CN101921724A - Recombinant engineering bacterium for monitoring Hg<2+> pollution in environment and application thereof - Google Patents
Recombinant engineering bacterium for monitoring Hg<2+> pollution in environment and application thereof Download PDFInfo
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- CN101921724A CN101921724A CN 201010166095 CN201010166095A CN101921724A CN 101921724 A CN101921724 A CN 101921724A CN 201010166095 CN201010166095 CN 201010166095 CN 201010166095 A CN201010166095 A CN 201010166095A CN 101921724 A CN101921724 A CN 101921724A
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Abstract
The invention belongs to a microbial luminescence reporter gene system for monitoring toxic substances in an environment and the application thereof to the monitoring of toxic environments and other residual substances. A recombinant engineering bacterium for monitoring the Hg<2+> pollution in the environment is a recombinant vector constructed by using a high-mercury resistant bacterium as a host and leading a merR-Pt contained mercury-resistant gene control region and EGFP (Enhanced Green Fluorescence Protein) genes into the host. Concretely, the pUT-P-G bacterial strain can monitor the pollution of heavy metal Hg<2+> with strongest toxicity in soil, rivers, lakes, underground water and municipal sewage processing systems with specificity and high sensitivity. Compared with the prior art, the bacterial strain has strong specificity and adaptability and high monitoring sensitivity, is convenient for multiplication and use and can realize high-flux on-line monitoring.
Description
Technical field
The invention discloses a kind of recombination system and construction process and purposes that contains anti-mercuri because of (merR-Pt) promotor and reporter gene.
Background technology
Mercury is a kind of toxic chemical substance of serious physiology that has.Because it has persistence, easily transport property and biomagnification highly, makes it become one of the most noticeable environmental pollutant in the present whole world; On the other hand, mercury and mercury salt use very wide in industry again.For these reasons, the detection of mercury causes people's very big concern in the environment, constantly inquires into its detection method.
Along with the progress in development of science and technology and epoch, the protection of environment for human survival has caused people's attention more and more with improvement.The protection of global village biology, the Sustainable development of environment for human survival is had higher requirement to the standard and the method for relevant environment quality and detection.According to the latest information statistics, the content of mercury has more improved 3 times in water, soil, the atmosphere at present, and well-known, mercury pollution has great harm to the mankind.Human body can't be drained food chain or other approach cumulative Trace Mercury by the metabolism of self, and the Trace Mercury accumulation will directly cause diseases such as heart, liver, Tiroidina, even cause neurological disorder and chronic mercury poisoning.In the industrialization engineering, serious big area mercury poisoning incident once appearred in relevant in the world area, and had had a strong impact on local people's life.For this reason, international community is high to the degree of concern of mercury, and very strict control requirement proposed, for example, in February, 2003, European Union proposed at China up to 27,000,000,000 dollars new electronics and electric installation in the objectionable impuritiess such as (Hg) of guaranteeing not have mercury, thereby require domestic relevant enterprise must find out counter-measure, improve detection method, improve examination criteria, its monitoring capacity and means to mercury element must improve in the department of quality examination simultaneously.
The toxicity of heavy metal medium depends on the concentration of biological available metal ion in the environment in the environment.The light emitting-type microbiological sensor has been widely used in studying the available metal ion of detection of biological.Ralstonia eutropha AE2515 is fabricated out, warmly constructs a complete cell sensor to noclilucence luxCDABE reporting system and is used for detecting the biological available Ni of soil by the cnrYXH regulatory gene is transcribed
2+And Co
2+Concentration (Tibazarwa C etal.2001).Comprise the regulation and control zone (merR) of mer gene and the fusion segment of luxCDABE in the engineering bacterium of some optical pickocffs in addition, this is used for Hg by development
2+Quantitative assay.Work as Hg
2+When being attached on the merR, the mer promotor is promptly expressed activity, then causes transcribing of lux reporter gene, sends light (Olga Selifonova et al.1993) subsequently.Biological available cupric ion also can be measured by the false unit cell engineering bacteria of fluorescence in the soil, and method is by cupric ion inductive gene and contains Tn5::lux promotor transposon probe and realize (Tom-Petersen et al.2004)
Come the testing environment pollutent that many advantages are arranged with luciferase as reporter gene, as less demanding to sample, less demanding to equipment and technology, the time period of detection, the precision of detection and sensitivity are also relatively higher or the like.
But because the determination of activity of used luciferase needs lysing cell in the experiment, can not detect by live body, the process of extracting enzyme relatively bothers, and its expression system can not stable existence in cell, thereby is unfavorable for the popularization and the utilization of method.
GFP is isolated a kind of natural fluoresence albumen from the Aequorea Victoria of multitube Medusa, contains 238 amino-acid residues.The GFP of Aequoria can absorb blue light at 395nm, is subjected to Ca
2+Or green-emitting fluorescence when UV-activated, maximum emission peak is 509nm.The chromophore of GFP is a stable ring-type tripeptides structure, forms by recirculation and oxidation by Ser65, Tyr66 and these 3 amino-acid residues of Gly67, and be to pass through covalent bonds between the chromophore.The fluorescence intensity of wild-type GFP is lower, in the tenuigenin about 1 * 10
4The fluorescence of individual GFP molecular emission could accurately be measured with common fluorescent microscope.Transform by structure and biochemical characteristic, obtained many mutant, make the GFP fluorescence intensity and improve greatly as the detection sensitivity of reporter gene with different excitation peaks and emission peak to GFP.
Because GFP gene expression product pair cell does not have toxic action, and very convenient simple by the fluorescent mark detection of GFP generation, be widely used in the scientific research.GFP is as the advantage of the marker gene of environmental microorganism: (1) fluorescent characteristic is stable.The fluorescence of GFP just can disappear when having only overheated (＞65 ℃), peracid (pH＜4), mistake alkali (pH is 12～13) or other denaturing agents to exist.(2) detection is very convenient.Detection to GFP only needs blue light and near ultraviolet excitation, without any need for external source matrix, also needn't carry out pre-treatment, fixing or dyeing by pair cell; Can observe with common fluorescent microscope or hand-held ultraviolet lamp (365nm) irradiation, use the laser scanning co-focusing microscope effect better, can also use cell sorter to detect.Can carry out online detection at the scene.(3) kind of the expression of GFP and recipient cell is irrelevant, and protokaryon and eukaryotic cell can both be expressed, and pair cell do not have toxicity, can not upset the normal function of recipient cell.(4) can carry out unicellular and the viable cell observation, also can carry out real-time in-situ observation, can not destroy observed cell and growing environment thereof.(5) do not contain GFP in the indigenous microorganism, thereby generally false positive results can not occur when detecting GFP.
Usually with GFP as molecule marker.Studies show that, the GFP gene can be transformed, remove that it is unnecessary, the regulation and control segment, artificially add controlling element, make up the reporter gene expression system that is applied to environmental monitoring.In addition, the GFP gene can be with many carriers, import to and make up the reporter gene expression system in the prokaryote by different way.As can also transferring in the host cell in the transduction mode with importing behind GFP gene and the plasmid formation recombinant chou by having a liking for thalline.The carrier that shifts the GFP gene also has a lot of transposon carriers, as Min-Mu, Tn series etc. except that plasmid commonly used.
Have not yet to see about making up the patent and the report of stable reorganization merR-Pt promotor and reporter gene and its purposes aspect.
Summary of the invention
Purpose of the present invention is exactly to disclose a kind of stable recombinant expression system that contains merR-Pt promotor and reporter gene, and the determination of activity of this report gene does not need lysing cell, can detect by live body, and the good stability operating process is quite convenient.
It is of the present invention that a purpose is arranged is to disclose a kind of method that produces the recombinant expression system of the above-mentioned merR-Pt of containing promotor and reporter gene.
A further object of the present invention is exactly the purposes that discloses the recombinant expression system of a kind of above-mentioned merR-Pt of containing promotor and reporter gene.
The present invention is achieved by the following technical solution.With enhanced green fluorescence protein EGFP is reporter gene, makes up the recombinant expression system that contains merR-Pt promotor and EGFP reporter gene, and this system is incorporated on the thalline karyomit(e) by swivel base; The swivel base thalline is stablized in screening; Pass through Ha
2+Induce the variation that causes the somatic cells fluorescent protein expression, come Hg in the testing environment with the variation of fluorescence intensity
2+Pollution.
The said monitoring of environmental Hg that is used for of the present invention
2+The recombinant bacterial strain that pollutes is to be the host with the anti-mercury bacterium of height, imports to contain the recombinant vectors of the anti-mercuri of merR-Pt because of control region and EGFP gene in the host.
The anti-mercuri of the above-mentioned merR-Pt of containing is pUT-P-G because of the recombinant vectors of control region and EGFP gene.The anti-mercury bacterium of described height is pseudomonas putida (Pseudomonas Putida).
Detect Hg with above-mentioned recombinant bacterial strain
2+The method of polluting is to pass through Hg
2+Induce the variation that causes the somatic cells fluorescent protein expression, come Hg in the testing environment with the variation of fluorescence intensity
2+Pollution.
Because of the reporter gene that adopts among the present invention is EGFP, it expresses the restriction that is not subjected to biotype, genotype or cell tissue type, and determination of activity does not need lysing cell, can carry out live body and detect, and the pair cell toxicological harmless need not the substrate or the coreaction factor.And the BM bacterial strain that carries merR-Pt promotor and EGFP reporter gene among the present invention has does not have an antibiotics resistance, makes this system be used for detecting Hg
2+Have during pollution easy to detect, reliable results, applied widely, ecotope is not caused the characteristics of secondary pollution.
Description of drawings
Fig. 1 is the building process figure of luminous reporter gene system
Fig. 2 is the pUT-P-G structural representation
The heavy metal ion of Fig. 3 different sorts and concentration is induced the intensity curve of MB bacterial strain luciferase expression
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described.
Concrete steps of the present invention are: make up the recombination system that contains merR-Pt promotor and EGFP reporter gene; This system is incorporated in the bacterial chromosome of high anti-mercury by the Tn5 transposon system.Pass through Hg
2+Induce and make egfp expression, come Hg in the testing environment according to the variation of fluorescence intensity
2+Pollute.
The bacterium of the anti-mercury of said height is meant pseudomonas putida (Pseudomonas Putida).
One, experiment material
1, plasmid: pEGFP-N1, pGEX4T-1, pUT-Km (plasmid is all available from Nanjing honor Microbial Technics Ltd)
2, bacterial strain: bacillus coli DH 5 alpha, pseudomonas putida (Pseudomonas Putida bacterium numbering 1.1003), intestinal bacteria SM10 (λ pair)
3, LB culture medium prescription: tryptone 10 grams, yeast extract 5 grams, sodium-chlor 10 grams, agar 15～20 grams, 1000 milliliters in water, pH7.2
4, biochemical reagents
1) Xho I, Not I, EcoRI, BamHI, T
4Toolenzyme such as dna ligase and DNA reclaim test kit available from TAKARA company
2) the PCR primer is synthetic by Nanjing Jin Site bio-engineering corporation.
Two, method steps
1, the structure of reorganization merR-Pt promotor and EGFP reporter gene system
(1) amplification of EGFP gene, enzyme are cut and purifying:
Transform the conventional bacillus coli DH 5 σ competent cell for preparing with the pEGFP-N1 plasmid, increase in a large number; Alkaline lysis is taken out plasmid in a large number, electrophoresis detection plasmid purity and content; According to plasmid sequence, the design primer:
Primer egfp-XhoI is upstream 5` end primer, wherein comprises 10 EGFP gene 5` end complementary bases, an XhoI recognition site, 3 protection bases;
Primer egfp-BamHI is a downstream 3` end primer, wherein comprises 10 and EGFP gene 3` end complementary base, a BamHI recognition site, 3 protection bases;
egfp-XhoI：5’-CCGCTCGAGATGGTGAGCA-3’
egfp-BamHI：5’-CGCGGATCCTTACTTGTAC-3’
With the pEGFP-N1 plasmid is masterplate, utilizes Ex Taq to carry out pcr amplification; Reaction conditions is: 95 ℃ of pre-sex change 5min, and 95 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 30s react 30 circulations, and 72 ℃ are extended 5min.Reaction is got 25 μ l reaction product after finishing, and behind the agarose gel electrophoresis, cuts glue, reclaims test kit with gel and reclaims the EGFP gene fragment.With the product that reclaims, with XhoI and BamHI double digestion.
(2) amplification of merR-Pt promotor, enzyme are cut and purifying:
Design primer according to the anti-mercuri of pseudomonas putida W619 ctg69 (Pseudomonas Putida W619 ctg69) because of (the gene accession number is AAVY01000006) control region:
mer-XhoI：5’-CCGCTCGAGCGTTTTTGGTTCAGACATG-3’
mer-EcoRI：5’-CCGGAATTCCTAAGGCATAGCCGAACCT-3’
Primer mer-XhoI is upstream 5` end primer, wherein comprises 19 EGFP gene 5` end complementary bases, an XhoI recognition site, 3 protection bases;
Primer mer-EcoRI is a downstream 3` end primer, wherein comprises 19 and EGFP gene 3` end complementary base, an EcoRI recognition site, 3 protection bases;
To be subjected to Hg
2+The Contaminated soil sample is a masterplate, utilizes Ex Taq to carry out pcr amplification; Reaction conditions is: 95 ℃ of pre-sex change 5min, and 95 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 60s react 30 circulations, and 72 ℃ are extended 5min.Reaction is got 25 μ l reaction product after finishing, and behind the agarose gel electrophoresis, cuts glue, reclaims test kit with gel and reclaims the merR-Pt promoter fragment.With the product that reclaims, with XhoI and EcoRI double digestion.
(3) preparation of pGEX4T-1 plasmid:
Transform the conventional bacillus coli DH 5 σ competent cell for preparing with the pGEX4T-1 plasmid, increase in a large number; Alkaline lysis is taken out plasmid in a large number, electrophoresis detection plasmid purity and content; With EcoRI and BamHI the pGEX4T-1 plasmid that extracts is carried out enzyme and cut, the test kit gel reclaims klenow fragment and cuts product.
(4) ligation:
Egfp fragment, merR-Pt promoter fragment and pGEX4T-1 fragment that enzyme is cut are carried out ligation with the T4 ligase enzyme in centrifuge tube, 0 ℃ connects 12h
(5) screening recon
Connect liquid and transform DH5 α competent cell, several bacterium colonies of picking at random from the transformant flat board extract plasmid on a small quantity with alkaline lysis after the amplification cultivation and carry out enzyme and cut, check order and identify and promptly obtain merR-Pt promotor and the pGEX4T-P-G of reporter gene system (Fig. 1)
2, can stablize the foundation of the MB bacterium of the antibiotic-free resistance of carrying merR-Pt promotor and reporter gene system
(1) carry the structure of the pUT-P-G plasmid of merR-Pt promotor and reporter gene system:
To contain 37 ℃ of incubated overnight of intestinal bacteria inoculation LB substratum of pGEX4T-P-G plasmid, increase in a large number; Alkaline lysis is taken out plasmid in a large number, electrophoresis detection plasmid purity and content; According to plasmid sequence, design following primer:
merR-NotI：5’-GTCAGTCACGAT-3’
pUT?BamHI：5’-CGCGGATCCGGCCTAGG-3’
pUT?NotI：5’-TCATTAAACGCG-3’
With pGEX4T-P-G is masterplate, and merR-NotI and egfp-BamHI are primer, utilizes Ex Taq to carry out pcr amplification; Reaction conditions is: 95 ℃ of pre-sex change 5min, and 95 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 60s react 30 circulations, and 72 ℃ are extended 5min.Reaction is got 25 μ l reaction product after finishing, and behind the agarose gel electrophoresis, cuts glue, reclaims test kit with gel and reclaims merR-Pt promotor and reporter gene system fragment.With the product that reclaims, cut the back agarose gel electrophoresis with BamHI and NotI enzyme, gel reclaims test kit and reclaims the 1.2Kb fragment.
Same quadrat method is a masterplate with pUT-Km, and pUT BamHI and pUT NotI are primer; The product that obtains after the amplification is cut the back agarose gel electrophoresis with BamHI and NotI enzyme, and gel reclaims test kit and reclaims the 5.3Kb fragment.
5.3Kb fragment behind merR-Pt promotor and reporter gene system endonuclease bamhi, the pUT-Km plasmid enzyme restriction is carried out ligation with the T4 ligase enzyme in centrifuge tube, 0 ℃ connects 12h and obtains carrying the pUT-P-G plasmid (Fig. 1) of merR-Pt promotor and reporter gene system.PUT-P-G plasmid Transformed E .coli SM10 (λ pir) is obtained SM10 (pUT-P-G)
(2) this filter membrane of parents is in conjunction with swivel base
Recipient bacterium Pseudomonas Putida of incubated overnight (available from China Committee for Culture Collection of Microorganisms common micro-organisms center, bacterium numbering 1.1003) and SM10 (pUT-P-G) in containing corresponding antibiotic LB substratum.Get each 1 μ L of each culture respectively, the centrifugal back 10mmol/L MgSO of sterilization
4Equal-volume is resuspended. and respectively get 100 μ L bacterium liquid mixings, drip the filter membrane central authorities of 0.45 μ m on the LB flat board, it is dried that placement 0.5～1h. treats that liquid oozes substantially, places 28 ℃ to cultivate 8～12h flat board: the 5mL 10mmol/L MgSO that the cell of growing on the filter membrane is used sterilization
4Be used to screen stability-enhanced mutant after washing.
(3) screening recon
Swivel base is engaged the 10mmol/L MgSO that the cell of growing on the filter membrane in the experiment is used sterilization
4After washing, transfer on the LB flat board.Several bacterium colonies of picking at random from the transformant flat board, dibbling is in LB and ammonia benzyl resistance LB substratum respectively.Select on the ammonia benzyl flat board not longly again, the bacterium colony of growing on the LB flat board further detects.Extracting plasmid on a small quantity with alkaline lysis after the choosing colony amplification cultivation carries out pcr amplification, checks order and identify the MB bacterial strain that promptly obtains stablizing the antibiotic-free resistance of carrying merR-Pt promotor and reporter gene system as primer with merR-NotI and egfp-BamHI.
3, utilization MB bacterial strain detects Hg
2+
Connect the single bacterium colony of MB bacterium in 5mL LB nutrient solution, incubated overnight is transferred in fresh LB nutrient solution by 1% inoculum size again.Treat thalli growth to logarithmic phase, add the HgCl of 10000PPM
2Solution makes Hg
2+Concentration is respectively 5,10,15,20,25nmol/mL.Under spectrophotofluorometer, measure fluorescence intensity behind the 12h.The data that obtain are used to set up green fluorescence expression intensity and different concns Hg
2+Relation curve.
4, different heavy metal ion are to the detection of inducing of MB bacterial strain
Bacterial strain MB strain culturing to logarithmic phase, is seeded to respectively and contains Cu
2+, Fe
3+, Mn
4+, Sn
2+, Zn
2+, Co
2+, Ag
+, Ba
2+And Hg
2+The LB substratum is behind the cultivation 12h.Under visible light and shortwave blue light, only be grown in and contain Hg
2+Bacterium colony on the LB substratum can send fluorescence.
To sum up respectively going on foot experimental result shows: the MB bacterial strain can have Hg
2+Expressing green fluorescent protein under the existence condition.Thalline is yellow-green colour under visible light; Be bright green at shortwave blue light hypothallus.Other heavy metal ion do not have the abduction delivering effect to the MB bacterial strain.The thalline luminous quantity within the specific limits (＜30nM/mL) increase with ion concentration of mercury and be certain relation and increase progressively (Fig. 3).In this scope, thalline can reflect the concentration of mercury ion sensitive, exactly by intensity of fluorescence.Illustrate and utilize reorganization bacterium of the present invention to implement qualitative and quantitative detection mercury ion.
Bacterial classification that relates in the above-described embodiments and plasmid source are as table 1.
Table 1. bacterial classification and plasmid
Ap
r: amicillin resistance, Cm
r: chlorampenicol resistant, Km
r: kalamycin resistance
CGMCC: China Committee for Culture Collection of Microorganisms common micro-organisms center
NJZW Corp.: Nanjing honor Microbial Technics Ltd.
Claims (4)
1. one kind is used for monitoring of environmental Hg
2+The recombinant bacterial strain that pollutes is characterized in that, is to be the host with the anti-mercury bacterium of height, imports to contain the recombinant vectors of the anti-mercuri of merR-Pt because of control region and EGFP gene in the host.
2. as the said monitoring of environmental Hg that is used for of claim 1
2+The recombinant bacterial strain that pollutes is characterized in that the anti-mercuri of the described merR-Pt of containing is pUT-P-G because of the recombinant vectors of control region and EGFP gene.
3. one kind by the described monitoring of environmental Hg that is used for of claim 1
2+The recombinant bacterial strain that pollutes is characterized in that the anti-mercury bacterium of described height is pseudomonas putida (Pseudomonas Putida).
4. one kind is detected Hg with the described recombinant bacterial strain of claim 1
2+The method of polluting is characterized in that, is to pass through Hg
2+Induce the variation that causes the somatic cells fluorescent protein expression, come Hg in the testing environment with the variation of fluorescence intensity
2+Pollution.
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| CN102604901A (en) * | 2012-03-22 | 2012-07-25 | 中山大学 | Heavy-metal mercury-resistance related protein DbsMerA and encoding genes and application thereof |
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Non-Patent Citations (2)
| Title |
|---|
| 《生态学报》 20091231 茆婷等 报告基因技术及其在土壤质量监测中的应用 第29卷, 第12期 2 * |
| 《第五次全国土壤生物与生物化学学术研讨会》 20091231 何伟等 基于细菌染色体的全细胞报告基因系统检测红壤中 , 2 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604901A (en) * | 2012-03-22 | 2012-07-25 | 中山大学 | Heavy-metal mercury-resistance related protein DbsMerA and encoding genes and application thereof |
| CN102604901B (en) * | 2012-03-22 | 2013-06-12 | 中山大学 | Heavy-metal mercury-resistance related protein DbsMerA and encoding genes and application thereof |
| CN103215214A (en) * | 2013-03-18 | 2013-07-24 | 中国科学院生态环境研究中心 | Double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone |
| CN104140943A (en) * | 2014-07-16 | 2014-11-12 | 清华大学 | Recombinant luminous bacteria and application thereof |
| CN104140943B (en) * | 2014-07-16 | 2016-07-13 | 清华大学 | A kind of restructuring photobacteria and application thereof |
| CN104313046A (en) * | 2014-10-15 | 2015-01-28 | 南京师范大学 | Microbial whole-cell luminescent reporting sensor for monitoring Hg<2+> pollution and kit prepared thereby and application |
| CN110241064A (en) * | 2019-07-05 | 2019-09-17 | 中国农业大学 | A kind of building and its application of the nucleic acid-protein compound allosteric type microbial whole-cell sensor for mercury ion detecting |
| CN114958612A (en) * | 2022-02-23 | 2022-08-30 | 武汉科技大学 | Screening method of chlamydomonas reinhardtii for specifically identifying heavy metal cadmium |
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Application publication date: 20101222 |