CN102250819A - Highly-sensitive biosensor cell for detecting heavy metal mercury and manufacturing method thereof - Google Patents
Highly-sensitive biosensor cell for detecting heavy metal mercury and manufacturing method thereof Download PDFInfo
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Abstract
The invention relates to a highly-sensitive biosensor cell for detecting heavy metal mercury and a manufacturing method thereof. A metalloregulatory protein MerR, a red fluorescent protein mCherry and a methylmercury lyase MerB are used and colon bacillus is used as host bacteria so that the biosensor cell capable of detecting inorganic mercury ions and methylmercury (0.1-5000 nM) is constructed. Through introducing mercury ion transport proteins MerT or a gene cascade expression system (a gene circuit) with an amplification function, a PRM-cI system containing bacteriophage and a Pm-XylS2 system containing pseudomonas putida can improve the detection sensitivity of the biosensor cell on detecting the low-concentration heavy metal mercury by 1-2 times. 23 MerR mutants are obtained through direct evolution and high throughout screening, and the biosensor cell constructed by the mutants MerR can greatly improve the detection sensitivity. When the biosensor cell is used for detecting the pollution of the mercury ions and the methylmercury, the biosensor cell has high specificity and wide adaptability and is not influenced by the presence of the other metal ions.
Description
Technical field
The invention belongs to the Environmental Biotechnology field, the biosensor cell and the manufacture method thereof of heavy metal Hg in the solution example in the particularly highly sensitive testing environment.
Background technology
Mercury (Hg) is main poisonous metal pollutent, can find in common industry and/or agricultural waste (Zhang and Wong, 2007).Occurring in nature is difficult to find pure liquid metal mercury, more is the form appearance with compound and inorganic salt.Mercury can unit price mercury or mercuric form and other compound in conjunction with (also being expressed as Hg (I) and Hg (II) or Hg respectively
2+).The chemical form of the mercury that is discharged (or type formation) along with Source Type with other factors and different.Because it is dissimilar mercury has different toxicity, therefore also different with the influence of other biological organism environment to human health.Mercury can be changed between different forms, forms various forms at circulation time, and human and environment are caused murder by poisoning.
When combining with carbon, mercury just forms organomercury compound or organo-metallic mercury.Modal organomercury compound is a methyl mercury in environment.It has been generally acknowledged that its formation in environment mainly is because the bioprocess of effects such as microorganism.Because the conversion of the various forms of mercury, the anthropogenic discharge just causes the formation of methyl mercury in the environment.At present in many edible fresh-water fishes, sea water fish and marine mammal, methyl mercury can increase to several thousand times (biological accumulation and biological magnifications) of content in the water body on every side, and it has been subjected to and has obtained the serious concern of people.
Accurately detect and monitoring environment in, particularly mercury exists and pollution directly has influence on people's daily production and life in the waters.The years of researches exploitation has produced the routine techniques means and the device of various detection mercury, and has excellent sensitivity, as atomic absorption spectrometry (0.02ppb), atomic power spectrography (0.001ppt), atomic emission spectrometry (0.01ppt) and inductively coupled plasma mass spectrometry (0.08ppt), but the use of these detection meanss and device has great limitation and cost is high, needs training professionals to operate pertinent instruments equipment (Knecht and Sethi, 2009) simultaneously.The biology that these conventional detection methods of what is more important can not be measured pollution metal can utilize concentration (Bioavailable concentration), is exactly the utilizable quantity of organism of living in the pollution metal sum in the environment.This just need will utilize biological detecting method in conjunction with conventional physical and chemical detection method.Based on this reason, the pollution of developing biosensor detection mercury and other metals in recent years more and more is subjected to people's extreme attention.
Biosensor is a kind of analysis and detection device, closely is made up of with a compatible with it transmitter an immobilized biomaterial usually, can be converted to bio signal and be easy to quantized electrical signal.Biomaterial is responsible for specificity ground identification analysans and is produced bio signal, and the physics and chemistry transmitter is then responded to and the biochemical signals and export and can survey electrical signal (Scheller etc., 1991) accordingly of increase.Biosensor not only can be measured the multiple metal pollutant that comprises mercury effectively, and they also are the ideal selections of measurable various toxic substances based on Basic of Biology.In addition, being easy to duplicate with relative low cost is that biosensor has bigger competitive power.
The whole-cell biological transmitter is to come specificity identification analysans and produce corresponding bio signal (Heim etc., 1999) as biomaterial with complete viable cell.The biosensor of full cell has than traditional analytical procedure and other biological transmitter, as protein, enzyme polysaccharide transmitter alive many advantages are arranged: 1) cell, especially microorganism cells is always more cheap than the protein of purifying or enzyme etc., and biomolecules has better activity in cellular environment.2) use of specific pathways metabolism can make testing compound or pollutent in the cell, and especially aromatics and heavy metal have better and discerned by selectivity.3) full cell sensor has better stability, can use the long relatively time.4) the complete high-throughout automated operation of the easier realization of cell system.5) have only the whole-cell biological transmitter can realize the environmental monitoring of eco-toxicity test and pollutent.
The whole-cell biological transmitter can be divided into two big classes, a class is based on constitutive expression, the another kind of abduction delivering that is based on.The constitutive expression system usually utilizes has highly active promotor under cell normal growth condition, from but reporter gene can have higher basal expression level.And under harmful or deleterious condition, this basal expression level just descends, and the decline of expression level is relevant with the toxicity of testing sample.Therefore the biosensor of constitutive expression system provides individual cells or culture metabolism state " big picture ", any reduction cell growth or cause cytotoxic compare the semaphore that all can reduce reporter gene with control group.And reporter gene merges mutually with inducible promoter in the abduction delivering system, and inducible promoter can respond specific coercing (as heat shock, oxidative damage, dna damage etc.) or compound (organic pollutant and heavy metal).Come the quantity levels of reaction induced thing by the expression level of measuring reporter gene.The key advantage of inducible system is a specificity response inductor (for the cls analysis thing), allows the single determinand of detection by quantitative.
Coerce along with increasing response is specific or the gene of compound is positioned and clones, people just may clone the promotor of these genes and they are merged mutually with many reporter genes.In addition, because unique specificity of these promotors, the combination of countless promotors and reporter gene is used to detect inductor (test analyte) widely.The fusion of narrow spectrum promotor and reporter gene can be cloned on the plasmid, also can be inserted in the genome of some bacterium.This new reorganization biosensor is commonly referred to as the biosensor cell of genetic engineering bacterium (GEM).
Recently the progress of research bacterial system toxic metal ion resistance causes relying on the discovery of the transcription factor MerR family of metal, and MerR family transcription factor all has existence in the various kinds of cell type.The prototype of MerR family transcription activator is the regulatory factor in gram negative bacterium mercury resistance (mer) operon, exists in transposon Tn21 and Tn501 usually (Brown et.al.1983).When mercuric salt existed, this regulon was the incitant of mer gene, and when not having dimercurion to exist, was a faint repressor (Lund and Brown, 1987).) other metal resistance in subsequently the various bacteria, some drug resistances and some these transcription regulaton factors that studies show that of coercing responsive genes also all belong to MerR family.
All MerR family proteins are activating transcription factors.They hold inductor in conjunction with clear separate in territory distinguish in conjunction with the territory with C by N end promoter DNA.They have higher amino acid sequence similarity in conjunction with the territory at DNA, but inductor then has low similarity in conjunction with the territory.When not having divalence mercury, MerR albumen combines with DNA with the repressor conformation, makes promotor keep the state of checking.Just cause conformational change when one of them of two binding sites of dimercurion and MerR albumen combines, make MerR be in the activation conformation.It also is to play a role by similar mechanism that other members of MerR family are considered to, but according to the difference of C end inductor in conjunction with the territory, needs different metal ions, as Cu (II), and Cd (II), inductor is transcribed in Pb (II) and Zn conducts such as (II).
When promotor that regulated by MerR or MerR proteinoid and the reporter gene that is easy to detect, as various fluorescins (as emGFP, eYFP, eBFP or mCherry) when merging mutually, the transcriptional activation to reporter gene after adjusting albumen combines with metal ion remains derivable.Just can prepare by the set out genetic modification biosensor of various detection metals of these reorganization reporter genes.These genetic modification biosensors are highly narrow spectrum when detecting metal pollutant, and recombinant bacteria is with can produce after the specificity metal ion contacts can quantitative fluorescent signal simultaneously.
Preparing the key factor that high-quality genetic modification biosensor is used to detect heavy metal is specificity and the affinity that relies on metal binding protein.Yet the more protein-bonded limitation of native metal have greatly limited genetic modification biosensor application potentiality: 1) natural MerR or MerR albuminoid often have only very narrow to the metal ion responding range; 2), they are subjected to the non-specific binding interference of other metal ions usually; 3) they are merely able to play a role at narrow temperature range activated transcription, the in-situ applications under not too suitable high temperature is regulated under the face of land.
In order to overcome above-mentioned shortcoming, make the whole-cell biological transmitter can detect and monitor the heavy metal contamination of various envrionment conditionss effectively, it is novel that we have at first adopted synthetic biology and protein orthogenesis technology to develop, and has the biosensor cell that mercury ion is had higher specificity and binding affinity.This reconstitution cell will be used to build microscale cheaply, can carry out the whole-cell biological transmitter of field monitoring and detection mercury contaminants.This technology can further be applied to design other biological transmitter cell and come specificity to detect other heavy metal contaminants or radionuclide.
Summary of the invention
Mercury is well-known serious environmental pollutant always in decades, and the mercury that is leaked in the environment may cause serious harm to HUMAN HEALTH.Environmental Protection Agency determines that the dimercurion safety range is below the 10nM (2ppb) in the tap water.Therefore needs can provide the following mercury ion method of detection 10nM, and preferably easy and low-cost, easy handling.The transmitter of the various detection mercury pollution of having developed at present all has relative merits separately, but and can convert microorganism cells and transmitter coupled whole-cell biological transmitter to the photometry signal to the cell induced signal, and be easy to duplicate, also have very big potentiality to reduce cost.Protein engineering in recent years in addition, the fast development of microbiology and photoelectric detecting technology has also greatly advanced the development and the exploitation of whole-cell biological transmitter.
Utilize metalloregulatory protein MerR gene and red fluorescent protein mCherry gene (Shaner etc., 2005) form the red fluorescent protein genetic expression regulation system that dimercurion is induced control, with intestinal bacteria (Escherichia coli) is host bacterium (down together), and we have made up the biosensor cell I (accompanying drawing 1) that can detect mercury pollution.This transmitter cell has very big sensing range, can detect 1 dimercurion to the 1000nM scope, still keeps the specificity of height simultaneously, not to other metal ions, as Mg
2+, Ca
2+, Mn
2+, Fe
3+, Co
2+, Ni
2+, Cu
2+, Zn
2+, Cd
2+, pb
2+And Mo
2+Deng producing response, thereby the detection of mercury ion is not exerted an influence.
On the red fluorescent protein genetic expression regulation system basis of above-mentioned mercury ion mediation, introduce methyl mercury lyase (MerB), under the effect of methyl mercury lyase, the dimercurion in the methyl mercury is released.Thereby the biosensor cell II that makes structure finishes the detection (accompanying drawing 2) to methyl mercury.If contain inorganic mercury and organic mercury (methyl mercury) in the testing sample simultaneously, just can measure the summation of inorganic dimercurion and methyl mercury earlier with biosensor cell II, measure inorganic dimercurion with biosensor cell I then, both differences are exactly the content of methyl mercury.
What the design transmitter often will be concerned about is the detection sensitivity of transmitter, just detects the ability of low concentration sample.From the synoptic diagram (accompanying drawing 1) of our transmitter cell design as can be known, there are three kinds may improve detection sensitivity by the approach that improves bio-identification.1) improves the interior ion concentration of mercury of born of the same parents fast.Our design is the transmitter cell, need be input to mercury ion in the born of the same parents, with the MerR protein-interacting, fluorescence protein gene is expressed, and generation can be surveyed signal; So the ability that increases in the mercury ion input born of the same parents might improve sensor detection sensitivity.2) improve MerR albumen relative content, increase the detection sensitivity that also may improve transmitter with mercury ion bonded chance.3) change the proteic performance of MerR, particularly identification and interact in conjunction with mercury ion and with RNA polymerase and to start the ability of downstream gene expression also may improve sensor detection sensitivity.Based on these hypothesis, we have further made again has the biosensor cell that various inspection sensitivity improve.
Mercury transport protein MerT can accelerate the transportation (Rossy etc., 2004) of mercury ion.Therefore on the basis of biosensor cell I, by introducing the MerT gene, just can make up the biosensor cell III (accompanying drawing 3) that makes new advances, the whole-cell biological transmitter that makes with this cell can be accelerated the transportation of mercury ion in the identical time, increase fluorescent protein expression, thereby improve the detection sensitivity (increasing 1-2 doubly) of transmitter.
Produce the proteic amount of MerR in order in the identical time, to increase cell, we have introduced gene cascade expression system, perhaps be the genetic circuit of biological enlarging function, by increasing the detection sensitivity that the proteic content of MerR in the cell improves the biosensor cell at short notice fast.In the bacterial lambda phage, P
RMThe operator gene on promotor and its right side is an eclipsed.This operator gene contains three action site Or1, Or2 and Or3.When Or3 produce some the sudden change after, repressor cI can combine with Or3 effectively, from but cI can keep transcriptional expression (Gussin etc., 1987 efficiently; Bushman, 1993), so when MerR and cI are formed operon for synthesizing, introduce some sudden changes at Or3 simultaneously, MerR and cI be overexpression simultaneously just, thereby increases the proteic ability of unit time interior production MerR.Such gene component is introduced biosensor cell I just obtain having P
RMThe biosensor cell IV Or3 mutant (accompanying drawing 4) of-cI cascade enlarging function.In the identical time, biosensor cell IV ratio biosensor cell I can produce more MerR albumen, thus the existence of (increase 1-2 is doubly) detection mercury ion more delicately.
Two transcription regulaton factor NahR and XylS2 from pseudomonas putida, Induced by Salicylic Acid can increase goal gene overexpression (Cebolla etc., 2001) by synergy.When we substitute NahR with MerR, when mercury ion and Whitfield's ointment all exist, coordinative role also can take place in new transcription regulaton factor, fluorescent protein expression is increased fast, the biosensor cell V (accompanying drawing 5 contains Pm-XylS2 cascade enlarging function) that makes up based on this principle has better detection sensitivity (increasing 1-2 doubly).
Use protein orthogenesis and high flux screening technology (Romero and Arnold, 2009), by orthogenesis and the screening of MerR, we obtain the MerR mutant of some performance changes.When mercury ion existed, the generation of these mutains can increase the expression of fluorescence protein gene fast.MerR substitute wild-type MerR among the biosensor cell I with these sudden changes, just obtain a series of biosensor cell I mutants.I compares with wild-type biology transmitter cell, the sensitivity of mutant biosensor cell I has raising in various degree, wherein best mutant can improve sensitivity and reach more than 10 times, this plant mutant body biosensor cell is named as intestinal bacteria TIB-Hg (Escherichia coli TIB-Hg), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 02 5th, 2010, preserving number is CGMCC No 3628.
Embodiment
The invention will be further described below by embodiment.
The substratum LB (peptone 20 grams per liters, yeast extract paste 10 grams per liters, sodium-chlor 20 grams per liters) that embodiment 1 usefulness is added kantlex (40 micrograms per litre) cultivates biosensor cell I (37 ℃/6 hours).Then cell is joined in M9 salt (Atlas and Parks, the 1997) solution that contains different concns mercury chloride, the concentration of cell is that OD600 is 0.1.37 ℃ cultivate 6 hours after, measure the relative intensity of fluorescence (RFU, the excitation wavelength of mensuration is 587nm, emission wavelength is 610nm) of each solution.The cell solution of different concns mercury chloride can obtain different RFU.Wherein in the scope of 1-10nM mercury chloride, concentration and RFU can present linear corresponding relation (accompanying drawing 6).This presentation of results biosensor cell I can be used for detecting dimercurion in the aqueous solution, and can quantitatively determine the degree of inorganic divalence mercury pollution within the specific limits.
The substratum LB that embodiment 2 usefulness are added kantlex (the same) cultivates biosensor cell II (37 ℃/6 hours).Then cell is joined in the M9 salts solution that contains the different concns methyl mercury, the concentration of cell is that OD600 is 0.1.After 6 hours, measure the relative intensity of fluorescence (the same) of each solution 37 ℃ of cultivations.The cell solution of different concns methyl mercury can obtain different RFU.Wherein in the scope of 1-10nM mercury chloride, concentration and RFU can present linear corresponding relation (accompanying drawing 7).This presentation of results biosensor cell II can be used for detecting methyl mercury in the aqueous solution, and can quantitatively determine the degree that methyl mercury pollutes within the specific limits.
The substratum LB that embodiment 3 usefulness are added kantlex (the same) cultivates biosensor cell I (37 ℃/6 hours).Then cell is joined and contain 500nM different metal muriate, the M9 salt (Atlas and the Parks that comprise Manganous chloride tetrahydrate, zinc chloride, iron trichloride, cupric chloride, nickelous chloride, cobalt chloride, molybdenum chloride, chromium trichloride, Cadmium chloride fine powder, gold trichloride, lead chloride and mercury chloride, 1997) in the solution, the concentration of cell is that OD600 is 0.1, simultaneously not add any metal chloride in contrast.After 6 hours, measure the relative intensity of fluorescence (the same) of each solution 37 ℃ of cultivations.Experiment shows that mercury chloride could produce fluorescent signal (compared with the control), and other metal chlorides can not produce (accompanying drawing 8) by induced fluorescence.This presentation of results biosensor cell I can detect dimercurion in the solution in specificity ground.
The substratum LB that embodiment 4 usefulness are added kantlex (the same) cultivates biosensor cell III (37 ℃/6 hours), then cell is joined in the M9 salts solution that contains 50nM mercury chloride, the concentration of cell is that OD600 is 0.1, compares with biosensor cell I simultaneously.After 0,2,4,6 hours, measure the relative intensity of fluorescence (the same) of each solution 37 ℃ of cultivations, obtain the time curve (accompanying drawing 9) that two kinds of biosensor cell fluorescences produce.Biosensor cell III contains mercury transport protein MerT, biosensor cell I does not then have MerT, experiment shows that the existence of MerT can increase the production of fluorescin within a certain period of time, thereby improves the detection sensitivity that biosensor cell III detects mercury chloride in the solution.
The substratum LB that embodiment 5 usefulness are added kantlex (the same) cultivates biosensor cell IV Or3 mutant (37 ℃/6 hours), then cell is joined contain 0,10, in the M9 salts solution of 50nM mercury chloride, the concentration of cell is that OD600 is 0.1, (does not have P with biosensor cell IV Or3 wild-type simultaneously
RM-cI cascade enlarging function) and biosensor cell I compare.After 6 hours, measure the relative intensity of fluorescence (the same) of each solution 37 ℃ of cultivations, obtain the concentration curve (accompanying drawing 10) of the corresponding mercury chloride of biosensor cell fluorescence generation.Two Or3 mutants (solid line among the figure) ratio biosensor cell IV Or3 wild-type and the biosensor cell I of biosensor cell IV have higher RFU.This result shows the P that contains the Or3 mutant
RM-cI cascade enlarging function can increase the production of fluorescin within a certain period of time, thereby improves the detection sensitivity that biosensor cell IV Or3 mutant detects mercury chloride in the solution.
The substratum LB that embodiment 6 usefulness are added kantlex (the same) cultivates biosensor cell V (37 ℃/6 hours), then cell is joined and contain a 50nM mercury chloride, only contain the 2mM sodium salicylate or contain simultaneously in both M9 salts solutions, the concentration of cell is that OD600 is 0.1, compares with biosensor cell I simultaneously.After 6 hours, measure the relative intensity of fluorescence (the same) of each solution 37 ℃ of cultivations, obtain the fluorescence production (accompanying drawing 11) of two kinds of biosensor cells in different solutions.Biosensor cell V contains the Pm-XylS2 cascade and amplifies genetic circuit, in solution, exist two kinds to transcribe inductor (mercury chloride and sodium salicylate) Pm-XylS2 cascade and amplify and to produce the cascade amplification simultaneously, increase the production of fluorescin within a certain period of time, thereby improve the detection sensitivity that biosensor cell V detects mercury chloride in the solution.
Accompanying drawing 1: detect the biosensor cell I sketch of mercury pollution, this cell can detect divalence inorganic mercury ion in the aqueous solution.Respective detection the results are shown in accompanying drawing 6.
Accompanying drawing 2: detect the biosensor cell II sketch of mercury pollution, this cell can detect methyl mercury and divalence inorganic mercury ion in the aqueous solution.Respective detection the results are shown in accompanying drawing 7.
Accompanying drawing 3: detect the biosensor cell III sketch of mercury pollution, this cell contains the mercury ion transport protein, can promote mercury ion to intracellular conveying, thereby increases the detection sensitivity of mercury ion.Respective detection the results are shown in accompanying drawing 9.
Accompanying drawing 4: detect the biosensor cell IV sketch of mercury pollution, contain the metalloregulatory protein MerR overexpression system of bacterial lambda phage repressor cI and operation gene mediated thereof in this cell.The sudden change of operation gene action site Or3 can make cell overexpression MerR, thereby improves the sensitivity of biosensor cell detection.Respective detection the results are shown in accompanying drawing 10.
Accompanying drawing 5: detect the biosensor cell V sketch of mercury pollution, this cell contains the transcription regulaton factor XylS2 from the pseudomonas putida Induced by Salicylic Acid, increases the detection sensitivity of biosensor cell by acting synergistically with MerR.Respective detection the results are shown in accompanying drawing 11.
Accompanying drawing 6: biosensor cell I detects the figure as a result of mercury chloride.
Accompanying drawing 7: biosensor cell II detects the figure as a result of methyl mercury.
Accompanying drawing 8: biosensor cell I detects specificity to the different metal ionic.
Accompanying drawing 9: the detection sensitivity of biosensor cell III and biosensor cell I relatively.The short side piece: biosensor cell I does not have the MerT transport protein; The solid yardage piece: biosensor cell III has the MerT transport protein.
Accompanying drawing 10: the gene expression system of bacterial lambda phage repressor cI and operation gene mediated thereof is to the influence of mercury detection sensitivity.Solid yardage piece solid line: biosensor cell IV, Or3 contains sudden change; Solid yardage piece dotted line: biosensor cell IV, but not sudden change of Or3, short side piece dotted line: biosensor cell I does not have the gene expression system of bacterial lambda phage repressor cI and operation gene mediated thereof.
Accompanying drawing 11: the gene expression system of the transcription regulaton factor XylS2 mediation of pseudomonas putida Induced by Salicylic Acid is to the influence of mercury detection sensitivity.White post figure: biosensor cell I does not have transcription regulaton factor XylS2; Black post figure: biosensor cell V has transcription regulaton factor XylS2.
Accompanying drawing 12: the influence of different MerR mutant biosensor cell mercury detection sensitivities.Mensuration is to 5nMHgCl
2The time relative intensity of fluorescence (RFU).
Reference
Atlas?RM，Parks?LC.Handbook?of?Microbiological?media(2
nd?Edition)，1997，CRCPress.
Brown?NL，Ford?SJ，Pridmore?RD，Fritzinger?DC.Nucleotide?sequence?of?a?gene?fromthe?Pseudomonas?transposon?Tn501encoding?mercuric?reductase.Biochemistry?198322(17)：4089-4095.
Bushman?FD.The?bacteriophage?434right?operator.Roles?of?O(R)1，O(R)2and?O(R)3.JMol?Biol.1993230(1)：28-40.
Cebolla?A，Sousa?C，de?Lorenzo?V.Rational?design?of?a?bacterial?transcriptional?cascadefor?amplifying?gene?expression?capacity.Nucleic?Acids?Res.200129(3)：759-66.
Gussin?GN，Brown?S，Ferm?J，Matz?K.New?mutations?in?the?Prm?promoter?ofbacteriophage?lambda.Gene?198754(2-3)：291-297.
Helmann，J.D.，Ballard，B.T.and?Walsh，C.T.The?MerR?metalloregulatory?protein?bindsmercuric?ion?as?a?tricoordinate，metal-bridged?dimer.Science?1990247，946-948.
Knecht?MR，Sethi?M.Bio-inspired?colorimetric?detection?of?Hg2+and?Pb2+heavy?metalions?using?Au?nanoparticles.Anal?Bioanal?Chem.2009394(1)：33-46.
Lund?PA，Brown?NL.Role?of?the?merT?and?merP?gene?products?of?transposon?Tn501inthe?induction?and?expression?of?resistance?to?mercuric?ions.Gene?198752(2-3)：207-14.
Romero?PA，Arnold?FH.Exploring?protein?fitness?landscapes?by?directed?evolution.NatRev?Mol?Cell?Biol.200910(12)：866-76.
Rossy?E；Seneque?O；Lascoux?D；Lemaire?D；Crouzy?S；Delangle?P；Coves?J.Is?thecytoplasmic?loop?of?MerT，the?mercuric?ion?transport?protein，involved?in?mercurytransfer?to?the?mercuric?reductase？FEBS?Letters?2004575(1-3)：86-90.
Shaner?NC，Steinbach?PA，Tsien?RY.A?guide?to?choosing?fluorescent?proteins，NatMethods.20052(12)：905-9.
Scheller?FW，Schubert?F，Neumann?B，Pfeiffer?D，Hintsche?R，Dransfeld?I，WollenbergerU，Renneberg?R，Warsinke?A，Johansson?G.Second?generation?biosensors.BiosensBioelectron.19916(3)：245-53.
Zhang?L，Wong?MH.Environmental?mercury?contamination?in?China：sources?andimpacts.Environ?Int.200733(1)：108-21.
Claims (7)
1. detect the biosensor cell and the biological manufacture method thereof of mercury pollution in the water-soluble sample, described manufacture method comprises as follows:
2. by claim 1, by with metalloregulatory protein MerR gene (comprising promoter region) and red fluorescent protein mCherry genomic constitution mercury ion inductive gene expression system and import in the host bacterium intestinal bacteria and obtain the biosensor cell.This transmitter cell can specificity ground at 15-40 ℃, in 0.5-8 hour, detect the inorganic dimercurion foul solution of 1-5000nM under the pH4-9 condition.
3. by claim 2, in MerR gene and mCherry genomic constitution mercury ion inductive gene expression system, introduce behind the methyl mercury lyase gene MerB resulting biosensor cell can specificity ground at 15-40 ℃, in 0.5-8 hour, detect 1-300nM methyl mercury foul solution under the pH4-9 condition.
4. press claim 2, resulting biosensor cell behind the introducing mercury ion transport protein gene M erT in MerR gene and mCherry genomic constitution mercury ion inductive gene expression system.This transmitter cell can specificity ground at 15-40 ℃, in 0.5-8 hour, detect the inorganic dimercurion foul solution of 1-5000nM under the pH4-9 condition.
5. by claim 2, in MerR gene and mCherry genomic constitution mercury ion inductive gene expression system, introduce origin and come from resulting biosensor cell after the gene cascade amplification system that bacterial lambda phage PRM-cI and MerR form.This transmitter cell can specificity ground at 15-40 ℃, in 0.5-8 hour, detect the inorganic dimercurion foul solution of 1-5000nM under the pH4-9 condition.
6. by claim 1, by the MerR gene, mCherry gene and the Pm-XylS2 that derives from pseudomonas putida form novel gene cascade amplification system and import in the host bacterium intestinal bacteria and obtain the biosensor cell.This transmitter cell can specificity ground at 15-40 ℃, in 0.5-8 hour, detect the inorganic dimercurion foul solution of 1-5000nM under the pH4-9 condition.
7. by claim 2, obtain MerR mutant in 23: S131L, S125P by protein orthogenesis and high flux screening technology, S125T, V124D, V124T, V109E, V109M, M106T, M106V, K99N, K99Q, K99T, S86C, T80S, E77K, L74Q, L74V, E72K, S66C, F56L, F53Q, A16V, K15E, by these metalloregulatory proteins MerR mutator gene respectively with mCherry genomic constitution mercury ion inductive gene expression system and import and obtain different biosensor cells in the host bacterium intestinal bacteria.These transmitter cells can specificity ground at 15-40 ℃, in 0.5-8 hour, detect the inorganic dimercurion foul solution of 0.1-5000nM under the pH4-9 condition.
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| CN104845996A (en) * | 2015-02-13 | 2015-08-19 | 温州医科大学 | Microbiological method for detecting metallic mercury in water body |
| 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 |
| CN110257415A (en) * | 2019-07-05 | 2019-09-20 | 中国农业大学 | A kind of full cell sensor building of nucleic acid-protein compound microbial for metal ion detection and threshold value control technique |
| CN110257466A (en) * | 2019-07-05 | 2019-09-20 | 中国农业大学 | A kind of nucleic acid-protein compound allosteric type heavy metal Hg test strip technology of preparing based on microbial whole-cell sensor |
| CN110873790A (en) * | 2018-09-03 | 2020-03-10 | 华南理工大学 | Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof |
| CN113234651A (en) * | 2021-03-31 | 2021-08-10 | 深圳市职业病防治院 | Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal |
| CN114152601A (en) * | 2021-12-28 | 2022-03-08 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method and kit for rapidly detecting mercury ions in water on site and application of kit |
| CN114560950A (en) * | 2022-03-15 | 2022-05-31 | 甘肃民族师范学院 | Genetic-coded organic mercury fluorescent probe and preparation method and application thereof |
-
2010
- 2010-05-18 CN CN2010101855456A patent/CN102250819A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| QINHONG WANG等: "Developing Highly Sensitive Micro-Biosensors for In Situ Monitoring of Mercury and Chromium (VI) Contaminants by Genetically Evolved and Computer-Designed Metal-Binding Proteins", 《4TH ANNUAL DOE-ERSP PI MEETING: ABSTRACTS》 * |
| 时国庆等: "汞化合物的生物检测技术", 《环境污染治理技术与设备》 * |
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| WO2014141066A3 (en) * | 2013-03-11 | 2014-12-31 | Consejo Nacional De Investigaciones Cientificas Y Tecnicas (Conicet) | Mutated polypeptide, bacteria strain comprising it and methods to simultaneously detect different metal cations |
| CN104007151A (en) * | 2014-05-05 | 2014-08-27 | 北京航空航天大学 | Gene circuit based on lung-cancer marker detection |
| CN104845996A (en) * | 2015-02-13 | 2015-08-19 | 温州医科大学 | Microbiological method for detecting metallic mercury in water body |
| CN110873790A (en) * | 2018-09-03 | 2020-03-10 | 华南理工大学 | Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof |
| 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 |
| CN110257415A (en) * | 2019-07-05 | 2019-09-20 | 中国农业大学 | A kind of full cell sensor building of nucleic acid-protein compound microbial for metal ion detection and threshold value control technique |
| CN110257466A (en) * | 2019-07-05 | 2019-09-20 | 中国农业大学 | A kind of nucleic acid-protein compound allosteric type heavy metal Hg test strip technology of preparing based on microbial whole-cell sensor |
| CN110257466B (en) * | 2019-07-05 | 2021-06-15 | 中国农业大学 | Preparation technology of nucleic acid protein compound allosteric heavy metal mercury detection test strip based on microbial whole-cell sensor |
| CN113234651A (en) * | 2021-03-31 | 2021-08-10 | 深圳市职业病防治院 | Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal |
| CN114152601A (en) * | 2021-12-28 | 2022-03-08 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method and kit for rapidly detecting mercury ions in water on site and application of kit |
| CN114152601B (en) * | 2021-12-28 | 2023-09-15 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method and kit for rapidly detecting mercury ions in water on site and application of kit |
| CN114560950A (en) * | 2022-03-15 | 2022-05-31 | 甘肃民族师范学院 | Genetic-coded organic mercury fluorescent probe and preparation method and application thereof |
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