CN101386828B

CN101386828B - Reporting bacterial strain sensitive to oxidation-reduction cycle reactant and preparation method thereof

Info

Publication number: CN101386828B
Application number: CN2008101331843A
Authority: CN
Inventors: 吕建新; 王茂峰; 童贞珍
Original assignee: Wenzhou Medical College
Current assignee: Wenzhou Medical College
Priority date: 2008-07-09
Filing date: 2008-07-09
Publication date: 2010-10-06
Anticipated expiration: 2028-07-09
Also published as: CN101386828A

Abstract

The invention relates to an Escherichia coli report bacterial strain and a preparation method thereof, in particular to a report bacterial strain with high sensitivity on an oxidation reduction cycle reactant and a preparation method thereof. The Escherichia coli report bacterial strain is prepared by deleting five genes, namely soda, sodB, katG, ahpCF and frdABCD, of wild Escherichia coli E.coliMC4100 and replacing an SoxS encoding gene with GFP by the gene substitution technology. The Escherichia coli report bacterial strain is named as E.coli WMC-002, and is preserved in the China GeneralMicrobiological Culture Collection Center, with a preserving number of CGMCC No.2464. The Escherichia coli report strain can solve the problem of the limitation of detection of the oxidation reduction cycle reactant by the growth curve method, the end-point method and the MIC metho, simultaneously can overcome the defects of high background of a report signal of the report bacterial strain provided with a report vector, relative difficulty in precise quantification, poor stability and so on, and can be used for biological detection of the oxidation reduction cycle reactant.

Description

A kind of reporting bacterial strain to the oxidation-reduction cycle reactant sensitivity and preparation method thereof

Technical field

The present invention relates to a kind of molecular biology method that passes through, utilize the bacterial isolates of metabolic engineering, be specifically related to a kind of intestinal bacteria reporting bacterial strain, preparation method and the application aspect the detection oxidation-reduction cycle reactant thereof the high sensitivity of oxidation-reduction cycle reactant.

Background technology

In recent years, industrial fast development, people's living condition is improved, and also more and more pays close attention to for environmental problem." environmental protection, ecology, green, health " has become the theme notion of 21 century people life.

Oxidation-reduction cycle reactant (redox cycling reagents) is the more serious industrial or agricultural pollutent of a class, such material has the characteristic of enzyme, make the cell persistence produce superoxide anion, cause reducing equivalent (NADH/NADPH) mass consumption in the cell simultaneously, the superoxide radical that is produced can cause the body oxidative stress, oxidative stress participates in the generating process of a lot of diseases such as neurodegeneration, cancer and aging etc., also participate in atherosclerosis and cardiopathic progress, also relevant with the adjusting of vasculogenesis and cardiovascular status.This material in the environment increases can worsen ecotope, polluted source food, and people's health in serious threat.

Some physico-chemical processes have been developed at present, utilize mass spectrograph, gas chromatograph and other modern instrument to oxygen also cycle reactant carry out precise monitoring, but, these physico-chemical processes need expensive instrument and special laboratory, the more important thing is, though these technology can the accurate quantification pollutent, can not provide potential modifications/detoxification and intravital working in coordination with/biology key messages such as antagonistic effect in the bioavaliability, cell of these pollutents.

Therefore, being necessary to develop a kind of biosensor comes these toxic substances are detected.In more than ten years in the past, the research that utilizes Measurement for Biotechnique to detect specific pollutant has caused widely to be paid close attention to, and the ultimate principle of Measurement for Biotechnique promptly is to utilize the molecular reaction mechanism of model animals to particular chemicals.For Measurement for Biotechnique, need three kinds of essential elements: 1) at inductor block specific or that have the chemical substance of similar quality; 2) promotor of controlling by inductor block; 3) reporter gene that controlled by this promotor.In design during Measurement for Biotechnique, the advantages such as quick, low cost and easy care that exist in a large number owing to bacterium has in environment, grow are subjected to the general parent of researchist and look at.

Use growth curve method, end-point method, MIC method to detect bacterial strain the susceptibility of oxidation-reduction cycle reactant is all had certain limitation, reacting bacteria is to the susceptibility of oxidation-reduction cycle reactant well, the microbial culture time is long, repeatedly revision test takes time and effort, and is unfavorable for rapid detection.Dull and stereotyped meter viable bacteria method bacterial detection is to the susceptibility of oxidation-reduction cycle reactant, bacterial growth state under then can not the effect of continuous monitoring oxidation-reduction cycle reactant, and it is longer that this method is measured the cycle, subjective error is big, accuracy is not high, so can not finely detect the susceptibility of bacterium to oxidation-reduction cycle reactant.So need further to reconstruct bacterial strain to detect the susceptibility of bacterium fast and sensitively to oxidation-reduction cycle reactant.

For the Study on Biosensor of oxidation-reduction cycle reactant mainly based on the research of SoxR in the intestinal bacteria and two transcription factors of OxyR.More clearly intestinal bacteria are at the molecule mechanism of oxidation-reduction cycle reactant reaction now, and two kinds of oxygen also react transcription factor, and SoxR and OxyR mainly are responsible for the reaction of mediated cell to oxidation-reduction cycle reactant.SoxR comprises the adjustment structure territory of the spiral-turnover-helical dna binding domains and the C end of two structural domain: N ends.Four cysteine residues (Cys) of SoxR C end are as the oxygen part of ergophore (2Fe-2S) also.The SoxR of purifying is a homology dimer, and each monomer comprises also ergophore (2Fe-2S) of an oxygen.Under common culture condition, SoxR (2Fe-2S) bunch is in reduced state, does not have any transcriptional activity.When adding oxidation-reduction cycle reactant in substratum, SoxR is oxidized immediately, and stimulates its expression because of SoxS of its unique target.Its product S oxS is that the ara-c associated retroviral activates son, induces 36 kinds of genes, comprises the superoxide dismutase that contains Mn, examine sour restriction endonuclease IV, glucose-6-phosphate dehydrogenase (G6PD), FURAMIC ACID C, the expression of aconitase A and NADPH-ferredoxin oxide-reductase gene.When intestinal bacteria catalytic oxidation reduction cycle reactant, the SoxR of reduced state (2Fe-2S) bunch oxidized immediately.Also the oxidation of ortho states (2Fe-2S) bunch promptly demonstrates the SoxR transcriptional activity, and the ability that stimulation target gene SoxS expresses can increase and reaches 100.Induce control can infer that SoxR and target SoxS promotor thereof can be as the excellent biological detection systems that detects oxidation-reduction cycle reactant by SoxR to SoxS is direct and strong.

SoxR and SoxS gene head to head are distributed in intestinal bacteria dyeing and go up (as Fig. 1), their the translation initiation position only 85bp of being separated by.The SoxS promotor comprises typical case-35 (TTTACC) and-10 (TATACT) element.SoxS promotor-35 and-10 interelements 19bp of being separated by, to the superoxide that is generated by oxidation-reduction cycle reactant, SoxR activation back stimulates the SoxS expression of gene.In order to detect the activation of SoxR, will merge a reporter gene mutually with the SoxS promotor.Normally used reporter gene comprises LacZ and LuxABCD gene.The beta-galactosidase enzymes of escherichia coli lacz gene coding catalysis-galactoside and analogue hydrolysis thereof.This enzyme turnover ratio is high and active strong.But, for the activity that detects beta-galactosidase enzymes must add enzyme substrates in detection system.Aerobic oxidation becomes oxidation state FMN and corresponding carboxylic acid with long-chain aldehyde to come from the bacterial luciferase catalytic reduction attitude vitamin B2 phosphate (FMN) of LuxABCD genes encoding of Vibrio fisher, and the latter has emmission spectrum about 490nm.If use complete LuxABCD gene, need not add the external source substrate and be used to generate detection signal, but bioluminescence technique not exclusively is equal in situ detection.In addition, even the bacillary luciferase of expression in escherichia coli is not when existing any oxidation-reduction cycle reactant, its enzymic activity also can produce oxidative stress in the significant cell.

Reporting bacterial strain of the present invention can overcome these deficiencies, forms more stable transgenation strain E.coliWMC-002 reporting bacterial strain, and the quality testing of making a living is surveyed oxidation-reduction cycle reactant and laid the foundation.The gene substitution reporting bacterial strain of this research and establishment does not all have bibliographical information at home and abroad by retrieval.

Summary of the invention

The objective of the invention is to have made up the reaction host of a kind of stable intestinal bacteria reporting bacterial strain as the biosensor that detects oxidation-reduction cycle reactant by gene knockout, gene substitution technique, come rapid sensitive to detect oxidation-reduction cycle reactant, detect to solve growth curve method, end-point method, MIC method that oxidation-reduction cycle reactant takes time and effort, subjective error is big, the not high defective of accuracy.

Intestinal bacteria reporting bacterial strain of the present invention, name is called E.coli WMC-002, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 25th, 2008, the address is the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, numbering CGMCC No.2464.This intestinal bacteria reporting bacterial strain is compared with wild-type e. coli E.coli MC4100, lacks five genes, is respectively sodA (sequence is shown in SEQ ID NO.1), sodB (sequence is shown in SEQ ID NO.2), katG (sequence

This bacterial strain is compared five genes of disappearance with wild-type e. coli E.coli MC4100, be respectively sodA (sequence is shown in SEQ ID NO.1), sodB (sequence is shown in SEQ ID NO.2), katG (sequence is shown in SEQ ID NO.3), ahpCF (sequence is shown in SEQ ID NO.4), frdABCD (sequence is shown in SEQ ID NO.5), sodA in these genes, sodB is the gene of coding superoxide dismutase, katG, the ahpCF catalase of encoding, the frdABCD fumaric reductase of encoding, produce endogenous superoxide and hydrogen peroxide, the deletion of these genes can increase bacterial strain to redox agent sensitivity that detects and the background values that reduces detection.On this basis, by intersecting the PCR reaction, formation contains reporter gene GFPmut2 (sequence is shown in SEQ ID NO.6) and the dna fragmentation of waiting to replace about one section 1.7kb that goal gene SoxS (sequence is shown in SEQ IDNO.7) both sides dna fragmentation N and C end merges mutually, as shown in Figure 2; Be cloned into pKOV condition plasmid replication, make up the pKOV-GFPmut2 recombinant vectors, as shown in Figure 3; This recombinant vectors is transformed among the E.coli MC4100 that has knocked out five genes, selection by temperature and sucrose, homologous recombination takes place twice, at last with gene SoxS displacement, obtains the high responsive intestinal bacteria reporting bacterial strain E.coli WMC-002 of oxidation-reduction cycle reactant.

The construction process of E.coli WMC-002 reporting bacterial strain of the present invention is: utilize condition plasmid replication pKOV as instrument, utilize the homologous recombination principle, knock out sodA, sodB, katG, ahpCF, five genes of frdABCD, by gene substitution technique the SoxS encoding gene on the genome being replaced becomes the GFPmut2 reporter gene again.Its method of replacing is: utilize polymerase chain reaction (PCR) technology to increase waiting to replace goal gene SoxS both sides dna fragmentation N and C in the bacillus coli gene group, according to experience, need bigger dna fragmentation N and C (every segment DNA length for～500bp) could utilize host's recombinase system successfully to recombinate; Again reporter gene GFPmut2 reporter gene is increased.For dna fragmentation N, primer SoxS-No is designed to contain the NotI restriction enzyme site, and primer SoxS-Ni 5 ' end contains and primer GFP-N complementary base sequence; The primer SoxS-Co of dna fragmentation C is designed to contain the SalI restriction enzyme site, and primer SoxS-Ci 5 ' end contains and primer GFP-C complementary base sequence.Synthetic DNA product is connected to form and contains reporter gene GFPmut2 and wait to replace goal gene SoxS both sides dna fragmentation N and the C end section of DNA fragment of fusion mutually by intersecting PCR from above-mentioned PCR reaction, as shown in Figure 2.

The dna fragmentation of GFPmut2 gene fusion is cloned double enzyme site in NotI, the SalI of pKOV plasmid into.Clone's plasmid imports the e. coli strains E.coliMC4100 that has knocked out 5 genes involveds by transforming.The intestinal bacteria that process transforms are coated to contain paraxin/LB flat board and put 42 ℃ and hatch.The bacterium colony of survival indication reorganization pKOV plasmid successfully is integrated into genomic dna on the flat board, because free plasmid can not duplicate propagation under non-allowable temperature.The bacterium colony of survival will be placed in immediately puts 30 ℃ of cultivations to select to take place the escherichia coli cloning of recombination event for the second time on the flat board that contains 5% (W/V) sucrose.To sucrose opposing and selected as further analyzing to the bacterial clone of paraxin sensitivity.

If insertion and integration incident hologony in the same side of goal gene SoxS, will produce large intestine bacterial strain as parental generation.On the other hand, if insert the not homonymy (as Fig. 6) that occurs in goal gene SoxS with integration, GFPmut2 displacement SoxS gene will take place.Therefore, positive colony is proved conclusively by the PCR method at the primer of goal gene SoxS both sides sequence.

This gene substitution method is implemented under the situation of any antibiotics resistance DNA sign in the quiding gene group not and realizes accurate gene substitution.

In order to overcome beta-galactosidase enzymes and noctilcent limitation, the GFP gene of selecting coding jellyfish AequoreaVicroria green fluorescent protein (GFP) for use is as reporter gene.Light emitting-type bacterium living beings transmitter is because can rapid detection receive an acclaim to poisonous substance.Therefore, introducing reporter gene in the strain gene group, to come rapid sensitive to detect oxidation-reduction cycle reactant be the characteristic of this patent.

GFP has just obtained in the time using widely at a few years as the tagged molecule that enjoys favor, and its reason is that it has the following advantages:

(1) is easy to detect.The GFP fluorescent reaction does not need to add substrate and cofactor, only needs UV-light or blue-light excited, can send green fluorescence, just can observe with fluorescent microscope even naked eyes, and highly sensitive, also can discern for the expression of unicellular level;

(2) fluorescence is stable.GFP has stronger tolerance to photobleaching, can tolerate long illumination; GFP also can be normally luminous in pH7～12 scopes; High temperature (70 ℃), alkalescence, scale remover, salt, organic solvent and great majority all there is strong resistance;

(3) toxicological harmless.From present result of study, GFP to the also not influence of function of goal gene, but transforms cell still continuous passage in back to the basic toxicological harmless of cell of life;

(4) versatility.The expression that shows GFP is subjected to the restriction of kind scope hardly, has all obtained successful expression in microorganism, plant, animal; Next is that GFP does not have cell category and locational restriction, can express at each position and send fluorescence;

(5) be easy to carrier construction.Because the GFP molecular weight is less, only is 27～30kD, the gene order of coding GFP is also very short, thus make up multiple plasmid together with other sequence easily, and be unlikely to make the excessive transformation frequency that influences of plasmid.

(6) can carry out regularly position observation of viable cell.To proteic functional study in the viable cell, more can be near the state of natural reality.Stable GFP albumen has higher quantum yield (0.88) than bacillary luciferase (～0.1).And, only need about 105～106 GFP molecules can detect fluorescent signal.

At this, use a kind of GFP albumen by the GFPmut2 genes encoding, it is than bright more than 100 times of wild-type GFP albumen.From pGFPmut2 plasmid (available from Clonetech) DNA, obtain to contain the dna fragmentation of GFPmut2 gene, utilize the intersection round pcr to carry out gene fusion by three step PCR, with goal gene SoxS two terminal sequences and GFPmut2 gene fusion, utilize pKOV plasmid characteristic, carry out gene substitution, formed the pattern that the SoxS promotor directly starts GFPmut2 genetic expression.In this made up, the SoxR protein binding that the SoxR genes encoding produces was in the SoxS promoter sequence.When having oxidation-reduction cycle reactant, the peroxide activated SoxR that is produced by oxidation-reduction cycle reactant under oxidative stress status also stimulates the expression of SoxS::GFPmut2 fusion gene, produces GFPmut2 albumen, as shown in Figure 4.

The invention provides a kind of to the high responsive intestinal bacteria reporting bacterial strain of oxidation-reduction cycle reactant, by wild-type e. coli E.coli MC4100 disappearance sodA, sodB, katG, ahpCF, five genes of frdABCD, utilize gene substitution technique again, the SoxS encoding gene is replaced with the GFPmut2 reporter gene, called after E.coli WMC-002, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, numbering CGMCC No.2464.

The present invention also provides the construction process of described intestinal bacteria reporting bacterial strain, comprises following steps:

(1) utilize condition plasmid replication pKOV as instrument, the method by homologous recombination knocks out frdABCD, sodB in the wild-type e. coli E.coli MC4100 genome, ahpCF, katG and five genes of sodA in proper order;

(2) utilize intersection PCR reaction to make up the fusion PCR segment that contains reporter gene GFPmut2 and wait to replace the two ends homology arm of goal gene SoxS;

(3) with step 2 gained fragment cloning to pKOV condition plasmid replication, make up the pKOV-GFPmut2 recombinant vectors;

(4) described recombinant vectors pKOV-GFPmut2 is transformed in the middle obtained strains genome of step (1), by twice homologous recombination, with obtained strains genome SoxS encoding gene displacement becoming GFPmut2 reporter gene in the step (1).

The present invention also provides the application of described intestinal bacteria reporting bacterial strain aspect the detection oxidation-reduction cycle reactant.

Intestinal bacteria reporting bacterial strain of the present invention is for typical oxidation-reduction cycle reactant VitK3 (Vitamin K3, vitamin K3), PMS (Phenazine methosulfate, phenazine methosulfate) and the susceptibility of PQ (Parquat, Paraquat) apparently higher than wild bacterium E.coli MC4100.Can solve simultaneously growth curve method, end-point method, MIC method and detect that oxidation-reduction cycle reactant takes time and effort, subjective error is big, the not high defective of accuracy.Also can overcome the reporting bacterial strain that has report carrier because the high copy number and the quantity heterogeneity of plasmid in the single bacterium, not have the background of report signal under the situation of inductor very high in bacterium basal metabolism, reduce the shortcoming of the susceptibility that pollutent is detected; The heredity of plasmid has relative independence, introduces the plasmid of microorganism cells, through the cultivation of several generations, loses probably, causes engineering bacteria to lose efficacy; The plasmid that enters environment also may pollute other bacterial classification, causes the potential ambient wind dangerous; Utilize plasmid also to have accurate quantification shortcomings such as difficulty, poor stability relatively in addition, and the transgenation strain E.coli WMC-002 reporting bacterial strain that makes up can overcome these deficiencies as the reporting system detection of contamination of carrier.

Utilize the reaction host bacterium of E.coli WMC-002 bacterial strain provided by the invention as the biosensor that detects oxidation-reduction cycle reactant, its susceptibility to oxidation-reduction cycle reactant obviously increases and bio signal can detect quick and objectively.Utilize bacterial strain of the present invention can in the detection of environmental pollutant-oxidation-reduction cycle reactant, be applied as the host bacterium.

Description of drawings

SoxS and soxR gene distribution figure in Fig. 1 E.coli karyomit(e);

Fig. 2 utilizes the intersection round pcr to carry out the gene fusion synoptic diagram;

The structure schema of Fig. 3 recombinant vectors pKOV-GFPmut2;

Fig. 4 SoxS::GFPmut2 gene fusion synoptic diagram;

Fig. 5 gene replacement carrier imports the host and hatches the PCR electrophorogram in 30 ℃;

1:DL2000DNA Marker; 5: blank; 2,3,4,6,7: gene replacement carrier imports the host and hatches the PCR product in 30 ℃;

Fig. 6 SoxS and GFPmut2 gene substitution make up the reporter gene synoptic diagram;

Fig. 7 gene replaces carrier and the host is hatched generation homologous recombination PCR electrophorogram at 42 ℃ of paraxin;

1:DL2000DNA Marker; 3: blank; 7: gene replacement carrier imports the host and hatches the PCR product in 30 ℃; 2,4,5,6,8: gene is replaced carrier and homologous recombination PCR product takes place in 42 ℃ of paraxin are hatched the host.

Fig. 8 SoxS and GFPmut2 gene producer are replaced the PCR electrophoresis and are identified figure;

1:DL2000DNA Marker; 2: gene replacement carrier imports the host and hatches the PCR product in 30 ℃; 3,4,6,7:SoxS and GFPmut2 gene producer are replaced the PCR product; 5,8-13: the host is the PCR product (negative control) of producer replacement not; 14: blank.

Fig. 9 gene substitution reporting bacterial strain observations figure under fluorescent microscope;

A left side: the gene substitution reporting bacterial strain is observations under fluorescent microscope; Right: Bacillus coli communis is observations (negative control) under fluorescent microscope;

Figure 10 reporting bacterial strain growth curve chart;

Figure 11 different concns PQ acts on reporting bacterial strain fluorescence microscope result (10 * 100 times) figure;

A：10μM；B：1μM；C：0.1μM；D：Blank

Figure 12 different concns PMS acts on reporting bacterial strain fluorescence microscope result (10 * 100 times) figure;

A：10μM；B：1μM；C：0.1μM；D：Blank

Figure 13 different concns VitK3 acts on reporting bacterial strain fluorescence microscope result (10 * 100 times) figure.

A：100μM；B：10μM；C：1μM；D：Blank

Embodiment

Of the present invention as follows to the high responsive colibacillary structure concrete grammar of oxidation-reduction cycle reactant:

The fusion of embodiment 1. goal gene

(1), primer information and synthetic

Provide the primer sequence of gene knockout and SoxS and the GFPmut2 gene order that GenBank announces according to the Harvard University website, the inboard primer (P2:soxS-Ni and P3:soxS-Ci) and the outside primer (P1:soxS-No and P4:soxS-Co) of design soxS gene N-terminal and C-terminal, make P2 and P5, there is complementary sequence between P3 and the P6, two outsides of gene knockout primer is constant, and P5 and P6 are a pair of amplification GFPmut2 gene primer.Specifying information sees Table-1, and primer is synthetic by the precious biotech firm in Dalian.With aseptic deionized water primer is dissolved, being made into concentration is 10 μ mol/L.

Table 1

(2), intersection PCR carries out gene fusion

Utilize the intersection round pcr to carry out gene fusion,, form the pattern that the SoxS promotor directly starts GFPmut2 genetic expression goal gene SoxS two terminal sequences and GFPmut2 gene fusion by three step PCR.

Embodiment two homology arms of 2. amplifications and GFPmut2 gene

1. the amplification of two homology arms:

With the dull and stereotyped E.coli MC4100 of inoculating needle picking LB one single bacterium colony a little, mix in the 200 μ LPCR reaction tubess that contain 15 μ L sterilized waters, boiling water boils 10min, the centrifugal 3～5min of 12000rpm is condensing in water droplet on the lid to the pipe end.Get that institute's part is that 5 μ L add in this pipe mixing in the table 2.

Table 2

The PCR reaction conditions is as follows: 94 ℃ of 1min; 88 ℃ of 4min; 94 ℃ of 10sec, 66 ℃ of 3min, 25 circulations; 72 ℃ of 10min, 4 ℃ of preservations.Getting 5 μ L products identifies with 1.0% Agarose gel electrophoresis.The PCR product carries out purifying with PCR product purification test kit, and it is standby to add an amount of ddH2O dissolving wash-out at last.

2.GFPmut2 the amplification of gene:

With the inoculating needle picking contain pGFPmut2 plasmid (Clonetech) single bacterium colony a little, mix in the 200 μ L PCR reaction tubess that contain 15 μ L sterilized waters, boiling water boils 10min, the centrifugal 3～5min of 12000rpm is condensing in water droplet on the lid to the pipe end.Get that institute's part is that 5 μ L add in this pipe mixing in the table 3.The PCR reaction conditions is as follows: 94 ℃ of 1min; 88 ℃ of 4min; 94 ℃ of 25sec, 55 ℃ of 35sec, 72 ℃ of 40sec, 25 circulations; 72 ℃ of 10min, 4 ℃ of preservations.Getting 5 μ L products identifies with 1.0% Agarose gel electrophoresis.The PCR product carries out purifying with PCR product purification test kit, and it is standby to add an amount of ddH2O dissolving wash-out at last.

Table 3

3. a side homology arm and GFPmut2 gene fusion

Press institute's part system reaction in the table 4, the PCR reaction conditions is as follows: 94 ℃ of 1min; 88 ℃ of 4min; 94 ℃ of 25sec, 55 ℃ of 35sec, 72 ℃ of 40sec, 25 circulations; 72 ℃ of 10min, 4 ℃ of preservations.Getting 5 μ L products identifies with 1.0% Agarose gel electrophoresis.The PCR product carries out purifying with PCR product purification test kit, and it is standby to add an amount of ddH2O dissolving wash-out at last.

Table 4

4. both sides homology arm and GFPmut2 gene fusion

Press institute's part system reaction in the table 5, the PCR reaction conditions is as follows: 94 ℃ of 1min; 88 ℃ of 4min; 94 ℃ of 10sec, 66 ℃ of 3min, 25 circulations; 72 ℃ of 10min, 4 ℃ of preservations.Getting 5 μ L products identifies with 1.0% Agarose gel electrophoresis.The result shows that both sides homology arm and GFPmut2 gene successfully merge.

Table 5

Embodiment 3, PCR product add the A reaction

The ratio adding Ex Taq that in above-mentioned PCR reaction product, adds 5U in per 50 μ L

Polysaccharase, 72 ℃ of 10min.

(4), PCR segment purifying reclaims

Add the A reaction product and carry out purifying with PCR product purification test kit, it is standby to add an amount of ddH2O dissolving wash-out at last.

Embodiment 4. purpose fragments are connected with pMD19-T simple carrier

Carry out being connected of T carrier and goal gene by the pMD19-T simple Vector test kit specification sheets of TaKaRa company.The ligation system sees Table 6, and ligation is spent the night in 16 ℃.

Table 6

Embodiment 5. transforms

By cold CaCl ₂Method will connect product and be transformed in the intestinal bacteria DH-5 α competent cell.

Embodiment 6. order-checkings

Select one through outside primer P1, P4PCR evaluation, plasmid enzyme restriction identifies that the order-checking of the precious biotech firm in Dalian, the canonical reference sequence alignment that provides on sequencing result and the NCBI are provided the mono-clonal that confirms.

The recovery of the double digestion of embodiment 7. recombinant clone pMD19T-GFPmut2 and purpose fragment GFPmut2 fusion gene

1. the alkaline lysis preparation of plasmid DNA in a small amount (being undertaken) by the method in " molecular cloning ".

2.pMD19T-GFPmut2 the plasmid DNA that the recovery of the double digestion of plasmid and purpose fragment GFPmut2 fusion gene is extracted is carried out double digestion, its reaction system sees Table 7,37 ℃, and enzyme is cut 3h.

Table 7

The glue of cutting of purpose fragment GFPmut2 fusion gene reclaims: the capable 0.8% low melting-point agarose gel electrophoresis of endonuclease reaction after product, under long-wave ultra violet lamp, cut target DNA fragment, and reclaim test kit with TaKaRa company glue and reclaim.Quantitative with Du-800 nucleic acid/protein analyzer at last to reclaiming product.

The preparation of embodiment 8.pKOV carrier segments

(1), amount preparation pKOV carrier in the alkaline lysis

Because plasmid pKOV is (5～10/cell), measure extraction method (being undertaken by the method in " molecular cloning ") in order to obtain enough amounts in the plasmid with regard to selecting for use in a low copy.

(2), the double digestion of pKOV plasmid and recovery thereof

System and enzyme tangent condition reclaim the cmy vector fragment with the pMD19T-GFPmut2 plasmid.

Structure and the preservation of embodiment 9. recombinant vectors pKOV-GFPmut2

(1), the ligation of purpose segment and carrier

Reaction system sees Table 8, and ligation is spent the night in 16 ℃.

Table 8

(2), the product that connects is changed in the DH5 α competent cell

(3), the evaluation of positive colony

1.PCR identify

With the dull and stereotyped single bacterium colony of inoculating needle picking LB CM+ a little, mix in getting the 200 μ LPCR reaction tubess that contain 14 μ L sterilized waters, boiling water boils 10min, the centrifugal 3～5min of 12000rpm the water vapour on the lid to the pipe end.Get in the table 9 institute's part system and add in this pipe mixing.

Table 9

The PCR reaction conditions is as follows: 94 ℃ of 1min; 88 ℃ of 4min; 94 ℃ of 10sec, 66 ℃ of 3min, 25 circulations; 72 ℃ of 10min, 4 ℃ of preservations.Getting 5 μ L products identifies with 1.0% Agarose gel electrophoresis.The mono-clonal of electrophoresis showed PCR fragment length about 1.7kb is selected, and carries out following plasmid extraction and double digestion and identifies.

2. the plasmid double digestion is identified

1) the middle amount of above-mentioned cloned plasmids is extracted (with above-mentioned)

2) system and enzyme tangent condition are with the pMD19T-GFPmut2 plasmid.The capable 0.8% low melting-point agarose gel electrophoresis of endonuclease reaction after product is identified.The enzyme section is broken and confirm the construction of recombinant vector success about 1.7kb, and bacterial classification is put among the LB CM+ that contains glycerine 15%, and-70 ℃ of preservations are standby.

The displacement of embodiment 10.SoxS and GFPmut2 gene

(1), the preparation of five genetically deficient bacterial strain competent cells

(1) with-70 ℃ of five frozen genetically deficient inoculation in the LB of about 2～3mL liquid nutrient medium, 37 ℃, 200rpm, shaking culture is spent the night.

(2) the bacterium 500 μ L that shaking culture spent the night add and contain in the Erlenmeyer flask of 50mL LB liquid nutrient medium shaking culture 3h～3.5h.

(3) bacterium liquid is moved in the 50mL polypropylene tube of ice precooling, place 10min on ice.

(4) 4 ℃, 4, the centrifugal 10min of 000rpm abandons supernatant, collects thalline.

(5) with the 0.1M CaCl of 10mL ice precooling ₂Resuspended precipitation places on ice.

(6) 4 ℃, 4, the centrifugal 10min of 000rpm abandons supernatant.

(7) with the 0.1M CaCl of 2mL ice precooling ₂Resuspended precipitation, packing (the every pipe of 100 μ L/).Be directly used in and transform or add 15% glycerine, be put in-70 ℃ frozen.

(2), recombinant vectors pKOV-GFPmut2 is transformed into five genetically deficient bacterial strain competent cells

1, the extraction of pKOV-GFPmut2 recombinant vectors among the DH5 α (test kit method TaKaRa MiniBESTPlasmid Purification Kit Ver.2.0)

2, recombinant vectors pKOV-GFPmut2 transforms

(1) gets 100 μ L competence bacteriums, add above-mentioned recombinant vectors, shake up ice bath 30min gently.

(2) 42 ℃ of heat-shocked 90～120s, ice bath 3～5min.

(3) add 800 μ L LB liquid nutrient mediums, shake up 37 ℃ of 100rpm shaking culture 1h.

(4), and respectively get 0.1mL and be applied to LB CM+ flat board respectively respectively with 10 times, 100 times of LB liquid nutrient medium dilutions

(5) treat that bacterium liquid absorbs fully after, be inverted culture dish, 37 ℃ are spent the night.

(3), the evaluation before the homologous recombination

1.30 the single bacterium colony PCR that ℃ LB CM+ flat board grows identifies

With the dull and stereotyped single bacterium colony of inoculating needle picking LB CM+ a little, mix in the 200 μ LPCR reaction tubess that contain 14 μ L sterilized waters, boiling water boils 10min, the centrifugal 3～5min of 12000rpm the water droplet on the lid that condenses to the pipe end.Get in the table 10 institute's part system and add in this pipe mixing.

Table 10

The PCR reaction conditions is as follows: 94 ℃ of 1min; 88 ℃ of 4min; 94 ℃ of 10sec, 66 ℃ of 3min, 25 circulations; 72 ℃ of 10min, 4 ℃ of preservations.Getting 5 μ L products identifies with 1.0% agarose gel electrophoresis.

2.PCR product is two bands, its brightness has notable difference, (darker about 1200bp as shown in Figure 5, brighter about 1700bp) getting 3～5 clones is diluted in 42 ℃ of preheating 1mL LB substratum, by 10 times, 100 times dilutions, and respectively get on the flat board of LB CM+ that 0.1mL is applied to 42 ℃ of preheatings respectively.After treating that bacterium liquid absorbs fully, be inverted culture dish, 42 ℃, 24-36h cultivates.

(4), the first step homologous recombination (GFPmut2 is incorporated in the colibacillary genome)

1.42 the PCR evaluation (method is the same) of the single bacterium colony of the dull and stereotyped growth of ℃ LB CM+,

2.PCR product is two bands, its brightness is suitable, and the length about about 1200bp and 1700bp as Fig. 7, proves the first step homologous recombination has taken place that segment and plasmid are incorporated on the genome.Choose 3～5 of above-mentioned bacterium colonies and be diluted in 30 ℃ of preheating 1mL LB substratum, doubling dilution, and respectively get on the 5% sucrose LB flat board that 0.1mL is applied to 30 ℃ of preheatings respectively.After treating that bacterium liquid absorbs fully, be inverted culture dish, 30 ℃, 24-36h cultivates.

(5), the second step homologous recombination (plasmid and gene element from and lose)

With 30 ℃ of inoculating needle pickings, the single bacterium colony of growing on the 5% sucrose LB flat board, be inoculated in respectively on the LBCM+ flat board and 5% sucrose LB flat board on, 30 ℃, 24-36h cultivates.

(6), the PCR screening of the positive bacterium colony of gene substitution

30 ℃ of LB CM+ flat boards are not grown, and the bacterium colony of growing on the LB 5% sucrose flat board is made bacterium colony tentative confirmation that PCR identifies that (method is seen above-mentioned) PCR product is about 1700bp and is producer metathetical positive bacterium colony (as Fig. 8).Divide pure bacterium colony, PCR further identifies and confirms the gene substitution success.

(7), fluorescent microscope is identified

Get PCR and identify the mono-clonal that confirms the gene substitution success, cultivate 4-6h in 37 ℃ of 1mL LB substratum, get medium centrifugal, PBS damping fluid washing 3 times, precipitation stay in fluorescent microscope and observe down, and the excitation wavelength of GFPmut2 is 488nm, wavelength of transmitted light is 512nm, observations such as Fig. 9.

Embodiment 11. reporting bacterial strain growth curves are measured

In order to detect the susceptibility of gene substitution reporting bacterial strain E.coli WMC-002 to oxidation-reduction cycle reactant, select representative oxidation-reduction cycle reactant VitK3, PMS and PQ (all available from sigma company) to do the experiment of oxidation-reduction cycle reactant susceptibility, specifically implement as follows:

1. seed liquor preparation

Get the single fresh bacterium colony on the LB flat board, be inoculated in 2ml LB liquid nutrient medium, 200rpm, 37 ℃ of shaken overnight are cultured to state of saturation.

2. adjust the OD600 value of starter bacteria liquid

(1) draw saturated bacterium liquid 200 μ l and join pressure-vaccum mixing in the cuvette that contains LB liquid nutrient medium 1,800 μ l, with blank LB liquid nutrient medium zeroing, mensuration OD600 value, result are taken advantage of the 10 OD600 values that are saturated bacterium liquid.

(2) according to formula V=1/OD600, calculate the bacterium liquid measure that need join in the 50ml LB liquid nutrient medium, make starter bacteria liquid OD600 value be about 0.02.

(3) draw in the seed liquor adding 50ml LB liquid nutrient medium according to calculating good amount, shake up gently, draw bacterium liquid 2ml simultaneously respectively and join mensuration OD600 value (as 0h OD600 value) in the cuvette.

(4) packing: the nutrient solution that above-mentioned dilution is good joins in the sterile test tube according to the 2ml/ pipe.

3. marker number

Reporting bacterial strain, wild type strain, parent strain are all according to 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h mark;

4. cultivate

With above-mentioned test tube 37 ℃ of following shaking culture.By the corresponding time test tube is taken out respectively then, put in the refrigerator immediately and store, wait to cultivate and together measure the OD value when finishing.

5. increment is measured

Nonvaccinated LB substratum is poured in the cuvette, select 600nm wavelength spectrophotometer adjusted zero point for use, as blank, and the different time nutrient solution measured successively from 0h, the big bacteria suspension of concentration is suitably diluted the back with nonvaccinated LB liquid nutrient medium to be measured, make its OD value in 0.10～0.65, the OD value that records after dilution will multiply by extension rate, is only the OD value of nutrient solution reality.Parallel pipe is set in the experiment, and last OD600 value is the mean value of institute's measured value, tests triplicate at least at every turn.

6. result treatment

With time is X-coordinate, and the OD600 value is an ordinate zou, draws colibacillary growth curve.

Estimate growth conditions after its gene substitution by the growth curve of measuring reporting bacterial strain, from growth curve as can be seen the parent's bacteria growing state before reporting bacterial strain and the gene substitution do not have significant difference, growth tendency basically identical (as Figure 10), illustrate that gene substitution does not influence the normal growth of bacterium, help as the detection of reporting bacterial strain to oxidation-reduction cycle reactant.

Embodiment 12. reporting bacterial strain WMC-002 are to the susceptibility experiment of oxidation-reduction cycle reactant

With the susceptibility of set time point method examining report bacterial strain WMC-002 to VitK3, PMS and PQ.

1. the preparation of seed liquor

The single fresh bacterium colony of picking from the LB flat board is inoculated in 20ml LB liquid nutrient medium, 200rpm, and 37 ℃ of shaken overnight are cultured to state of saturation.

2. adjust the OD600 value of starter bacteria liquid

1) draw saturated bacterium liquid 200 μ l and join pressure-vaccum mixing in the cuvette that contains LB liquid nutrient medium 1800 μ l, with blank LB liquid nutrient medium zeroing, mensuration OD600 value, result are taken advantage of the 10 OD600 values that are saturated bacterium liquid.

2) according to formula V=1/OD600, calculate the bacterium liquid measure that need join in the 50ml LB liquid nutrient medium, make starter bacteria liquid OD600 value be about 0.02.

3. add inductor (being respectively VitK3, PMS and PQ)

Drawing an amount of seed liquor adds in the 50ml LB liquid nutrient medium, shake up gently, draw bacterium liquid 2ml simultaneously respectively and join mensuration OD600 value (as 0h OD600 value) in the cuvette, the every pipe of bacterium liquid packing 2ml joins in the test tube, 200rpm behind 37 ℃ of shaking culture 2h, adds an amount of inductor according to different induced concentration demands, 200rpm, 37 ℃ of shaking culture.

4. reporting bacterial strain was cultivated after 6 hours, got medium centrifugal, and PBS damping fluid washing 3 times adds the resuspended precipitation of 1mlPBS, gets 5 μ l bacterium liquid film-makings, adds anti-fluorescent quenching mountant during film-making, observed down in fluorescent microscope.

3. experimental result

The most common in the oxidation-reduction cycle reactant is PQ, PMS, V itK3, and the different concns of these three kinds of oxidation-reduction cycle reactants of this experimental selection acts on reporting bacterial strain and estimates its susceptibility.Experimental result shows (as Figure 11,12,13): reporting bacterial strain is to the susceptibility difference of three kinds of oxidation-reduction cycle reactants, the fluorescence intensity of inducing reporting bacterial strain to send under the identical concentration differs, fluorescence intensity is the strongest under the effect of PMS, and fluorescence intensity is the most weak under the effect of VitK3.This is that strong oxidation-reduction cycle reactant is consistent with PMS in theory also.Come the detection of contamination oxidation-reduction cycle reactant will overcome growth curve method, end-point method, MIC method with this reporting bacterial strain as the host of biosensor and detect oxidation-reduction cycle reactant and have defectives such as time and effort consuming, for the research and development of biosensor are had laid a good foundation.

The above is preferred embodiment of the present invention only, is not to be used to limit protection scope of the present invention.

SEQUENCE?LISTING

＜110〉Wenzhou Medical College

＜120〉a kind of reporting bacterial strain to the oxidation-reduction cycle reactant sensitivity and preparation method thereof

<130>081646-I-CP-NZJ

<160>13

<170>PatentIn?version?3.5

<210>1

<211>621

<212>DNA

<213>sodA

<400>1

atgagctata?ccctgccatc?cctgccgtat?gcttacgatg?ccctggaacc?gcacttcgat 60

aagcagacca?tggaaatcca?ccacaccaaa?caccatcaga?cctacgtaaa?caacgccaac 120

gcggcgctgg?aaagcctgcc?agaatttgcc?aacctgccgg?ttgaagagct?gatcaccaaa 180

ctggaccagc?tgccagcaga?caagaaaacc?gtactgcgca?acaacgctgg?cggtcacgct 240

aaccacagcc?tgttctggaa?aggtctgaaa?aaaggcacca?ccctgcaggg?tgacctgaaa 300

gcggctatcg?aacgtgactt?cggctccgtt?gataacttca?aagcagaatt?tgaaaaagcg 360

gcagcttccc?gctttggttc?cggctgggca?tggctggtgc?tgaaaggcga?taaactggcg 420

gtggtttcta?ctgctaacca?ggattctccg?ctgatgggtg?aagctatttc?tggcgcttcc 480

ggcttcccga?ttatgggcct?ggatgtgtgg?gaacatgctt?actacctgaa?attccagaac 540

cgccgtccgg?actacattaa?agagttctgg?aacgtggtga?actgggacga?agcagcggca 600

cgttttgcggc?gaaaaaataa 621

<210>2

<211>582

<212>DNA

<213>sodB

<400>2

atgtcattcg?aattacctgc?actaccatat?gctaaagatg?ctctggcacc?gcacatttct 60

gcggaaacca?tcgagtatca?ctacggcaag?caccatcaga?cttatgtcac?taacctgaac 120

aacctgatta?aaggtaccgc?gtttgaaggt?aaatcactgg?aagagattat?tcgcagctct 180

gaaggtggcg?tattcaacaa?cgcagctcag?gtctggaacc?atactttcta?ctggaactgc 240

ctggcaccga?acgccggtgg?cgaaccgact?ggaaaagtcg?ctgaagctat?cgccgcatct 300

tttggcagct?ttgccgattt?caaagcgcag?tttactgatg?cagcgatcaa?aaactttggt 360

tctggctgga?cctggctggt?gaaaaacagc?gatggcaaac?tggctatcgt?ttcaacctct 420

aacgcgggta?ctccgctgac?caccgatgcg?actccgctgc?tgaccgttga?tgtctgggaa 480

cacgcttatt?acatcgacta?tcgcaatgca?cgtcctggct?atctggagca?cttctgggcg 540

ctggtgaac?tgggaattcg?tagcgaaaaat?ctcgctgcat?aa 582

<210>3

<211>582

<212>DNA

<213>KatG

<400>3

atgtcattcg?aattacctgc?actaccatat?gctaaagatg?ctctggcacc?gcacatttct 60

gcggaaacca?tcgagtatca?ctacggcaag?caccatcaga?cttatgtcac?taacctgaac 120

aacctgatta?aaggtaccgc?gtttgaaggt?aaatcactgg?aagagattat?tcgcagctct 180

gaaggtggcg?tattcaacaa?cgcagctcag?gtctggaacc?atactttcta?ctggaactgc 240

ctggcaccga?acgccggtgg?cgaaccgact?ggaaaagtcg?ctgaagctat?cgccgcatct 300

tttggcagct?ttgccgattt?caaagcgcag?tttactgatg?cagcgatcaa?aaactttggt 360

tctggctgga?cctggctggt?gaaaaacagc?gatggcaaac?tggctatcgt?ttcaacctct 420

aacgcgggta?ctccgctgac?caccgatgcg?actccgctgc?tgaccgttga?tgtctgggaa 480

cacgcttatt?acatcgacta?tcgcaatgca?cgtcctggct?atctggagca?cttctgggcg 540

ctggtgaac?tgggaattcg?tagcgaaaaat?ctcgctgcat?aa 582

<210>4

<211>2181

<212>DNA

<213>ahpCF

<400>4

atgagcacgt?cagacgatat?ccataacacc?acagccactg?gcaaatgccc?gttccatcag 60

ggcggtcac?gaccagagtg?cgggggcgggcacaaccactc?gcgactggtg?gccaaatcaa 120

cttcgtgttg?acctgttaaa?ccaacattct?aatcgttcta?acccactggg?tgaggacttt 180

gactaccgca?aagaattcag?caaattagat?tactacggcc?tgaaaaaaga?tctgaaagcc 240

ctgttgacag?aatctcaacc?gtggtggcca?gccgactggg?gcagttacgc?cggtctgttt 300

attcgtatgg?cctggcacgg?cgcggggact?taccgttcaa?tcgatggacg?cggtggcgcg 360

ggtcgtggtc?agcaacgttt?tgcaccgctg?aactcctggc?cggataacgt?aagcctcgat 420

aaagcgcgtc?gcctgttgtg?gccaatcaaa?cagaaatatg?gtcagaaaat?ctcctgggcc 480

gacctgttta?tcctcgcggg?taacgtggcg?ctagaaaact?ccggcttccg?taccttcggt 540

tttggtgccg?gtcgtgaaga?cgtctgggaa?ccggatctgg?atgttaactg?gggtgatgaa 600

aaagcctggc?tgactcaccg?tcatccggaa?gcgctggcga?aagcaccgct?gggtgcaacc 660

gagatgggtc?tgatttacgt?taacccggaa?ggcccggatc?acagcggcga?accgctttct 720

gcggcagcagctatccgcgc?gaccttcggc?aacatgggca?tgaacgacga?agaaaccgtg 780

gcgctgattg?cgggtggtca?tacgctgggt?aaaacccacg?gtgccggtcc?gacatcaaat 840

gtaggtcctg?atccagaagc?tgcaccgatt?gaagaacaag?gtttaggttg?ggcgagcact 900

tacggcagcg?gcgttggcgc?agatgccatt?acctctggtc?tggaagtagt?ctggacccag 960

acgccgaccc?agtggagcaa?ctatttcttc?gagaacctgt?tcaagtatga?gtgggtacag 1020

acccgcagcc?cggctggcgc?aatccagttc?gaagcggtag?acgcaccgga?aattatcccg 1080

gatccgtttg?atccgtcgaa?gaaacgtaaa?ccgacaatgc?tggtgaccga?cctgacgctg 1140

cgttttgatc?ctgagttcga?gaagatctct?cgtcgtttcc?tcaacgatcc?gcaggcgttc 1200

aacgaagcct?ttgcccgtgc?ctggttcaaa?ctgacgcaca?gggatatggg?gccgaaatct 1260

cgctacatcg?ggccggaagt?gccgaaagaa?gatctgatct?ggcaagatcc?gctgccgcag 1320

ccgatctaca?acccgaccga?gcaggacatt?atcgatctga?aattcgcgat?tgcggattct 1380

ggtctgtctg?ttagtgagct?ggtatcggtg?gcctgggcat?ctgcttctac?cttccgtggt 1440

ggcgacaaacgcggtggtgc?caacggtgcg?cgtctggcat?taatgccgca?gcgcgactgg 1500

gatgtgaacg?ccgcagccgt?tcgtgctctg?cctgttctgg?agaaaatcca?gaaagagtct 1560

ggtaaagcct?cgctggcgga?tatcatagtg?ctggctggtg?tggttggtgt?tgagaaagcc 1620

gcaagcgccg?caggtttgag?cattcatgta?ccgtttgcgc?cgggtcgcgt?tgatgcgcgt 1680

caggatcaga?ctgacattga?gatgtttgag?ctgctggagc?caattgctga?cggtttccgt 1740

aactatcgcg?ctcgtctgga?cgtttccacc?accgagtcac?tgctgatcga?caaagcacag 1800

caactgacgc?tgaccgcgcc?ggaaatgact?gcgctggtgg?gcggcatgcg?tgtactgggt 1860

gccaacttcg?atggcagcaa?aaacggcgtc?ttcactgacc?gcgttggcgt?attgagcaat 1920

gacttcttcg?tgaacttgct?ggatatgcgt?tacgagtgga?aagcgaccga?cgaatcgaaa 1980

gagctgttcg?aaggccgtga?ccgtgaaacc?ggcgaagtga?aatttacggc?cagccgtgcg 2040

gatctggtgt?ttggttctaa?ctccgtcctg?cgtgcggtgg?cggaagttta?cgccagtagc 2100

gatgcccacg?agaagtttgt?taaagacttc?gtggcggcat?gggtgaaagt?gatgaacctc 2160

gaccgtttcgacctgctgtaa 2181

<210>5

<211>3300

<212>DNA

<213>frdABCD

<400>5

gtgcaaacct?ttcaagccga?tcttgccatt?gtaggcgccg?gtggcgcggg?attacgtgct 60

gcaattgctg?ccgcgcaggc?aaatccgaat?gcaaaaatcg?cactaatctc?aaaagtatac 120

ccgatgcgta?gccataccgt?tgctgcagaa?gggggctccg?ccgctgtcgc?gcaggatcat 180

gacagcttcg?aatatcactt?tcacgataca?gtagcgggtg?gcgactggtt?gtgtgagcag 240

gatgtcgtgg?attatttcgt?ccaccactgc?ccaaccgaaa?tgacccaact?ggaactgtgg 300

ggatgcccat?ggagccgtcg?cccggatggt?agcgtcaacg?tacgtcgctt?cggcggcatg 360

aaaatcgagc?gcacctggtt?cgccgccgat?aagaccggct?tccatatgct?gcacacgctg 420

ttccagacct?ctctgcaatt?cccgcagatc?cagcgttttg?acgaacattt?cgtgctggat 480

attctggttg?atgatggtca?tgttcgcggc?ctggtagcaa?tgaacatgat?ggaaggcacg 540

ctggtgcaga?tccgtgctaa?cgcggtcgtt?atggctactg?gcggtgcggg?tcgcgtttat 600

cgttacaaca?ccaacggcgg?catcgttacc?ggtgacggta?tgggtatggc?gctaagccac 660

ggcgttccgc?tgcgtgacat?ggaattcgtt?cagtatcacc?caaccggtct?gccaggttcc 720

ggtatcctga?tgaccgaagg?ttgccgcggt?gaaggcggta?ttctggtcaa?caaaaatggc 780

taccgttatc?tgcaagatta?cggcatgggc?ccggaaactc?cgctgggcga?gccgaaaaac 840

aaatatatgg?aactgggtcc?acgcgacaaa?gtctctcagg?ccttctggca?cgaatggcgt 900

aaaggcaaca?ccatctccac?gccgcgtggc?gatgtggttt?atctcgactt?gcgtcacctc 960

ggcgagaaaa?aactgcatga?acgtctgccg?ttcatctgcg?aactggcgaa?agcgtacgtt 1020

ggcgtcgatc?cggttaaaga?accgattccg?gtacgtccga?ccgcacacta?caccatgggc 1080

ggtatcgaaa?ccgatcagaa?ctgtgaaacc?cgcattaaag?gtctgttcgc?cgtgggtgaa 1140

tgttcctctg?ttggtctgca?cggtgcaaac?cgtctgggtt?ctaactccct?ggcggaactg 1200

gtggtcttcg?gccgtctggc?cggtgaacaa?gcgacagagc?gtgcagcaac?tgccggtaat 1260

ggcaacgaag?cggcaattga?agcgcaggca?gctggcgttg?aacaacgtct?gaaagatctg 1320

gttaaccagg?atggcggcgaaaactgggcg?aagatccgcg?acgaaatggg?cctggctatg 1380

gaagaaggct?gcggtatcta?ccgtacgccg?gaactgatgc?agaaaaccat?cgacaagctg 1440

gcagagctgc?aggaacgctt?caagcgcgtg?cgcatcaccg?acacttccag?cgtgttcaac 1500

accgacctgc?tctacaccat?tgaactgggc?cacggtctga?acgttgctga?atgtatggcg 1560

cactccgcaatggcacgtaa?agagtcccgc?ggcgcgcacc?agcgtctgga?cgaaggttgc 1620

accgagcgtg?acgacgtcaa?cttcctcaaa?cacaccctcg?ccttccgcga?tgctgatggc 1680

acgactcgcc?tggagtacag?cgacgtgaag?attactacgc?tgccgccagc?taaacgcgtt 1740

tacggtggcgaagcggatgcagccgataaggcggaagcagccaataagaa?ggagaaggcg 1800

aatggctgaa?tggctgagat?gaaaaacctg?aaaattgagg?tggtgcgcta?taacccggaa 1860

gtcgataccg?caccgcatag?cgcattctat?gaagtgcctt?atgacgcaac?tacctcatta 1920

ctggatgcgc?tgggctacat?caaagacaac?ctggcaccgg?acctgagcta?ccgctggtcc 1980

tgccgtatgg?cgatttgtgg?ttcctgcggc?atgatggtta?acaacgtgcc?aaaactggca 2040

tgtaaaacct?tcctgcgtga?ttacaccgac?ggtatgaagg?ttgaagcgtt?agctaacttc 2100

ccgattgaac?gcgatctggt?ggtcgatatg?acccacttca?tcgaaagtct?ggaagcgatc 2160

aaaccgtaca?tcatcggcaa?ctcccgcacc?gcggatcagg?gtactaacat?ccagaccccg 2220

gcgcagatgg?cgaagtatca?ccagttctcc?ggttgcatca?actgtggttt?gtgctacgcc 2280

gcgtgcccgc?agtttggcct?gaacccagag?ttcatcggtc?cggctgccat?tacgctggcg 2340

catcgttata?acgaagatag?ccgcgaccac?ggtaagaagg?agcgtatggc?gcagttgaac 2400

agccagaacg?gcgtatggag?ctgtactttc?gtgggctact?gctccgaagt?ctgcccgaaa 2460

cacgtcgatc?cggctgcggc?cattcagcag?ggcaaagtag?aaagttcgaa?agactttctt 2520

atcgcgaccc?tgaaaccacg?ctaaatgacg?actaaacgta?aaccgtatgt?acggccaatg 2580

acgtccacct?ggtggaaaaa?attgccgttt?tatcgctttt?acatgctgcg?cgaaggcacg 2640

gcggttccgg?ctgtgtggtt?cagcattgaa?ctgattttcg?ggctgtttgc?cctgaaaaat 2700

ggcccggaag?cctgggcggg?attcgtcgac?tttttacaaa?acccggttat?cgtgatcatt 2760

aacctgatca?ctctggcggc?agctctgctg?cacaccaaaa?cctggtttga?actggcaccg 2820

aaagcggcca?atatcattgt?aaaagacgaa?aaaatgggac?cagagccaat?tatcaaaagt 2880

ctctgggcgg?taactgtggt?tgccaccatc?gtaatcctgt?ttgttgccct?gtactggtaa 2940

atgattaatc?caaatccaaa?gcgttctgac?gaaccggtat?tctggggcct?cttcggggcc 3000

ggtggtatgt?ggagcgccat?cattgcgccg?gtgatgatcc?tgctggtggg?tattctgctg 3060

ccactggggt?tgtttccggg?tgatgcgctg?agctacgagc?gcgttctggc?gttcgcgcag 3120

agcttcattg?gtcgcgtatt?cctgttcctg?atgatcgttc?tgccgctgtg?gtgtggttta 3180

caccgtatgc?accacgcgat?gcacgatctg?aaaatccacg?tacctgcggg?caaatgggtt 3240

ttctacggtc?tggctgctat?cctgacagtt?gtcacgctga?ttggtgtcgt?tacaatctaa 3300

<210>6

<211>717

<212>DNA

<213>gfpmut2

<400>6

atgagtaaag?gagaagaact?tttcactgga?gttgtcccaa?ttcttgttga?attagatggt 60

gatgttaatg?ggcacaaatt?ttctgtcagt?ggagagggtg?aaggtgatgc?aacatacgga 120

aaacttaccc?ttaaatttat?ttgcactact?ggaaaactac?ctgttccatg?gccaacactt 180

gtcactactt?tctcttatgg?tgttcaatgc?ttttcccgtt?atccggatca?tatgaaacgg 240

catgactttt?tcaagagtgc?catgcccgaa?ggttatgtac?aggaacgcac?tatatctttc 300

aaagatgacg?ggaactacaa?gacgcgtgct?gaagtcaagt?ttgaaggtga?tacccttgtt 360

aatcgtatcg?agttaaaagg?tattgatttt?aaagaagatg?gaaacattct?cggacacaaa 420

ctcgagtaca?actataactc?acacaatgta?tacatcacgg?cagacaaaca?aaagaatgga 480

atcaaagcta?acttcaaaat?tcgccacaac?attgaagatg?gatccgttca?actagcagac 540

cattatcaac?aaaatactcc?aattggcgat?ggccctgtcc?ttttaccaga?caaccattac 600

ctgtcgacac?aatctgccct?ttcgaaagat?cccaacgaaa?agcgtgacca?catggtcctt 660

cttgagtttg?taactgctgc?tgggattaca?catggcatgg?atgagctcta?caaataa 717

<210>7

<211>324

<212>DNA

<213>soxs

<400>7

atgtcccatc?agaaaattat?tcaggatctt?atcgcatgga?ttgacgagca?tattgaccag 60

ccgcttaaca?ttgatgtagt?cgcaaaaaaa?tcaggctatt?caaagtggta?cttgcaacga 120

atgttccgca?cggtgacgca?tcagacgctt?ggcgattaca?ttcgccaacg?ccgcctgtta 180

ctggccgccg?ttgagttgcg?caccaccgag?cgtccgattt?ttgatatcgc?aatggacctg 240

ggttatgtct?cgcagcagac?cttctcccgc?gttttccgtc?ggcagtttga?tcgcactccc 300

agcgattatcgccaccgcctgtaa 324

<210>8

<211>35

<212>DNA

<213>p1

<400>8

aaggaaaaaagcggccgctcgcccgggttacgcaa 35

<210>9

<211>59

<212>DNA

<213>P2

<400>9

acaactccag?tgaaaagttc?ttctccttta?ctcataaatc?tgcctctttt?cagtgttca 59

<210>10

<211>60

<212>DNA

<213>P3

<400>10

tgggattaca?catggcatgg?atgaactata?caaataattt?tattgcccgc?gcgttaactc 60

<210>11

<211>41

<212>DNA

<213>P4

<400>11

cgcacgcatgtcgaccgattttcatacgtc?acggttgata?g 41

<210>12

<211>20

<212>DNA

<213>P5

<400>12

atgagtaa?aggagaagaact 20

<210>13

<211>25

<212>DNA

<213>P6

<400>13

ttatttgtatagttcatccatgcca 25

Claims

1. one kind high responsive intestinal bacteria reporting bacterial strain to oxidation-reduction cycle reactant, it is characterized in that, knock out sodA, sodB, katG, ahpCF, frdABCD gene by wild-type e. coli E.coli MC4100, utilize gene substitution technique that the SoxS encoding gene is replaced with the GFPmut2 reporter gene again and obtain.

2. according to the described intestinal bacteria reporting bacterial strain of claim 1, it is characterized in that described intestinal bacteria reporting bacterial strain called after E.coli WMC-002 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, numbering CGMCC No.2464.

3. the preparation method of the described intestinal bacteria reporting bacterial strain of claim 1 is characterized in that, comprises following steps:

(1) utilizes condition plasmid replication pKOV as instrument, knock out frdABCD, sodB, ahpCF, katG and sodA gene in the wild-type e. coli E.coliMC4100 genome;

(2) design soxS gene N-terminal and the inboard primer of C-terminal and the primer of outside primer and GFPmut2 gene, utilize intersection PCR reaction to make up the fusion PCR fragment that contains reporter gene GFPmut2 and wait to replace the two ends homology arm of goal gene SoxS;

(3) with step (2) gained fragment cloning to pKOV condition plasmid replication, make up the pKOV-GFPmut2 recombinant vectors;

(4) described recombinant vectors pKOV-GFPmut2 is transformed in the coli strain genome of step (1) gained, by twice homologous recombination, with coli strain genome SoxS encoding gene displacement the becoming GFPmut2 reporter gene of step (1) gained.

4. the preparation method of the described intestinal bacteria reporting bacterial strain of claim 3 is characterized in that, the outside primer sequence of soxS gene N-terminal is shown in SEQ ID NO:8 in the described step (2), and inboard primer sequence is shown in SEQ ID NO:9; The inboard primer sequence of soxS gene C end is shown in SEQ ID NO:10, and outside primer sequence is shown in SEQ ID NO:11, and the N-terminal primer sequence of GFPmut2 gene is shown in SEQ IDNO:12, and the C-terminal primer sequence is shown in SEQ ID NO:13.

5. the application of the described intestinal bacteria reporting bacterial strain of claim 1 aspect the detection oxidation-reduction cycle reactant.