CN110241064A - A kind of building and its application of the nucleic acid-protein compound allosteric type microbial whole-cell sensor for mercury ion detecting - Google Patents
A kind of building and its application of the nucleic acid-protein compound allosteric type microbial whole-cell sensor for mercury ion detecting Download PDFInfo
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- CN110241064A CN110241064A CN201910604605.4A CN201910604605A CN110241064A CN 110241064 A CN110241064 A CN 110241064A CN 201910604605 A CN201910604605 A CN 201910604605A CN 110241064 A CN110241064 A CN 110241064A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/09—Recombinant DNA-technology
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
The invention discloses a kind of microorganism fluorescent optical sensor of novel detection mercury ion, the detection of the dustiness of mercury ion suitable for water body and liquid food.It includes the microorganism fluorescent optical sensor and its application method for detecting mercury ion.The microorganism fluorescent optical sensor includes: Escherichia coli, and Escherichia coli carry recombinant plasmid as host cell；Recombinant plasmid, recombinant plasmid are the pENTR plasmid that system mer promoter, mercury resistance system regulation gene merR, green fluorescence protein gene egfp and constitutive promoter tandem sequence are resisted containing mercury.Sensor application method includes: strain method for resuscitation, sensor detecting method, sample detection methods.This sensor is 2h to the response time of mercury ion, and minimal detectable concentration 1nM, highest detection concentration is 100nM.The present invention can be widely used in the detection and risk assessment of pollution Mercury In Environment.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of microorganism of novel detection mercury ion is entirely thin
Born of the same parents' fluorescent optical sensor.
Background technique
Mercury is common heavy metal, and symbol of element Hg is located at the 6th period, group iib in the periodic table of chemical element, is
Uniquely with metal existing for liquid under normal temperature and pressure.Mercury is the glittering heavy liquid of silvery white, and chemical property is stablized, insoluble in acid
It is also insoluble in alkali.It can be evaporated under mercury room temperature, the compound of mercury vapour and mercury has hypertoxic (chronic) more.The usage history of mercury is long-drawn-out
Long, widely used, pollution condition is more severe.Mercury metal constantly gathers in the natural environment, causes water body, soil, atmosphere dirty
Dye.Mercury cannot can not also be participated in the chemical degradation of environment as other heavy metals by microbial metabolism, to deposit for a long time
It is to cause great harm in environment and to the ecosystem.Mercury present in environment equally threatens human health, they can lead to
It crosses two ways and enters human body: first is that the water and food of heavy metal pollution are consumed directly by humans；Another kind is by food chain richness
Collection, finally enters human body.Mercury vapour and mercury salt (in addition to some solubility are minimum such as mercuric sulphide) are all hypertoxic, oral, suctions
It can lead to brain and hepatic injury after entering or contacting.Mercury destroys central nervous system, and counterpart, mucous membrane and tooth have adverse effect.It is long
Time, which is exposed in high mercury environment, can lead to cerebral injury and death.In recent years, accident of mercury pollution takes place frequently, and endanger wide, rule
Mould is big, it is difficult to administer, has a far reaching influence.Fast and accurately detection technique has mercury poisoning, accident of mercury pollution important
Prevention effect.Currently, highly sensitive spectral technique meets the needs of metal accurately detected, such as atomic absorption spectrography (AAS)
(FAAS, ETAAS), inductively coupled plasma body light emitting (ICP-AES), Inductively coupled plasma-mass spectrometry (ICP-MS), gas
Phase chromatography etc..Although these analysis methods can effectively analyze the mercury ion in environment, and measurement accuracy is reachable
To mg/kg.However, these analysis methods are at high cost, time-consuming, the support of expensive instrument is not only needed, but also needs complexity
Preprocessing process and biggish sample size, be not suitable for scene, on-line analysis detection, it is difficult to adapt to showing for environment and market product
Field selective examination, manufacturing enterprise are checked oneself and product imports and exports the requirement to speed passage through customs.Therefore, researcher be dedicated to exploring easily use,
High, quick, the sensitive heavy metal analysis alternative of cost performance.
Microbiological sensor is a kind of biosensor, it is to utilize microorganism sheet using microbial cell as sensing element
The mechanism of body combines its Reports component, to detect various physics, chemistry and biological substance [8].Microbiological sensor can be with
Measurable signal, which is converted, by biological information in the detection process carries out qualitative or quantitative analysis.Because microorganism has type
The advantages that more, distribution is extensively, surface area is big, fertility is strong, adaptable, metabolism enlivens, is developed as have more and more
The sensor of detectability.With the development of correlation theory and technology, microbiological sensor has been applied to medicine, agricultural and life
Object technical field etc..
Research finds there is natural heavy metal ion resistance mechanisms (Heavy Metal Resistance in microbial body
System), this mechanism can identify intracellular specific metal ion, and open the expression of corresponding function gene, by urging
Change, three kinds of modes of outlet and chelating release heavy metal ion to the toxicity of microorganism.Heavy metal resistance mechanisms are microbial sensitive
The building of device provides recognition component.Using this mechanism, researcher constructs the microbial sensitive for heavy metal ion
Device, and have original performance in the application of trace detection.By optimizing the technology and methods of mercury ion microbiological sensor,
The detection effect of sensor is improved, can not only be made during it is applied to more harsh environment and more accurately detects, and
This technical method generalization can be produced the sensor for being more directed to different heavy metals, preferably be prevented heavy metal-polluted
The generation of dye event and the deterioration of natural environment.
Summary of the invention
The object of the present invention is to provide it is a kind of it is simple and easy, easy to carry, quick and precisely, low-cost novel detection
The microorganism fluorescent optical sensor and its application method of mercury ion.
The present invention provides microbial whole-cell sensor, the fluorescin reporter plasmid comprising a kind of induction of mercury ion,
It is a MerR gene by constitutive promoter regulatory transcription, the polynucleotide sequence that is combined by MerR protein-specific and should
Recombinant plasmid of the fluorescence protein gene of sequence direct regulation and control as core element.
In some specific embodiments of the invention, fluorescin reporter plasmid is transformed into host bacteria, host
Bacterium includes but is not limited to Escherichia coli.
In some specific embodiments of the invention, the constitutive promoter sequence includes but is not limited to sequence SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3.
In some specific embodiments of the invention, the MerR gene, sequence is SEQ ID NO:4.
In some specific embodiments of the invention, the MerR protein-specific combination polynucleotide sequence, sequence
It is classified as SEQ ID NO:5.
In some specific embodiments of the invention, the fluorescin, including but not limited to green fluorescent protein are red
Color fluorescin.
The present invention also provides the detection method of the microbial whole-cell sensor, MerR albumen and specific more is used
The compound that nucleotide sequence is formed, controls the transcription of fluorescence protein gene, converts fluorescence intensity for ion concentration of mercury signal
The method that signal obtains ion concentration of mercury in sample to be tested.It is preferred that sample to be tested is milk.
In some specific embodiments of the invention, the reaction system of the detection method is as follows:
Microbiological sensor culture solution (OD600 0.2-0.6) 1-8mL
Fresh culture medium 1-8mL
Test sample 100-200 μ L
Incubation time is 1h-5h.
It is in some specific embodiments of the invention, the fluorescence intensity of microbial whole-cell sensor and mercury ion is dense
Working curve is made in degree, to achieve the purpose that ion concentration of mercury in test sample by fluorescence intensity.
The microbiological sensor of detection mercury ion of the invention can be realized by establishing standard curve to the mercury in sample
Ion is fast and accurately qualitative and quantitative detection.
The present invention using synthetic biology be transformed Escherichia coli, by make its respond mercury ion, expressing green fluorescent protein,
To have the function that detect mercury ion.Application method of the invention remains the quick advantage of Microbial cell-based biosensors,
The shortcomings that it is vulnerable to interference is compensated for simultaneously, improves the accuracy of sample detection.
The present invention realizes the features such as sample requirements are few, detection time is short, high sensitivity, and detection range is wide, convenient for taking
Band and field detection, it is low in cost, it can be mass.Also, within the scope of a certain range of mercury ion, meat is can be achieved in the present invention
Eye detection, will can further use under the detection environment in field, bring change to traditional environment detection.
Microorganism fluorescent optical sensor of the invention, which is characterized in that including bacillus coli DH 5 alpha, Escherichia coli are as host
Cell carries recombinant plasmid.
The recombinant plasmid is by resisting system mer promoter, mercury resistance system regulation gene merR, green containing mercury
The pENTR plasmid of fluorescence protein gene egfp and constitutive promoter tandem sequence.
Construct the concrete operation step of the microorganism fluorescent optical sensor are as follows:
1. constitutive promoter, merR gene and Pmer import pEGFP plasmid
Constitutive promoter and PmerT, merR gene and egfp reporter gene are synthesized by company.In recombination matter of the invention
In grain, using gene from BglII and HindIII enzyme to the insertion synthesis of the pMBG comprising egfp reporter gene and promoter, adjust
Control protein gene merR is controlled by constitutive promoter, and egfp gene is controlled by specificity promoter PmerT.
The application method of the novel microbial sensor, preparation method, strain method for resuscitation including engineered strain
And detection method.
2. the conversion of recombinant plasmid
Competent E.coli is taken out from -80 DEG C of refrigerators, is placed in ice chest, and 10 μ L recombinant plasmids are added.By large intestine bar
Bacterium is placed in ice chest after 5min, is put into 42 DEG C of water-bath 90s.After taking out competence 200 μ LLB culture mediums of addition, in 37 DEG C of shaking tables
Grow 40min.Escherichia coli after conversion are coated on the solid LB media that kanamycins is added, are incubated overnight.Take sun
Property bacterium colony carry out bacterium colony PCR, sequence verification.Obtain the microorganism fluorescent optical sensor of detection mercury ion.
The pre-treating method of the test sample, which is characterized in that the impurity in removal sample that can be quick and easy, no
The content of mercury ion in sample is influenced, mercury ion microbiological sensor is helped to realize accurate detection.
Detailed description of the invention
Fig. 1 recombinant plasmid map；
Relation curve of Fig. 2 relative intensity of fluorescence from various concentration Hg2+ under different testing conditions；
Relation curve of Fig. 3 relative intensity of fluorescence from various concentration Hg2+ under different antibiotic concentrations；
The relation curve of various concentration Hg2+ in Fig. 4 relative intensity of fluorescence and milk and ultrapure water.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1: the performance detection of the microorganism fluorescent optical sensor of novel detection mercury ion
Step 1: inoculation sensor cell single bacterium falls within 50mL and fills in the triangular flask of LB culture medium, kanamycins is added
To final concentration of 50 μ g/mL, 37 DEG C, 200rpm is incubated overnight；
Step 2: taking the above-mentioned bacterium solution of 1mL into the fresh LB of 20mL, 37 DEG C, 200rpm is cultivated to OD600=
0.6；
Step 3: taking 5mL bacterium solution, 5mL fresh LB is mixed with 100 μ L mercury standard solutions, and 37 DEG C, 200rpm is lured
Lead 2h；
Step 4: taking the bacterium solution after the above-mentioned induction of 2mL in 2mL centrifuge tube, 12000g is centrifuged 2min, and 200 μ L are added
0.9% physiological saline, piping and druming are vortexed；
Step 5: taking saturating 96 orifice plate in completely black bottom, induction stoste and each 200 μ L of concentrate are added thereto, uses microplate reader
It is detected；
Step 6: according to standard sample induction lower sensor cell relative intensity of fluorescence, production relative intensity of fluorescence and
The standard curve of ion concentration of mercury.
Embodiment 2: detection of the microorganism fluorescent optical sensor of novel detection mercury ion to mercurous milk standard sample
Step 1: inoculation sensor cell single bacterium falls within 50mL and fills in the triangular flask of LB culture medium, kanamycins is added
To final concentration of 50 μ g/mL, 37 DEG C, 200rpm is incubated overnight；
Step 2: taking the above-mentioned bacterium solution of 1mL into the fresh LB of 20mL, 37 DEG C, 200rpm is cultivated to OD600=
0.6；
Step 3: taking milk sample to be measured, 12000g is centrifuged 5min, the milk soln after taking separation, for use；
Step 4: taking 5mL bacterium solution, 5mL fresh LB is mixed with 100 μ L mercury standard solutions, milk sample to be measured respectively
It closes, 37 DEG C, 200rpm induces 2h；
Step 5: taking the bacterium solution after the above-mentioned induction of 2mL in 2mL centrifuge tube, 12000g is centrifuged 2min, and 200 μ L are added
0.9% physiological saline, piping and druming are vortexed；
Step 6: taking saturating 96 orifice plate in completely black bottom, induction stoste and each 200 μ L of concentrate are added thereto, uses microplate reader
It is detected；
Step 7: according to standard sample induction lower sensor cell relative intensity of fluorescence, production relative intensity of fluorescence and
The standard curve of ion concentration of mercury calculates the ion concentration of mercury in milk sample using standard curve.
Sequence table
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Claims (9)
1. microbial whole-cell sensor, the fluorescin reporter plasmid comprising a kind of induction of mercury ion, which is characterized in that be one
A MerR gene by constitutive promoter regulatory transcription, the polynucleotide sequence combined by MerR protein-specific and the sequence
Recombinant plasmid of the fluorescence protein gene of direct regulation and control as core element.
2. microbial whole-cell sensor as described in claim 1, which is characterized in that fluorescin reporter plasmid to be transformed into
In host bacteria, host bacteria includes but is not limited to Escherichia coli.
3. microbial whole-cell sensor as claimed in claim 2, which is characterized in that the constitutive promoter sequence includes
But it is not limited to sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3.
4. microbial whole-cell sensor as claimed in claim 2, which is characterized in that the MerR gene, sequence SEQ
ID NO:4。
5. microbial whole-cell sensor as claimed in claim 2, which is characterized in that the MerR protein-specific combines more
Nucleotide sequence, sequence are SEQ ID NO:5.
6. microbial whole-cell sensor as claimed in claim 2, which is characterized in that the fluorescin, including but it is unlimited
In green fluorescent protein, red fluorescent protein.
7. the detection method based on any one of claim 1 to 6 microbial whole-cell sensor, which is characterized in that use
The compound that MerR albumen and specific polynucleotide sequence are formed, controls the transcription of fluorescence protein gene, by ion concentration of mercury
Signal is converted into the method that fluorescence intensity signals obtain ion concentration of mercury in sample to be tested.
8. detection method as claimed in claim 7, which is characterized in that the reaction system of the detection method is as follows:
Microbiological sensor culture solution (OD600 0.2-0.6) 1-8mL
Fresh culture medium 1-8mL
Test sample 100-200 μ L
Incubation time is 1h-5h.
9. detection method as claimed in claim 7, which is characterized in that by the fluorescence intensity and mercury of microbial whole-cell sensor
Working curve is made in ion concentration, to achieve the purpose that ion concentration of mercury in test sample by fluorescence intensity.
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Cited By (4)
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| CN110684789A (en) * | 2019-10-24 | 2020-01-14 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method 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 |
| CN113999864A (en) * | 2020-07-28 | 2022-02-01 | 中国科学院微生物研究所 | Heavy metal adsorption and escape control engineering strain and construction method and application thereof |
| 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 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684789A (en) * | 2019-10-24 | 2020-01-14 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method and application thereof |
| CN110684789B (en) * | 2019-10-24 | 2021-08-24 | 南京林业大学 | Fusion gene, recombinant vector and preparation method thereof, cadmium ion whole-cell biosensor and preparation method and application thereof |
| CN113999864A (en) * | 2020-07-28 | 2022-02-01 | 中国科学院微生物研究所 | Heavy metal adsorption and escape control engineering strain and construction method 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 |
| 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 |
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Application publication date: 20190917 |