CN106191049A - Promoter 32M6A and application thereof - Google Patents
Promoter 32M6A and application thereof Download PDFInfo
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- CN106191049A CN106191049A CN201510212572.0A CN201510212572A CN106191049A CN 106191049 A CN106191049 A CN 106191049A CN 201510212572 A CN201510212572 A CN 201510212572A CN 106191049 A CN106191049 A CN 106191049A
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Abstract
The invention discloses promoter 32M6A and application thereof.The promoter that the present invention provides, named 32M6A, for the sequence 1 of sequence table from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end.The present invention also protects a kind of DNA molecular (fusion gene), includes that from upstream to downstream the sequence 1 of sequence table is from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end and reporter gene successively.The present invention also protects the recombiant plasmid containing described fusion dna.The present invention also protects the recombinant bacterium obtained by described recombiant plasmid importing Host Strains.The present invention also protects the application in detecting 2,4,6-trinitrotoluene and/or 2,6-dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT and/or 1,4-dinitro benzene of the described recombinant bacterium.The biosensor that the present invention provides can be applied not only to the Testing and appraisal of the explosive position such as wartime and postwar Rhizoma Anemones flaccidae, also detects the method providing new for the TNT in soil and water environment.
Description
Technical field
The present invention relates to promoter 32M6A and application thereof.
Background technology
2,4, (English name is 2 to 6-trinitrotoluene, 4,6-Trinitrotoluene, it is called for short 2,4,6-TNT or TNT) it is a kind of colourless or light yellow crystal shape Nitrobenzol explosive, it is a kind of important industrial chemicals, all plays an important role in fields such as national defense industry, mining, capital constructions.In explosive production, using enterprise zone, and many ruins, battlefield, Military Training Area, TNT pollutes one of main environmental problem.TNT can penetrate in the middle of soil and water system, participates in ecological circulation for a long time, even produces " pink water ", and its cleaning is the most difficult and expensive.Have been reported show TNT to microorganism, the most toxic effect of green algae plant and animal, TNT and degradation product thereof can be introduced in food chain simultaneously, and then human health causes serious harmful effect.After contacting a large amount of trinitrotoluene in a short time, can cause acute poisoning, severe patient can cause respiratory failure and dead.After Long Term Contact trinitrotoluene, can cause chronic poisoning, the target organ of its infringement is mainly liver, eyes, blood system and nervous system, causes immunologic function to reduce simultaneously, and the probability suffering from anemia and cancer also can be greatly increased.
Owing to TNT has obvious toxic action, it is currently based on the physicochemical property of TNT, have been developed for the detection technique of many TNT, as high performance liquid chromatography (HPLC), ultraviolet, gas chromatography-mass spectrum (GC-MS), laser surface strengthen the methods such as Raman spectrum (SERS), nuclear magnetic resonance, NMR, ion mobility spectrometry, but they are required for costly and complicated instrument or complicated sample preparation methods.Biosensor is as a study hotspot of present field of engineering technology, response signal is produced using biological activity unit (such as enzyme, antibody, nucleic acid, cell etc.) after interacting as biosensor with object to be detected, response signal is received, processes, changes, exports by Signal Processing Element, thus realizes the qualitative and quantitative analysis to object.
At present, based on the bioactive biosensor technology of object be widely studied for some special compounds, chemical group and some can cause the detection of toxic compounds of environmental pollution.Conventional sensing element is the promoter region that take part in cell response, and in numerous selectable response elements (usually reporter gene), chemiluminescence (such as luxCDABE of Photorhabdusluminescens) and fluorescin (such as green fluorescent protein, GFP) are the most universal two kind.Currently, there is the report of a large amount of biological sensing element for heavy metallic poison compound, such as, DNA enzymatic (deoxyribose enzyme) has high specificity and susceptiveness and is applied to heavy metal ion sensor due to heavy metal ion such as Pb (II), Cu (II) and Zn (II).Use the DNA enzymatic of fluorophor and light group labelling of quenching detection limit can be reduced to 10nM with the DNA enzymatic biosensor of Pb (II), Hg (II) and the functionalization of Cu (II) complexation.Liao etc. are with cadherin as controlling gene, with green fluorescent protein as reporter gene, success builds e.colidh5αcell biosensor mensuration of heavy metal in soils and sediments, can detect that the Pb (II) of the Sb (III) of 0.1nmol/L, Cd (II) and 10nmol/L, highly sensitive and low price, has application prospect in actual sample.
Summary of the invention
It is an object of the invention to provide promoter 32M6A and application thereof.
The promoter that the present invention provides, derives from e. coli k-12 MG1655, named 32M6A, for the sequence 1 of sequence table from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end.
The present invention also protects the sequence 1 of sequence table from the application in starting destination gene expression of the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end.Described genes of interest can be reporter gene, concretely fluorescent marker gene, can be more specifically GFP gene.GFP gene specifically can be as shown in the sequence 2 of sequence table.Described startup destination gene expression concretely starts destination gene expression under the inducing action of 2,4,6-trinitrotoluene and/or 2,6-dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT and/or 1,4-dinitro benzene.
The present invention also protects a kind of DNA molecular (fusion gene), includes that from upstream to downstream the sequence 1 of sequence table is from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end and reporter gene successively.In described DNA molecular, the sequence 1 of sequence table start the expression of described reporter gene from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end.Described reporter gene concretely fluorescent marker gene, can be more specifically GFP gene.GFP gene specifically can be as shown in the sequence 2 of sequence table.
The present invention also protects the recombiant plasmid containing described fusion dna.Described recombiant plasmid concretely pET-24 (+) recombiant plasmid that the sequence 1 of the multiple clone site insertion sequence table of carrier obtains from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end and GFP gene, described recombiant plasmid is started from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end the expression of described GFP gene by the sequence 1 of sequence table.GFP gene specifically can be as shown in the sequence 2 of sequence table.Described recombiant plasmid concretely pET-24 (+) between Bgl II and Xba I restriction enzyme site of carrier the sequence 1 of insertion sequence table from inserting the recombiant plasmid that GFP gene obtains between the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end, BamHI and XhoI restriction enzyme site.
The present invention also protects the recombinant bacterium obtained by described recombiant plasmid importing Host Strains.Described Host Strains concretely escherichia coli, can be more specifically e. coli bl21 (DE3).Described recombinant bacterium is for detecting 2,4,6-trinitrotoluene and/or 2,6-dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT and/or the biosensor of 1,4-dinitro benzene.
The present invention also protects the application in detecting 2,4,6-trinitrotoluene and/or 2,6-dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT and/or 1,4-dinitro benzene of the described recombinant bacterium.
Synthetic biology is the brand-new cross discipline proposed at the beginning of 21 century, the Endy of MIT in 2005 has delivered " basis of bionics " review paper, clearly proposing to introduce " standardization ", " complication system solution Rhizoma Nelumbinis ", " conceptual abstraction " way conventional in engineering in synthetic biology, the biosystem related to by synthetic biology is divided into DNA, part, device, such 4 levels of system.The research of synthetic biology currently mainly towards both direction develop: one be design, build have element such as biomolecule or response system, biological device and the idiotype network of biological function, multicomponent form functional unit and the assembling etc. of higher level complication system.Two is the technology that exploitation sets up required for biology manufactures, and is combined into technology including such as htrb gene, the analysis and test technology of biological function element, the capture of Biont information and treatment technology, system simulation and control technology etc..Synthesising biological educational circles proposes Bio-Brick thought on this basis, has both built standard, it is simple in living cell body, uses the combination of existing biological elements to build new genetic circuits and biosystem.The invention provides the non-existent brand-new starting element of nature, utilize the promoter that the present invention provides, according to Bio-brick thought, as the detecting element of core the most in biosensor, other biological scholar can assemble the sense line of various dress mine detection, and provide indispensable stock element for building of the genetic circuits of New function and biosystem.
First the present inventor screens the starting element that response is the sensitiveest from escherichia coli, then starting element carries out random mutation and builds random start sublibrary, it is thus achieved that a collection of brand-new promoter responding target molecules TNT and derivant thereof.The present inventor uses 32M6A, with green fluorescent protein as reporter gene, construct the whole-cell biological sensor with escherichia coli as chassis, and embodied good specificity and sensitivity in the detection, provide good element deposit for the deeper exploitation of later stage biosensor.The biosensor that the present invention provides can be applied not only to the Testing and appraisal of the explosive position such as wartime and postwar Rhizoma Anemones flaccidae, also detects the method providing new for the TNT in soil and water environment.
Accompanying drawing explanation
Fig. 1 is the result of embodiment 3.
Fig. 2 is the result of embodiment 4.
Fig. 3 is the EC of 32M6A in embodiment 5200Value.
Fig. 4 is the EC compareing fragment in embodiment 5200Value.
Fig. 5 is the result of embodiment 6.
Fig. 6 is the result of embodiment 7.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.All statistical analysis all use SPSS 18.0 statistics software.
PET24-GFP carrier: the GFP gene shown in the sequence 2 of sequence table is inserted pET-24 (+) between BamHI and the XhoI restriction enzyme site of carrier, obtain pET24-GFP carrier.PET-24 (+) carrier: EMD Biosciences (Novagen), catalog number is 69749-3.E. coli bl21 (DE3): Bo Maide biotech firm (Beijing, China).Carrier pMD18-T:Takara Biotechnology (Dalian, China).
null2,4,6-Trinitrotoluene(TNT)、2,4-Dinitrotoluene(2,4-DNT)、2,6-Dinitrotoluene(2,6-DNT)、1,3-Dinitrobenzene(1,3-DNB)、1,4-Dinitrobenzene(1,4-DNB)、2-Nitrotoluene(2-NT)、3-Nitrotoluene(3-NT)、4-Nitrotoluene(4-NT)、Benzene (Benzene)、Toluene (Toluene)、Nitrobenzol (Nitrobenzene) is purchased from Sigma-Aldrich company.The dissolving of TNT uses ultrasonic method, is added by TNT powder in distilled water, ultrasonic 15 minutes of 800W.Other compound is all first dissolved in methanol, then dilution in Tris buffer (200mM, pH7.0).
Embodiment 1, the discovery of promoter sequence
1, the chromogene group of e. coli k-12 MG1655 is extracted.
2, chromogene group step 1 obtained, as template, uses NEB Q5MIX High fidelity PCR system to amplify starting element.
3, all amplified productions step 2 obtained add dATP respectively through Taq enzyme polyreaction, form polyA, then are connected into carrier pMD18-T by DNA attended operation, obtain recombiant plasmid.
4, the recombiant plasmid obtained by restricted enzyme Xba I and Bgl II double digestion step 3, reclaims small fragment.
5, with restricted enzyme Xba I and Bgl II double digestion pET24-GFP carrier, carrier framework is reclaimed.
6, the carrier framework that small fragment step 4 obtained obtains with step 5 is connected, and obtains recombiant plasmid.
7, recombiant plasmid step 6 obtained imports e. coli bl21 (DE3), obtains recombinant bacterium.
8, recombinant bacterium step 7 obtained is investigated under different TNT concentration, find that wherein part starting element can excite the expression of downstream reporter gene GFP under the TNT concentration of variable concentrations in various degree, and responsing reaction is the most obvious under the TNT concentration of 15mg/L.
9, on the basis of step 8, extract each starting element concensus sequence in function, carry out random mutation and build random start sublibrary, each DNA molecular in new promoter library is carried out step 3,4,5 and 6 successively, obtains the promoter::GFP library (i.e. recombiant plasmid storehouse) that storage capacity is 4500.
10, the recombiant plasmid that step 9 obtains is directed respectively into e. coli bl21 (DE3), obtains each recombinant bacterium.
11, take the recombinant bacterium that step 10 obtains, cultivate to OD600nmAdd TNT when=0.2 and to make its concentration be 15mg/L, then 30 DEG C, 200rpm shaken cultivation 8 hours, draw 200 μ l bacterium solution to black 96 orifice plate, carry out detecting that (GFP testing conditions is excitation/emission at Pekin Elmer2300Multilablel Reader, 485/535nm), numerical value is set to positive colony more than 200000, fluorescent value detection is again carried out after being transferred by positive colony, under 15mg/L TNT concentration, finally filter out 1 regulated and controled the clone that response is strong by TNT, by named for corresponding promoter 32M6A.
The nucleotide sequence of 32M6A is if the sequence 1 of sequence table is from shown in the 7th to 94 nucleotide of 5 ' end.
Embodiment 2, recombinant bacterium and the acquisition of comparison bacterium
One, the acquisition of recombinant bacterium
1, the double chain DNA molecule shown in sequence 1 of composition sequence table.
2, the double chain DNA molecule obtained by restricted enzyme Bgl II and Xba I double digestion step 1, reclaims digestion products.
3, with restricted enzyme Bgl II and Xba I double digestion pET24-GFP carrier, the carrier framework of about 6000bp is reclaimed.
4, the digestion products of step 2 and the carrier framework of step 3 are connected, obtain recombiant plasmid.
5, recombiant plasmid step 4 obtained imports e. coli bl21 (DE3), obtains recombinant bacterium.
Two, the acquisition of bacterium is compareed
1, T7 promoter (double chain DNA molecule shown in the sequence 3 of sequence table) is inserted between Bgl II and Xba I restriction enzyme site of pET24-GFP carrier, obtain recombiant plasmid.
2, recombiant plasmid step 1 obtained imports e. coli bl21 (DE3), obtains compareing fungus beetle.
3, N1 fragment (double chain DNA molecule shown in the sequence 4 of sequence table) is inserted between Bgl II and Xba I restriction enzyme site of pET24-GFP carrier, obtain recombiant plasmid.
4, recombiant plasmid step 3 obtained imports e. coli bl21 (DE3), obtains compareing bacterium second.
5, N2 fragment (double chain DNA molecule shown in the sequence 5 of sequence table) is inserted between Bgl II and Xba I restriction enzyme site of pET24-GFP carrier, obtain recombiant plasmid.
6, recombiant plasmid step 5 obtained imports e. coli bl21 (DE3), obtains compareing bacterium third.
7, recA fragment (double chain DNA molecule shown in the sequence 6 of sequence table) is inserted between Bgl II and Xba I restriction enzyme site of pET24-GFP carrier, obtain recombiant plasmid.
8, recombiant plasmid step 7 obtained imports e. coli bl21 (DE3), obtains compareing bacterium A.
9, umuDC fragment (double chain DNA molecule shown in the sequence 7 of sequence table) is inserted between Bgl II and Xba I restriction enzyme site of pET24-GFP carrier, obtain recombiant plasmid.
10, recombiant plasmid step 9 obtained imports e. coli bl21 (DE3), obtains compareing bacterium B.
11, SulA fragment (double chain DNA molecule shown in the sequence 8 of sequence table) is inserted between Bgl II and Xba I restriction enzyme site of pET24-GFP carrier, obtain recombiant plasmid.
12, recombiant plasmid step 11 obtained imports e. coli bl21 (DE3), obtains compareing bacterium C.
Embodiment 3, the functional verification of promoter
The processing method of recombinant bacterium TNT group: the recombinant bacterium that the step one of embodiment 2 obtains is seeded to LB fluid medium, cultivates to OD600nmAdd TNT when=0.6 and to make its concentration be 15mg/L, then 30 DEG C, 200rpm shaken cultivation 12h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
The processing method of recombinant bacterium matched group: the recombinant bacterium that the step one of embodiment 2 obtains is seeded to LB fluid medium, cultivates to OD600nm=0.6, then 30 DEG C, 200rpm shaken cultivation 12h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
The processing method of comparison bacterium TNT group: the comparison fungus beetle step 2 of embodiment 2 obtained is seeded to LB fluid medium, cultivates to OD600nmAdd TNT when=0.6 and to make its concentration be 15mg/L, then 30 DEG C, 200rpm shaken cultivation 12h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
The processing method of comparison bacterium IPTG group: the comparison fungus beetle step 2 of embodiment 2 obtained is seeded to LB fluid medium, cultivates to OD600nmAdd IPTG when=0.6 and to make its concentration be 1mM, then 30 DEG C, 200rpm shaken cultivation 12h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
The processing method of comparison bacterium matched group: the comparison fungus beetle step 2 of embodiment 2 obtained is seeded to LB fluid medium, cultivates to OD600nm=0.6, then 30 DEG C, 200rpm shaken cultivation 12h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
Carry out the indication promoter induction to target chemical combination by the fluorescence intensity indicated by instrument, be used in the fluorescence value difference (Δ RFU) in the case of inducer exists and be non-existent and embody intensity (the Δ RFU=RFU of inductionTNT Or IPTG-RFUComparison)。
Result is shown in Fig. 1.Under the induction of TNT, 32M6A can start the expression of GFP gene, and IPTG does not has inducing action to 32M6A.
Embodiment 4, the further functional verification of promoter
The comparison bacterium third that the step 2 of comparison fungus beetle that recombinant bacterium that the step one of Example 2 obtains, the step 2 of embodiment 2 obtain, the comparison bacterium second that the step 2 of embodiment 2 obtains or embodiment 2 obtains, is seeded to LB fluid medium, cultivates to OD600nm=2, then it is diluted to OD with LB fluid medium600nm=0.6, then draw 1 μ l bacterium solution to drip in the hole of culture medium flat plate (the LB culture medium flat plate containing 1mM IPTG, the LB culture medium flat plate containing 15mg/L TNT or LB culture medium flat plate) (punching on culture medium flat plate with the card punch of a diameter of 3mm), 30 DEG C of quiescent culture 8 hours, then carry out flat board imaging in ClinX fluorescence imaging system.
Fig. 2 (the picture left above: LB culture medium flat plate is shown in by photo；Top right plot: the LB culture medium flat plate containing 1mM IPTG；Lower-left figure: the LB culture medium flat plate containing 15mg/L TNT；Bottom-right graph: the promoter of bacterium corresponding to each hole in each flat board).Under conditions of without inducer, the bacterium colony corresponding to 32M6A, T7, N1, N2 does not has obvious fluorescence reaction.In the case of TNT induces, the bacterium colony that 32M6A, T7 are corresponding all shows obvious fluorescence, and the startup activity of 32M6A is higher than T7.In the case of IPTG induces, bacterium colony corresponding for only T7 presents fluorescence.Result shows, 32M6A has certain selectivity to starting inducer, and its induced activity can reach the activity suitable with strong promoter T7.
Embodiment 5, sensitivity
EC200Value be that moment sensor builds the more commonly used sensitivity Detection index in field, EC200Refer to the concentration of inducer when twice indication index can be caused to react, EC200Value the least, then illustrate reaction sensitivity the strongest.
One, the EC of 32M6A200Value
The recombinant bacterium that the step one of Example 2 obtains, is seeded to LB fluid medium, cultivates to OD600nmAdd TNT when=0.6 and to make its concentration be the gradient concentration control treatment of TNT (setting be added without), then 30 DEG C, 200rpm shaken cultivation 8h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer2300Multilablel Reader.
Result is shown in Fig. 3 (Δ RFU=RFUTNT-RFUComparison)。
Record multiple change reaches the least concentration of TNT during 2 times of comparison.Result shows, the EC of 32M6A200Value reaches 0.01mg/L.
Two, the EC of fragment is compareed200Value
Comparison bacterium A that the step 2 of Example 2 obtains, comparison bacterium B or comparison bacterium C, be seeded to LB fluid medium, cultivate to OD600nmAdd TNT when=0.6 and be the gradient concentration control treatment of TNT (setting be added without), then 30 DEG C, 200rpm shaken cultivation 8h, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
Result is shown in Fig. 4 (Δ RFU=RFUTNT-RFUComparison).Result shows, in the case of TNT induces, each comparison fragment is the lowest as the activity of promoter.
Embodiment 6, ageing
The recombinant bacterium that the step one of Example 2 obtains, is seeded to LB fluid medium, cultivates to OD600nmAdd TNT when=0.6 and to make its concentration be the 0.10mg/L control treatment of TNT (setting be added without), then 30 DEG C, 200rpm shaken cultivation certain time, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
Result is shown in Fig. 5 (Δ RFU=RFUTNT-RFUComparison).TNT induction about 50min 32M6A opens the expression of reporter gene, TNT induction about 90min reaction reach one obvious to specific strength, reach the change in fluorescence of more than 2 times, after TNT induction 8 hours, fluorescence reaction tends towards stability substantially.
Embodiment 7, specificity
The recombinant bacterium that the step one of Example 2 obtains, is seeded to LB fluid medium, cultivates to OD600nmAdd target compound when=0.2 and to make its concentration be 66.04 μm/L, then 30 DEG C, 200rpm shaken cultivation 8 hours, then 200 μ l bacterium solution are drawn to 96 hole polystyrenes detection plate (Bio-rad), carry out detecting (GFP testing conditions is excitation/emission, 485/535nm) at Pekin Elmer 2300Multilablel Reader.
Target compound is 2,4,6-TNT, 2,4-DNT, 2,6-DNT, 1,3-DNB, 1,4-DNB, 2-NT, 3-NT, 4-NT, Toluene or Nitrobenzol.
Result is shown in Fig. 6 (Δ RFU=RFUTNT-RFUComparison).32M6A all has significant difference to reaction relatively other compounds of TNT, 2,6-DNT, Toluene, 2,4-DNT, 1,4-DNB.
Claims (10)
1. the sequence 1 of sequence table is from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end.
2. the sequence 1 of sequence table is starting purpose base from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end
Because of the application in expressing.
3. a DNA molecular, includes to downstream successively that from upstream the sequence 1 of sequence table is from 5 ' ends the 7th to 94
Position DNA molecular shown in nucleotide and reporter gene.
4. DNA molecular as claimed in claim 3, it is characterised in that: described reporter gene is fluorescent marker gene.
5. DNA molecular as claimed in claim 4, it is characterised in that: described fluorescent marker gene is GFP gene.
6. contain the recombiant plasmid of arbitrary described DNA molecular in claim 3 to 5.
7. recombiant plasmid as claimed in claim 6, it is characterised in that: described recombiant plasmid be pET-24 (+)
The sequence 1 of the multiple clone site insertion sequence table of carrier is from the DNA molecular shown in the 7th to 94 nucleotide of 5 ' end
The recombiant plasmid obtained with GFP gene, by the sequence 1 of sequence table from 5 ' ends the 7th to 94 in described recombiant plasmid
Position DNA molecular shown in nucleotide starts the expression of described GFP gene.
8. recombiant plasmid described in claim 6 or 7 is imported the recombinant bacterium that Host Strains obtains.
9. recombinant bacterium as claimed in claim 8, it is characterised in that: described Host Strains is escherichia coli.
10. recombinant bacterium described in claim 8 or 9 is at detection 2,4,6-trinitrotoluene and/or 2,6-dinitrotoluene (DNT)
And/or the application in toluene and/or 2,4-DNT and/or 1,4-dinitro benzene.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021220A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院生态环境研究中心 | Improved technology of microbial cell sensor for detecting toluene organic pollutants |
| CN102021219A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院生态环境研究中心 | Microbial cell sensor for detecting toluene organic pollutants |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021220A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院生态环境研究中心 | Improved technology of microbial cell sensor for detecting toluene organic pollutants |
| CN102021219A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院生态环境研究中心 | Microbial cell sensor for detecting toluene organic pollutants |
Non-Patent Citations (3)
| Title |
|---|
| JUNJIE TAN ET AL.: "Construction of 2,4,6-Trinitrotoluene Biosensors with Novel Sensing Elements from Escherichia coli K-12 MG1655", 《CELL BIOCHEM BIOPHYS》 * |
| SHARON YAGUR-KROLL ET AL.: "Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene", 《APPL MICROBIOL BIOTECHNOL》 * |
| 阚乃鹏: "以构建启动子文库的方法筛选新的2,4,6-三硝基甲苯感应元件", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
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