEQ ID:8 and SEQ ID:9, and the nucleotides sequence of gained PCR product is classified SEQID NO:10 as.

5. method according to claim 1 is characterized in that the PCR primer that adopts in the step (4) is the nucleotide sequence of SEQ ID:11 and SEQ ID:12, and the nucleotides sequence of gained PCR product is classified SEQ ID NO:13 as.

6. the expression vector of the described method gained of claim 1 is characterized in that the nucleotides sequence of described expression vector is classified SEQ ID NO:14 as.

7. the expression vector of the described method gained of claim 1 is characterized in that described expression vector has an extraneous sources IPTG and an O simultaneously ₂The strong hybrid promoter of concentration regulation and control.

8. extraneous sources IPTG and the O described method gained of claim 1 time ₂The expression vector of concentration regulation and control, it is characterized in that described expression vector in the red bacterium of class ball the people for inducing pucB, pucA and pucC expression of gene.