CN106191054B - Promoter 3M1F and its application - Google Patents
Promoter 3M1F and its application Download PDFInfo
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- CN106191054B CN106191054B CN201510214182.7A CN201510214182A CN106191054B CN 106191054 B CN106191054 B CN 106191054B CN 201510214182 A CN201510214182 A CN 201510214182A CN 106191054 B CN106191054 B CN 106191054B
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Abstract
The invention discloses promoter 3M1F and its applications.Promoter provided by the invention, is named as 3M1F, is the DNA molecular shown in the nucleotide of 5 ' end the 7th to 93 of sequence 1 of sequence table.The present invention also protects a kind of DNA molecular (fusion), successively includes sequence 1 DNA molecular and the reporter gene shown in the nucleotide of 5 ' end the 7th to 93 of sequence table from upstream to downstream.The present invention also protects the recombinant plasmid containing the fusion dna.The present invention also protects the recombinant bacterium for obtaining recombinant plasmid importing host strain.The present invention also protects application of the recombinant bacterium in detection 2,4,6- trinitrotoluene and/or 2,6- dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT.Biosensor provided by the invention can be applied not only to the Testing and appraisal of the explosion such as wartime and postwar land mine object location, also provide new method for the TNT detection in soil and water environment.
Description
Technical field
The present invention relates to promoter 3M1F and its applications.
Background technique
2,4,6-trinitrotoluene (English name 2,4,6-Trinitrotoluene, referred to as 2,4,6-TNT or TNT)
It is a kind of colourless or light yellow crystal shape nitrobenzene explosive, is a kind of important industrial chemicals, is opened in national defense industry, mine
It adopts, the fields such as infrastructure play an important role.In explosive production, enterprise zone and many battlefield ruins, military training are used
Area, TNT pollution are always one of main environmental problem.TNT can be penetrated into soil and water system, long-term to participate in ecology
Circulation, or even " pink water " is generated, it clears up very difficult and expensive.It has been reported and shows TNT to microorganism, green algae plant
With the toxic effect of animal, while TNT and its degradation product can be introduced into food chain, and then be caused seriously to human health
Adverse effect.After contacting a large amount of trinitrotoluenes in a short time, acute poisoning can be caused, serious person can cause respiratory failure and dead
It dies.After Long Term Contact trinitrotoluene, slow poisoning can be caused, the target organ of damage be mainly liver, eyes, hematological system and
Nervous system, while immune function being caused to reduce, the probability for suffering from anaemia and cancer can also greatly increase.
Since there is TNT apparent toxic action to have developed the inspection of many TNT currently based on the physicochemical property of TNT
Survey technology, as high performance liquid chromatography (HPLC), ultraviolet, gas chromatography-mass spectrum (GC-MS), laser surface enhance Raman spectrum
(SERS), the methods of nuclear magnetic resonance, ion mobility spectrometry, however they require costly and complicated instrument or complicated sample system
Preparation Method.A research hotspot of the biosensor as present field of engineering technology, with bioactivity unit (such as enzyme, antibody,
Nucleic acid, cell etc.) as response signal is generated after biosensor and object to be detected interaction, signal processing is first
Part receives response signal, is processed, is converted, is exported, to realize the qualitative and quantitative analysis to object.
Currently, the biosensor technology based on target microbic activity has been widely studied for some special changes
Close the detection of object, chemical group and some toxic compounds for causing environmental pollution.Common sensing element is to take part in
The promoter region of cell response, and in numerous selectable response elements (usually reporter gene), chemiluminescence is (such as
LuxCDABE of Photorhabdusluminescens) and fluorescin (such as green fluorescent protein,
It GFP) is the most universal two kinds.Currently, there are the report of a large amount of biological sensing elements for heavy metallic poison compound, example
Such as, DNA enzymatic (deoxyribose enzyme) is due to having high specificity and spirit to heavy metal ion such as Pb (II), Cu (II) and Zn (II)
Quick property and be applied to heavy metal ion sensor.The DNA enzymatic and Pb (II), Hg marked using fluorophor and light group of quenching
(II) and detection limit can be reduced to 10nM by the DNA enzymatic biosensor of the functionalization of Cu (II) complexing.Liao etc. is viscous with calcium
Albumen is controlling gene, using green fluorescent protein as reporter gene, successfully constructs e.colidh5αcell biosensor and uses
The Sb (III) of 0.1nmol/L, the Pb of Cd (II) and 10nmol/L can be detected in the measurement of heavy metal in soils and sediments
(II), high sensitivity and cheap has application prospect in actual sample.
Summary of the invention
The object of the present invention is to provide promoter 3M1F and its applications.
Promoter provided by the invention derives from e. coli k-12 MG1655, is named as 3M1F, is the sequence of sequence table
The DNA molecular shown in the nucleotide of 5 ' end the 7th to 93 of column 1.
The present invention also protects the sequence 1 of sequence table DNA molecular shown in the nucleotide of 5 ' end the 7th to 93 in starting mesh
Gene expression in application.The target gene can be reporter gene, concretely fluorescent marker gene, more specifically can be
GFP gene.GFP gene specifically can be as shown in the sequence 2 of sequence table.The starting destination gene expression is concretely in 2,4,6-
Start purpose under the inducing action of trinitrotoluene and/or 2,6- dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT
Gene expression.
The present invention also protects a kind of DNA molecular (fusion), from upstream to downstream successively include sequence table sequence 1 from
DNA molecular and reporter gene shown in the 5 ' nucleotide of end the 7th to 93.In the DNA molecular, by sequence table sequence 1 from
DNA molecular shown in the 5 ' nucleotide of end the 7th to 93 starts the expression of the reporter gene.The reporter gene is concretely
Fluorescent marker gene more specifically can be GFP gene.GFP gene specifically can be as shown in the sequence 2 of sequence table.
The present invention also protects the recombinant plasmid containing the fusion dna.The recombinant plasmid is concretely in pET-24 (+)
The sequence 1 of the multiple cloning sites insetion sequence table of carrier DNA molecular and GFP base shown in the nucleotide of 5 ' end the 7th to 93
Because of obtained recombinant plasmid, in the recombinant plasmid as the sequence 1 of sequence table shown in the nucleotide of 5 ' end the 7th to 93
DNA molecular starts the expression of the GFP gene.GFP gene specifically can be as shown in the sequence 2 of sequence table.The recombinant plasmid tool
Body can between I restriction enzyme site of Bgl II and Xba of pET-24 (+) carrier insetion sequence table sequence 1 from 5 ' ends the 7th to
The recombinant plasmid that GFP gene obtains is inserted into shown in 93 nucleotide between DNA molecular, BamHI and XhoI restriction enzyme site.
The present invention also protects the recombinant bacterium for obtaining recombinant plasmid importing host strain.The host strain is concretely big
Enterobacteria more specifically can be e. coli bl21 (DE3).The recombinant bacterium be used to detect 2,4,6- trinitrotoluene and/
Or the biosensor of 2,6- dinitrotoluene (DNT) and/or toluene and/or 2,4-DNT.
The present invention also protect the recombinant bacterium detection 2,4,6- trinitrotoluene and/or 2,6- dinitrotoluene (DNT) and/or
Application in toluene and/or 2,4-DNT.
Synthetic biology is the completely new cross discipline proposed at the beginning of 21 century, and the Endy of MIT in 2005 has delivered " work
The basis of journey biology " review paper clearly proposes to be introduced into engineering common " standardization ", " complexity in synthetic biology
System solution lotus root ", " conceptual abstraction " way, the biosystem that synthetic biology is related to be divided into DNA, part, device, system this
4 levels of sample.The research of synthetic biology mainly develops towards both direction at present: first is that design, construction have biological function
The functional unit and its more advanced multiple of element such as biomolecule or reaction system, biological device and idiotype network, multicomponent composition
The assembling etc. of miscellaneous system.Second is that technology required for biology manufactures is established in exploitation, including as htrb gene is combined into technology, it is raw
The analysis and test technology of object function element, the capture of Biont information and processing technique, system simulation and control technology etc..?
Synthesising biological educational circles proposes Bio-Brick thought on the basis of this, both constructs standard, convenient in living cell body, use is existing
New genetic circuits and life system are built in biological elements combination.The present invention provides the completely new starting members that nature is not present
Part, using promoter provided by the invention, according to Bio-brick thought, the detection as core the most in biosensor is first
Part, other biological scholar can assemble the sense line of various dress mine detections, and be the genetic circuits and life of new function
Building for system provides indispensable reserve element.
The present inventor screens the more sensitive starting element of response first from Escherichia coli, then to starting member
Part carries out random mutation and simultaneously constructs random start sublibrary, and the complete of target molecules TNT and its derivative can be responded by obtaining a batch
New promoter.The present inventor is constructed using green fluorescent protein as reporter gene using Escherichia coli the bottom of as using 3M1F
The whole-cell biological sensor of disk, and good specificity and sensitivity have been embodied in the detection, it is later period biosensor
Deeper exploitation provides good element deposit.Biosensor provided by the invention can be applied not only to wartime and war
The Testing and appraisal of the explosion such as land mine object location afterwards also provides new method for the TNT detection in soil and water environment.
Detailed description of the invention
Fig. 1 is the result of embodiment 3.
Fig. 2 is the result of embodiment 4.
Fig. 3 is the EC of 3M1F in embodiment 5200Value.
Fig. 4 is the EC that segment is compareed in embodiment 5200Value.
Fig. 5 is the result of embodiment 6.
Fig. 6 is the result of embodiment 7.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.All statistical analysis are all made of 18.0 statistics software of SPSS.
PET24-GFP carrier: GFP gene shown in the sequence 2 by sequence table be inserted into pET-24 (+) carrier BamHI and
Between XhoI restriction enzyme site, pET24-GFP carrier is obtained.PET-24 (+) carrier: EMD Biosciences (Novagen) is produced
Product catalog number (Cat.No.) is 69749-3.E. coli bl21 (DE3): Bo Maide biotech firm (Beijing, China).Carrier pMD18-T:
Takara Biotechnology(Dalian,China)。
2,4,6-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), nitrobenzene (Nitrobenzene) are purchased from Sigma-Aldrich company.The dissolution of TNT
Using ultrasonic method, TNT powder is added in distilled water, 800W ultrasound 15 minutes.Other compounds are first dissolved in methanol, then exist
Dilution in Tris buffer (200mM, pH7.0).
The discovery of embodiment 1, promoter sequence
1, the DNA sequence of e. coli k-12 MG1655 is extracted.
2, the DNA sequence for obtaining step 1 is amplified as template using NEB Q5 MIX High fidelity PCR system
Starting element.
3, all amplified productions for obtaining step 2 pass through Taq enzyme polymerization reaction addition dATP respectively, form polyA, then
It is connected into carrier pMD18-T by DNA attended operation, obtains recombinant plasmid.
4, the recombinant plasmid obtained with restriction enzyme Xba I and II double digestion step 3 of Bgl recycles small fragment.
5, with II double digestion pET24-GFP carrier of restriction enzyme Xba I and Bgl, carrier framework is recycled.
6, the small fragment that step 4 obtains is connect with the carrier framework that step 5 obtains, obtains recombinant plasmid.
7, the recombinant plasmid for obtaining step 6 imports e. coli bl21 (DE3), obtains recombinant bacterium.
8, the recombinant bacterium that step 7 obtains is investigated under different TNT concentration, it is found that part of starting element exists
Under the TNT concentration of various concentration can excitation downstream reporter gene GFP in various degree expression, and 15mg/L's
Responsing reaction is the most obvious under TNT concentration.
9, on the basis of step 8, concensus sequence of each starting element in functioning is extracted, carries out random mutation
And random start sublibrary is constructed, each DNA molecular in new promoter library is successively subjected to step 3,4,5 and 6, obtains library
The library promoter::GFP (i.e. recombinant plasmid library) that capacity is 4500.
10, the recombinant plasmid for obtaining step 9 is directed respectively into e. coli bl21 (DE3), obtains each recombinant bacterium.
11, the recombinant bacterium for taking step 10 to obtain, culture to OD600nmTNT is added when=0.2 and makes its concentration 15mg/L,
Then 30 DEG C, 200rpm shaken cultivation 8 hours draw 200 μ l bacterium solutions to 96 orifice plate of black, in Pekin Elmer2300
Multilablel Reader is detected (GFP testing conditions are excitation/emission, 485/535nm), and numerical value exists
200000 or more are set to positive colony, carry out fluorescent value detection after positive colony is transferred again, finally in 15mg/L TNT
1 clone strong by TNT regulation response has been filtered out under concentration, and corresponding promoter is named as 3M1F.
The sequence 1 of the nucleotide sequence of 3M1F such as sequence table is from shown in the nucleotide of 5 ' end the 7th to 93.
Embodiment 2, recombinant bacterium and the acquisition for compareing bacterium
One, the acquisition of recombinant bacterium
1, double chain DNA molecule shown in the sequence 1 of composition sequence table.
2, the double chain DNA molecule obtained with restriction enzyme Bgl II and I double digestion step 1 of Xba, recycling digestion produce
Object.
3, with I double digestion pET24-GFP carrier of restriction enzyme Bgl II and Xba, the carrier bone of about 6000bp is recycled
Frame.
4, the digestion products of step 2 are connected with the carrier framework of step 3, obtains recombinant plasmid.
5, the recombinant plasmid for obtaining step 4 imports e. coli bl21 (DE3), obtains recombinant bacterium.
Two, the acquisition of bacterium is compareed
1, by the Bgl II of T7 promoter (double chain DNA molecule shown in the sequence 3 of sequence table) insertion pET24-GFP carrier
Between I restriction enzyme site of Xba, recombinant plasmid is obtained.
2, the recombinant plasmid for obtaining step 1 imports e. coli bl21 (DE3), obtains control fungus beetle.
3, by II He of Bgl of N1 segment (double chain DNA molecule shown in the sequence 4 of sequence table) insertion pET24-GFP carrier
Between I restriction enzyme site of Xba, recombinant plasmid is obtained.
4, the recombinant plasmid for obtaining step 3 imports e. coli bl21 (DE3), obtains control bacterium second.
5, by II He of Bgl of N2 segment (double chain DNA molecule shown in the sequence 5 of sequence table) insertion pET24-GFP carrier
Between I restriction enzyme site of Xba, recombinant plasmid is obtained.
6, the recombinant plasmid for obtaining step 5 imports e. coli bl21 (DE3), obtains control bacterium third.
7, by the Bgl II of recA segment (double chain DNA molecule shown in the sequence 6 of sequence table) insertion pET24-GFP carrier
Between I restriction enzyme site of Xba, recombinant plasmid is obtained.
8, the recombinant plasmid for obtaining step 7 imports e. coli bl21 (DE3), obtains control bacterium A.
9, by the Bgl II of umuDC segment (double chain DNA molecule shown in the sequence 7 of sequence table) insertion pET24-GFP carrier
Between I restriction enzyme site of Xba, recombinant plasmid is obtained.
10, the recombinant plasmid for obtaining step 9 imports e. coli bl21 (DE3), obtains control bacterium B.
11, by the Bgl II of SulA segment (double chain DNA molecule shown in the sequence 8 of sequence table) insertion pET24-GFP carrier
Between I restriction enzyme site of Xba, recombinant plasmid is obtained.
12, the recombinant plasmid for obtaining step 11 imports e. coli bl21 (DE3), obtains control bacterium C.
The functional verification of embodiment 3, promoter
The processing method of recombinant bacterium TNT group: one obtained recombinant bacterium the step of embodiment 2 is seeded to LB Liquid Culture
Base, culture to OD600nmTNT is added when=0.6 and makes its concentration 15mg/L, then 30 DEG C, 200rpm shaken cultivation 12h, so
After draw 200 μ l bacterium solutions to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer 2300Multilablel
Reader is detected (GFP testing conditions are excitation/emission, 485/535nm).
The processing method of recombinant bacterium control group: one obtained recombinant bacterium the step of embodiment 2 is seeded to LB Liquid Culture
Base, culture to OD600nm=0.6, then then 30 DEG C, 200rpm shaken cultivation 12h draw 200 μ l bacterium solutions to 96 hole polyphenyl second
Alkene detection plate (Bio-rad), is detected that (GFP testing conditions are in 2300 Multilablel Reader of Pekin Elmer
excitation/emission,485/535nm)。
It compares the processing method of bacterium TNT group: the control fungus beetle that the step of embodiment 2 two obtain is seeded to LB Liquid Culture
Base, culture to OD600nmTNT is added when=0.6 and makes its concentration 15mg/L, then 30 DEG C, 200rpm shaken cultivation 12h, so
After draw 200 μ l bacterium solutions to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer 2300Multilablel
Reader is detected (GFP testing conditions are excitation/emission, 485/535nm).
It compares the processing method of bacterium IPTG group: the control fungus beetle that the step of embodiment 2 two obtain is seeded to the training of LB liquid
Support base, culture to OD600nmIPTG is added when=0.6 and makes its concentration 1mM, then 30 DEG C, 200rpm shaken cultivation 12h, so
After draw 200 μ l bacterium solutions to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer 2300Multilablel
Reader is detected (GFP testing conditions are excitation/emission, 485/535nm).
It compares the processing method of bacterium control group: the control fungus beetle that the step of embodiment 2 two obtain is seeded to the training of LB liquid
Support base, culture to OD600nm=0.6, then then 30 DEG C, 200rpm shaken cultivation 12h draw 200 μ l bacterium solutions to 96 hole polyphenyl
Ethylene detection plate (Bio-rad) is detected (GFP testing conditions in 2300 Multilablel Reader of Pekin Elmer
For excitation/emission, 485/535nm).
The fluorescence intensity indicated by instrument, to the induction of target chemical combination, is used in inducer and exists come indication promoter
Intensity (the Δ RFU=RFU of induction is embodied with the fluorescence value difference (Δ RFU) in the case where being not presentTNT or IPTG-RFUControl)。
The result is shown in Figure 1.Under the induction of TNT, 3M1F can star the expression of GFP gene, and IPTG does not induce 3M1F
Effect.
The further functional verification of embodiment 4, promoter
Control fungus beetle that the step of recombinant bacterium that the step of Example 2 one obtains, embodiment 2 two obtains, embodiment 2
The step of control bacterium second that step 2 obtains or embodiment 2 two obtained control bacterium third, be seeded to LB liquid medium, culture is extremely
OD600nm=2, then OD is diluted to LB liquid medium600nm=0.6, it then draws 1 μ l bacterium solution and is added dropwise in culture medium flat plate
In the hole of (the LB culture medium flat plate containing 1mM IPTG, LB culture medium flat plate or LB culture medium flat plate containing 15mg/L TNT)
(being punched on culture medium flat plate with the punch that diameter is 3mm), 30 DEG C stationary culture 8 hours, then in ClinX fluorescence imaging
Plate imaging is carried out in system.
Photo is shown in Fig. 2 (A:LB culture medium flat plate；B: the LB culture medium flat plate containing 1mM IPTG；C: contain 15mg/L
The LB culture medium flat plate of TNT；D: the promoter of bacterium corresponding to each hole in each plate).Under conditions of no inducer,
Bacterium colony corresponding to 3M1F, T7, N1, N2 does not have apparent fluorescence reaction.In the case where TNT induction, the corresponding bacterium of 3M1F, T7
It falls and shows apparent fluorescence, the starting activity of 3M1F is higher than T7.In the case where IPTG induction, the corresponding bacterium of only T7
It falls and shows fluorescence.The result shows that 3M1F has certain selectivity to starting inducer, and its induced activity can achieve
With the comparable activity of strong promoter T7.
Embodiment 5, sensitivity
EC200Value be the more commonly used sensitivity Detection index in moment sensor building field, EC200Two can be caused by referring to
The concentration of inducer when the reaction of times indication index, EC200Value it is smaller, then illustrate reaction sensibility it is stronger.
One, the EC of 3M1F200Value
The recombinant bacterium that the step of Example 2 one obtains is seeded to LB liquid medium, culture to OD600nmAdd when=0.6
Enter TNT and makes its concentration gradient concentration (control treatment that setting is added without TNT), then 30 DEG C, 200rpm shaken cultivation 8h,
Then 200 μ l bacterium solutions are drawn to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer2300 Multilablel
Reader is detected (GFP testing conditions are excitation/emission, 485/535nm).
As a result see Fig. 3 (Δ RFU=RFUTNT-RFUControl)。
The minimum concentration of TNT when record multiple variation reaches 2 times of control.The result shows that the EC of 3M1F200Value reaches
0.01mg/L。
Two, the EC of segment is compareed200Value
Control bacterium A, the control bacterium B or control bacterium C that the step of Example 2 two obtains, are seeded to LB liquid medium, train
It supports to OD600nmTNT is added when=0.6 and makes its gradient concentration (control treatment that setting is added without TNT), then 30 DEG C,
Then 200rpm shaken cultivation 8h draws 200 μ l bacterium solutions to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer
2300 Multilablel Reader are detected (GFP testing conditions are excitation/emission, 485/535nm).
As a result see Fig. 4 (Δ RFU=RFUTNT-RFUControl).The result shows that each segment that compares is made in the case where TNT induction
It is all very low for the activity of promoter.
Embodiment 6, timeliness
The recombinant bacterium that the step of Example 2 one obtains is seeded to LB liquid medium, culture to OD600nmAdd when=0.6
Enter TNT and makes its concentration 0.10mg/L (control treatment that setting is added without TNT), then 30 DEG C, 200rpm shaken cultivation one
It fixes time, then draws 200 μ l bacterium solutions to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer 2300
Multilablel Reader is detected (GFP testing conditions are excitation/emission, 485/535nm).
As a result see Fig. 5 (Δ RFU=RFUTNT-RFUControl).TNT induces the expression of 50min or so 3M1F unlatching reporter gene,
TNT induction 90min or so reaction reach one it is obvious to specific strength, reach the change in fluorescence of 2 times or more, TNT induction 8
Fluorescence reaction tends towards stability substantially after hour.
Embodiment 7, specificity
The recombinant bacterium that the step of Example 2 one obtains is seeded to LB liquid medium, culture to OD600nmAdd when=0.2
Enter target compound and make 66.04 μm/L of its concentration, then then 30 DEG C, 200rpm shaken cultivation 8 hours draw 200 μ l
Bacterium solution is examined to 96 hole polystyrene detection plates (Bio-rad) in 2300 Multilablel Reader of Pekin Elmer
Survey (GFP testing conditions are excitation/emission, 485/535nm).
Target compound be 2,4,6-TNT, 2,4-DNT, 2,6-DNT, 1,3-DNB, 1,4-DNB, 2-NT, 3-NT, 4-NT,
Toluene or nitrobenzene.
As a result see Fig. 6 (Δ RFU=RFUTNT-RFUControl).Reaction of the 3M1F to TNT, 2,6-DNT, Toluene, 2,4-DNT
There is significant difference compared with other compounds.
Claims (10)
1. the sequence 1 of sequence table DNA molecular shown in the nucleotide of 5 ' end the 7th to 93.
2. the sequence 1 of sequence table DNA molecular shown in the nucleotide of 5 ' end the 7th to 93 is in 2,4,6- trinitrotoluene or 2,
Start the application in destination gene expression under the induction of 6- dinitrotoluene (DNT) or toluene.
It successively include the sequence 1 of sequence table from the nucleotide institute of 5 ' end the 7th to 93 from upstream to downstream 3. a kind of DNA molecular
The DNA molecular and reporter gene shown.
4. DNA molecular as claimed in claim 3, it is characterised in that: the reporter gene is fluorescent marker gene.
5. DNA molecular as claimed in claim 4, it is characterised in that: the fluorescent marker gene is GFP gene.
6. the recombinant plasmid containing the DNA molecular any in claim 3 to 5.
7. recombinant plasmid as claimed in claim 6, it is characterised in that: the recombinant plasmid be in pET-24(+) carrier it is more
The sequence 1 of cloning site insetion sequence table DNA molecular and GFP gene shown in the nucleotide of 5 ' end the 7th to 93 obtains
Recombinant plasmid, as the sequence 1 of sequence table, the DNA molecular shown in the nucleotide of 5 ' end the 7th to 93 is opened in the recombinant plasmid
Move the expression of the GFP gene.
8. the recombinant plasmid of claim 6 or 7 is imported the recombinant bacterium that host strain obtains.
9. recombinant bacterium as claimed in claim 8, it is characterised in that: the host strain is Escherichia coli.
10. the recombinant bacterium of claim 8 or 9 is in detection 2,4,6- trinitrotoluene or 2,6- dinitrotoluene (DNT) or toluene
Using.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
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| Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene;Sharon Yagur-Kroll et al.;《Appl Microbiol Biotechnol》;20130425;第98卷;第891页左栏第2段-右栏第1段 * |
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