CN106544345B - Promoter 5M6A and its application - Google Patents
Promoter 5M6A and its application Download PDFInfo
- Publication number
- CN106544345B CN106544345B CN201610816647.0A CN201610816647A CN106544345B CN 106544345 B CN106544345 B CN 106544345B CN 201610816647 A CN201610816647 A CN 201610816647A CN 106544345 B CN106544345 B CN 106544345B
- Authority
- CN
- China
- Prior art keywords
- dnt
- trinitrotoluene
- dinitrobenzene
- detection
- contain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/60—Vectors containing traps for, e.g. exons, promoters
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses promoter 5M6A and its applications.Promoter 5M6A provided by the invention is DNA molecular shown in the sequence 1 of sequence table.The present invention passes through building promoter library and Screening Platform, the new sensing element to work to target compound is searched out, realize detection more highly sensitive to target contaminant TNT and specific, good element deposit is provided for the deeper exploitation of later period biosensor, it 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 invention belongs to field of biotechnology, and in particular to promoter 5M6A and its application.
Background technique
Synthetic biology is a 21 century emerging cross discipline, is referred to according to certain rule and existing information
New biological elements, device and system are designed and constructed, or redesign existing natural biological system.Its core is to set
Biological elements, metabolic pathway are rebuild in meter, transformation, or even create the cell with vital movement ability and bion.Biology
Element, device and system are the three basic elements of synthetic biology.Biological elements as one of element refer to genetic system
In most simple, most basic biological block (BioBrick), be the amino acid or nucleotide sequence with specific function, can be with
Say it is the foundation stone of synthetic biology.And synthetic biology is also just being with the most significant difference of genetic engineering and metabolic engineering
A large amount of biological elements can be carried out quickly, arbitrarily assembling, to form the new device with special biological.And it is real
The premise of this existing target is to need largely to excavate, identify and be transformed more biological elements, and therefore, biological elements are still at this stage
Old is the significant concern point of synthetic biology.
Biosensor based on the building of synthetic biology technology is that a kind of pair of biological substance is sensitive and convert its concentration
For the instrument that electric signal is detected, it is made of biomolecule recognition component and all kinds of physics, chemical energy converter, is used for various lifes
Order the analysis and detection of substance and chemical substance.Molecular recognition elements are can to identify and combine measured matter and cause itself to change
Reaction is learned, and then downstream signal is caused to change, it can be said that molecular recognition part is the base that biosensor selectively measures
Plinth, and according to the difference of recognition component, enzyme biologic sensor, immunosensor, tissus sensor and microorganism can be subdivided into and passed
Sensor etc., and microbiological sensor is most studied.It is to use micro-organisms living cell as sensitive material, in vivo using it
Metabolic system measure and identify analyte；Recent study direction is that Reports component (such as lux, gfp, lacZ) is same
A derivable biological elements (such as gene promoter) blend, to detect interested target substance.Microbiological sensor
Can distinctive signal in feedback environment, can be used to identify the pathogenic microorganisms in water and in food, or monitoring soil, air,
Hazardous chemical in water；Accordingly, it can be said that the detection that biosensor is environmental contaminants opens new path.
2,4,6-trinitrotoluene (English name 2,4,6-Trinitrotoluene, referred to as 2,4,6-TNT or TNT)
It is the main fill of land mine, the life security of people is seriously threatened with the long-term effect of its explosion property.It is counted according to the United Nations,
The whole world at least 78 countries, which are still left mine fields and explosive by war, at present is influenced；And TNT can penetrate into soil
In earth and water system, ecological circulation is participated in for a long time, and degradation product can also be introduced into food chain, cause to environment and human health
Serious adverse effect.Therefore the detection of land mine in military affairs, explosive can not only be improved by developing effective TNT detection method
Effect, at the same it is all significant to pollution detection, prevention and treatment, anti-terrorism and maintenance on civil etc..
TNT also includes inevitable by-product in a variety of industrial manufacturing process in addition to itself in industrial processes,
Including 1,3- dinitrobenzene (1,3-DNB) and 2,4- dinitrotoluene (DNT) (2,4-DNT), these compounds in usual land mine can lead to
It crosses plastic components slowly to leak, with its vapor migration to surface, therefore usually with TNT, 1,3-DNB and 2,4-DNT is target
Molecule indicates the positions of the explosives such as land mine.Currently based on the physicochemical property of TNT, the detection skill of many TNT has been exploited
Art, such as high performance liquid chromatography, ultraviolet detection, however they require costly and complicated instrument or complicated sample preparation side
Method, it is still horizontal in laboratory testing.
Summary of the invention
The first purpose of the invention is to provide a kind of DNA fragmentations.
DNA fragmentation provided by the invention is following 1) -3) in any DNA molecular:
1) DNA molecular shown in sequence 1 in sequence table；
2) hybridize and have under strict conditions the DNA molecular of promoter function with the 1) DNA molecular；
3) with 1) described in DNA molecular have 90% or more homology, and with promoter function DNA molecular.
A second object of the present invention is to provide biomaterials relevant to above-mentioned DNA molecular.
Biomaterial relevant to above-mentioned DNA molecular provided by the invention is following A 1) any one of to A7):
A1) contain the expression cassette of above-mentioned DNA molecular；
A2) contain the recombinant vector of above-mentioned DNA molecular；
A3) contain A1) recombinant vector of the expression cassette；
A4) contain the recombinant microorganism of above-mentioned DNA molecular；
A5) contain A1) recombinant microorganism of the expression cassette；
A6) contain A2) recombinant microorganism of the recombinant vector；
A7) contain A3) recombinant microorganism of the recombinant vector.
In above-mentioned biomaterial,
The expression cassette from upstream to downstream successively include DNA molecular shown in the sequence 1 of sequence table and target gene.
In above-mentioned biomaterial,
The target gene is reporter gene.
In above-mentioned biomaterial,
The reporter gene is fluorescin encoding gene.
In above-mentioned biomaterial,
The fluorescin encoding gene is specially GFP gene.
Third object of the present invention is to provide above-mentioned DNA fragmentation or the new applications of above-mentioned relevant biological material.
The present invention provides above-mentioned DNA fragmentations or above-mentioned relevant biological material in following a1)-a8) in it is any in answer
With:
A1) start destination gene expression；
A2 molecular recognition elements) are used as；
A3 biosensor) is prepared；
A4) identify or assist in identifying 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4- dinitro first
Benzene；
A5) detect or assist detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4- dinitro first
Benzene；
A6) preparation detection or auxiliary detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4- dinitro
The product of toluene；
A7) detect or assist whether to contain 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene in detection sample to be tested
And/or 2,4-DNT；
A8) whether contain 2,4,6- trinitrotoluene and/or 1,3- dinitro in preparation detection or auxiliary detection sample to be tested
The product of base benzene and/or 2,4-DNT.
Fourth object of the present invention is to provide a kind of recombinant bacterium.
Recombinant bacterium provided by the invention is by target gene and the above-mentioned DNA molecular of the destination gene expression to be driven to import
In host strain, recombinant bacterium is obtained.
In above-mentioned recombinant bacterium,
The target gene is reporter gene.
In above-mentioned recombinant bacterium,
The reporter gene is fluorescin encoding gene.
In above-mentioned recombinant bacterium,
The fluorescin encoding gene is specially GFP gene.
In above-mentioned recombinant bacterium,
The host strain is Escherichia coli.
Fifth object of the present invention is to provide the new applications of above-mentioned recombinant bacterium.
The present invention provides above-mentioned recombinant bacteriums in following b1)-b6) in it is any in application:
B1 biosensor) is prepared；
B2) identify or assist in identifying 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4- dinitro first
Benzene；
B3) detect or assist detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4- dinitro first
Benzene；
B4) preparation detection or auxiliary detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4- dinitro
The product of toluene；
B5) detect or assist whether to contain 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene in detection sample to be tested
And/or 2,4-DNT；
B6) whether contain 2,4,6- trinitrotoluene and/or 1,3- dinitro in preparation detection or auxiliary detection sample to be tested
The product of base benzene and/or 2,4-DNT.
Sixth object of the present invention is to provide whether contain 2,4,6- tri- in a kind of detection or auxiliary detection sample to be tested
The method of nitrotoleune and/or 1,3- dinitrobenzene and/or 2,4-DNT.
Whether contain 2,4,6- trinitrotoluene and/or 1,3- in detection provided by the invention or auxiliary detection sample to be tested
Dinitrobenzene and/or 2,4- dinitrotoluene (DNT) include the following steps: to mix above-mentioned recombinant bacterium and sample to be tested, and culture obtains
Culture solution；Detect whether reporter gene in the culture solution expresses；
Contain if there is reporter gene expression in culture solution, in sample to be tested or candidate contains 2,4,6-trinitrotoluene
And/or 1,3- dinitrobenzene and/or 2,4-DNT；
It is not contained if there is no reporter gene expression in culture solution, in sample to be tested or candidate without containing 2,4,6- trinitro-s
Toluene and/or 1,3- dinitrobenzene and/or 2,4-DNT.
In the above method,
The time of the culture is at least 50min；
The sample to be tested derives from soil or water environment.
7th purpose of the invention is to provide the new application of the above method.
The present invention provides application of the above method in detection explosion object location.
The present invention has searched out the new sense worked to target compound by building promoter library and Screening Platform
Element, i.e. 5M6A promoter are answered, and using green fluorescent protein as reporter gene, constructs the full cell using Escherichia coli as chassis
Biosensor realizes detection more highly sensitive to target contaminant TNT and specific, detects in battlefield or postwar mine fields
In, can whether exceeded with quick lock in region, or report water quality condition, it saves manpower and time.Sensing element of the invention and
It detects the method for TNT and provides good element deposit for the deeper exploitation of later period biosensor, can be applied not only to
The Testing and appraisal of the explosion such as wartime and postwar land mine object location also provides new method for the TNT detection in soil and water environment.
Detailed description of the invention
Fig. 1 is the EC of 5M6A promoter200Value.
Fig. 2 is the timeliness of 5M6A promoter.
Fig. 3 is the specificity of 5M6A promoter.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
All statistical analysis in following embodiment are all made of 18.0 statistics software of SPSS.
Bacillus coli DH 5 alpha in following embodiments is the product of Beijing Bo Maide Bioisystech Co., Ltd.
In following embodiments carrier pPROBE-TT (carrier carry sequence table sequence 2 shown in GFP gene) be
The product of addgene company, catalog number 37822.
2,4,6-Trinitrotoluene (TNT), 2,4-Dinitrotoluene (2,4-DNT) in following embodiments,
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) and nitrobenzene (Nitrobenzene) are the products of Sigma-Aldrich company.
The dissolution of TNT in following embodiments uses ultrasonic method: TNT powder being added in distilled water, 15 points of 800W ultrasound
Clock.Other compounds in following embodiments are first dissolved in methanol, the then dilution in Tris buffer (200mM, pH7.0).
The acquisition of embodiment 1, promoter 5M6A
1, concensus sequence of each starting element in functioning is analyzed, -35 and -10th area are retained, rest part carries out
Completely random synthesis, constructs random start sublibrary, and nucleotides sequence is classified as TGTTGAATTAGATGGTGATGCGAAGCTTNN
NNNNNNNNNTTGACANNNNNNNNNNNNNNNNNNNTATAATNNNNNNNNNNGGATCC CGATAGCTTACCGTACC (by
Shanghai English fine horse (invitrogen) Biotechnology Co., Ltd synthesis).Wherein, N is A or G or C or T.
2, the random start sublibrary of synthesis is annealed, and with restriction enzyme Hind III and BamH I to library
In each DNA molecular carry out double digestion, recycle digestion products.
3, screening vector pPROBE-TT (carrier carries GFP gene shown in the sequence 2 of sequence table) is taken, use is restricted
Restriction endonuclease Hind III and BamH I carries out double digestion, recycles carrier framework.
4, the DNA fragmentation that step 2 recycling obtains is connect with the carrier framework that step 3 recycling obtains, obtaining storage capacity is
5000 library promoter::GFP, i.e. recombinant plasmid library.
The promoter sequence of each recombinant plasmid in recombinant plasmid library is located at the upstream of GFP gene in skeleton carrier.
5, the recombinant plasmid that step 4 obtains is converted into bacillus coli DH 5 ɑ respectively, obtains each recombinant bacterium.
6, the recombinant bacterium for taking step 5 to obtain respectively is added TNT when culture is to OD600nm=0.6, and makes its concentration
15mg/L, then 37 DEG C, 200rpm shaken cultivation 8 hours draw 200 μ l bacterium solutions to 96 orifice plate of black, in Pekin Elmer
2300Multilablel Reader is detected (GFP testing conditions are excitation/emission, 488/507nm), number
Value is set to positive colony 200000 or more.Fluorescent value detection is carried out after positive colony is transferred again, first uses 15mg/L
TNT concentration is screened, then is screened using 1mg/L TNT concentration, and the clone of false positive clones and constitutive expression is removed,
It finally screens to obtain 1 clone strong by TNT regulation response, and promoter corresponding in the clone is named as 5M6A,
For the nucleotide sequence of promoter 5M6A as shown in the sequence 1 of sequence table, which is the recombinant bacterium containing 5M6A.When being deposited in environment
In TNT, the promoter 5M6A in the clone can star the expression of downstream reporter gene GFP gene.
The functional verification of embodiment 2,5M6A promoter
One, the EC of 5M6A promoter200Value
The obtained recombinant bacterium containing 5M6A is screened in Example 1, is seeded to LB culture medium, culture to OD600nm=0.6
When TNT is added into bacterium solution respectively, and its concentration is made to be respectively 5mg/L, 10mg/L, 15mg/L, 20mg/L, 30mg/L, 50mg/
L, 75mg/L, 100mg/L, 200mg/L and 300mg/L, and the bacterium solution to be added without TNT as control.37 DEG C, 200rpm oscillation
8h is cultivated, then draws 200 μ l bacterium solutions to 96 hole polystyrene detection plates (Bio-rad), in Pekin Elmer
The relative fluorescence (RFU) in each hole, testing conditions excitation/ are detected on 2300Multilablel Reader
Emission, 488/507nm, and calculate EC200, EC200Refer to the concentration of inducer when can cause twice of indication index reaction,
That is the concentration of Δ RFU corresponding TNT when being 2RFU (control), EC200Value it is smaller illustrate reaction sensibility it is stronger.Wherein, Δ
RFU=RFU (TNT)-RFU (control), RFU (TNT) represent the relative fluorescence that the bacterium solution of TNT is added, and RFU (control) is represented not
The relative fluorescence of the bacterium solution (control) of TNT is added.
Testing result such as Fig. 1.The result shows that the EC200 value of 5M6A promoter is 4.8 ± 1.32mg/L.
Two, timeliness
The obtained recombinant bacterium containing 5M6A is screened in Example 1, is seeded to LB culture medium, culture to OD600nm=0.6
When TNT is added into bacterium solution, and make its concentration 15mg/L, and the bacterium solution to be added without TNT is as control, 37 DEG C, 200rpm
Shaken cultivation certain time, and 200 μ l bacterium solutions to 96 hole polystyrene detection plate (Bio- are drawn in the different time of culture
Rad), the relative fluorescence (RFU) in each hole, detector bar are detected on Pekin Elmer 2300Multilablel Reader
Part is excitation/emission, 488/507nm, and calculates Δ RFU, Δ RFU=RFU (TNT)-RFU (control), wherein
RFU (TNT) represents the relative fluorescence that the bacterium solution of TNT is added, and RFU (control) represents the phase for being added without the bacterium solution (control) of TNT
To fluorescent value.
As a result as shown in Figure 2.The result shows that TNT induction 50min or so can open the expression of reporter gene, TNT induction
120min or so is observed that apparent fluorescence reaction, and fluorescence reaction tends towards stability substantially after TNT induction 4 hours.Illustrate
When detecting TNT in environment, the change in fluorescence detected again after at least reaction 50min can just detect whether contain in environment
TNT。
Three, specific
The obtained recombinant bacterium containing 5M6A is screened in Example 1, is inoculated in LB culture medium, culture to OD600nm=0.6
When following respectively into bacterium solution target compound: 2,4,6-TNT, 2,4-DNT, 2,6-DNT, 1,3-DNB, 1,4-DNB, 2- is added
NT, 3-NT, 4-NT, Toluene and nitrobenzene, and make its concentration be respectively 15mg/L, and to be added without the bacterium of target compound
For liquid as control, then 37 DEG C, 200rpm shaken cultivation 8 hours draw 200 μ l bacterium solutions to 96 hole polystyrene detection plates
(Bio-rad), the relative fluorescence (RFU) in each hole is detected on Pekin Elmer 2300Multilablel Reader,
Testing conditions are excitation/emission, 488/507nm, and calculate Δ RFU, and Δ RFU=RFU (TNT)-RFU is (right
According to), wherein RFU (TNT) represents the relative fluorescence of the bacterium solution containing TNT, and RFU (control) represents the bacterium solution for being added without TNT
The relative fluorescence of (control).
As a result as shown in Figure 3.The result shows that 2,4,6-TNT fluorescent value be higher than 1,3-DNB and 2, the fluorescent value of 4-DNT,
The fluorescent value of 1,3-DNB and 2,4-DNT are apparently higher than other target compounds, illustrate 5M6A promoter to target contaminant TNT
Have more highly sensitive and specific, there are higher sensitivity and specificity to 1,3-DNB and 2,4-DNT.
Claims (12)
1.DNA segment is DNA molecular shown in sequence 1 in sequence table.
2. it is following A 1 biomaterial relevant to DNA molecular described in claim 1) any one of to A7):
A1) contain the expression cassette of DNA molecular described in claim 1；
A2) contain the recombinant vector of DNA molecular described in claim 1；
A3) contain A1) recombinant vector of the expression cassette；
A4) contain the recombinant microorganism of DNA molecular described in claim 1；
A5) contain A1) recombinant microorganism of the expression cassette；
A6) contain A2) recombinant microorganism of the recombinant vector；
A7) contain A3) recombinant microorganism of the recombinant vector.
3. DNA fragmentation described in claim 1 or relevant biological material as claimed in claim 2 are in following a1)-a5) in it is any
Application in kind:
A1 biosensor) is prepared；
A2) identify or assist in identifying 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4-DNT；
A3) detect or assist detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4-DNT；
A4) preparation detection or auxiliary detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4-DNT
Product；
A5) whether contain 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene in preparation detection or auxiliary detection sample to be tested
And/or the product of 2,4-DNT.
4. DNA fragmentation described in claim 1 is in following a1) or a2) in application:
A1) start destination gene expression；
A2 molecular recognition elements) are used as.
5. a kind of recombinant bacterium, to lead the DNA molecular described in claim 1 of target gene and the driving destination gene expression
Enter in host strain, obtains recombinant bacterium；The target gene is reporter gene.
6. recombinant bacterium according to claim 5, it is characterised in that:
The reporter gene is fluorescin encoding gene.
7. recombinant bacterium according to claim 6, it is characterised in that: the fluorescin encoding gene is GFP gene.
8. according to any recombinant bacterium of claim 5-7, it is characterised in that: the host strain is Escherichia coli.
9. any recombinant bacterium is in following b1 in claim 5-8)-b5) in it is any in application:
B1 biosensor) is prepared；
B2) identify or assist in identifying 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4-DNT；
B3) detect or assist detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4-DNT；
B4) preparation detection or auxiliary detection 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene and/or 2,4-DNT
Product；
B5) whether contain 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene in preparation detection or auxiliary detection sample to be tested
And/or the product of 2,4-DNT.
10. whether containing 2,4,6- trinitrotoluene and/or 1,3- dinitrobenzene in a kind of detection or auxiliary detection sample to be tested
And/or 2, the method for 4- dinitrotoluene (DNT), include the following steps: will in claim 5-8 any recombinant bacterium with it is to be measured
Sample blending, culture, obtains culture solution；Detect whether reporter gene in the culture solution expresses；
Contain if there is reporter gene expression in culture solution, in sample to be tested or it is candidate containing 2,4,6-trinitrotoluene and/or
1,3- dinitrobenzene and/or 2,4-DNT；
It is not contained if there is no reporter gene expression in culture solution, in sample to be tested or candidate without containing 2,4,6-trinitrotoluene
And/or 1,3- dinitrobenzene and/or 2,4-DNT.
11. according to the method described in claim 10, it is characterized by:
The time of the culture is at least 50min；
The sample to be tested derives from soil or water environment.
12. application of the method described in claim 10 or 11 in detection explosion object location.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610816647.0A CN106544345B (en) | 2016-09-12 | 2016-09-12 | Promoter 5M6A and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610816647.0A CN106544345B (en) | 2016-09-12 | 2016-09-12 | Promoter 5M6A and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106544345A CN106544345A (en) | 2017-03-29 |
| CN106544345B true CN106544345B (en) | 2019-07-19 |
Family
ID=58367856
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610816647.0A Active CN106544345B (en) | 2016-09-12 | 2016-09-12 | Promoter 5M6A and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106544345B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725339B (en) * | 2020-12-31 | 2022-03-25 | 青岛农业大学 | Promoter element for improving sensitivity of explosive molecule detection and screening method and application thereof |
| CN113373102B (en) * | 2021-07-05 | 2022-09-09 | 青岛农业大学 | Photosynthetic bacteria for detecting explosives and preparation method and application thereof |
-
2016
- 2016-09-12 CN CN201610816647.0A patent/CN106544345B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Construction of 2,4,6-Trinitrotoluene Biosensors with Novel Sensing Elements from Escherichia coli K-12 MG1655;Junjie Tan等;《Cell Biochem Biophys》;20150106;第72卷(第2期);第417-428页 * |
| Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene;Sharon Yagur-Kroll等;《Appl Microbiol Biotechnol》;20130425;第98卷(第2期);第885-895页 * |
| 可用于TNT 检测的新的生物传感元件topAp4的发现;阚乃鹏等;《军事医学》;20150813;第39卷(第7期);第523-527页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106544345A (en) | 2017-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Couradeau et al. | Probing the active fraction of soil microbiomes using BONCAT-FACS | |
| Biniarz et al. | Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review | |
| Czechowska et al. | Use of flow cytometric methods for single-cell analysis in environmental microbiology | |
| He et al. | Application of genetically engineered microbial whole-cell biosensors for combined chemosensing | |
| Kaushik et al. | Methods and approaches used for detection of cyanotoxins in environmental samples: a review | |
| Akondi et al. | Emerging trends in genomic approaches for microbial bioprospecting | |
| US11169152B2 (en) | Function-based probes for environmental microbiome analysis and methods of making and using the same | |
| CN106544345B (en) | Promoter 5M6A and its application | |
| Bovee et al. | Tailored microarray platform for the detection of marine toxins | |
| Tan et al. | Construction of 2, 4, 6-trinitrotoluene biosensors with novel sensing elements from Escherichia coli K-12 MG1655 | |
| Cai et al. | Evolved biosensor with high sensitivity and specificity for measuring cadmium in actual environmental samples | |
| Patel et al. | Development of fluorescent protein-based biosensing strains: A new tool for the detection of aromatic hydrocarbon pollutants in the environment | |
| Fuhrman et al. | Prokaryotic and viral diversity patterns in marine plankton | |
| Godheja et al. | Advances in molecular biology approaches to guage microbial communities and bioremediation at contaminated sites | |
| Pandey et al. | Variations in T-RFLP profiles with differing chemistries of fluorescent dyes used for labeling the PCR primers | |
| Elad et al. | Toxicant identification by a luminescent bacterial bioreporter panel: application of pattern classification algorithms | |
| Barret et al. | Exploiting New Systems‐Based Strategies to Elucidate Plant–Bacterial Interactions in the Rhizosphere | |
| Nazirkar et al. | Development of tracking tool for p-nitrophenol monooxygenase genes from soil augmented with p-Nitrophenol degrading isolates: Bacillus, Pseudomonas and Arthrobacter | |
| CN106191051B (en) | Promoter 7M4D and its application | |
| Mohanta et al. | Role of biotechnology in bioremediation | |
| CN106191054B (en) | Promoter 3M1F and its application | |
| CN106191050B (en) | Promoter 6M3B and its application | |
| Das et al. | Molecular tools for monitoring and validating bioremediation | |
| Nguyen et al. | Toxicity detection using lysosomal enzymes, glycoamylase and thioredoxin fused with fluorescent protein in Saccharomyces cerevisiae | |
| CN106191052B (en) | Promoter 17M11D and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190620 Address after: 100850 No. 27 Taiping Road, Beijing, Haidian District Applicant after: Academy of military medicine of the PLA Academy of Military Sciences Applicant after: Anhui University Address before: 100071 No. 20 East Main Street, Beijing, Fengtai District Applicant before: Bioengineering Inst., Academy of Military Medical Sciences, PLA Applicant before: Anhui University |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |