CN108949659A - Bioengineered strain and its preparation method and application for detecting dimercurion - Google Patents
Bioengineered strain and its preparation method and application for detecting dimercurion Download PDFInfo
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- CN108949659A CN108949659A CN201810732044.1A CN201810732044A CN108949659A CN 108949659 A CN108949659 A CN 108949659A CN 201810732044 A CN201810732044 A CN 201810732044A CN 108949659 A CN108949659 A CN 108949659A
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- ompa
- merr
- psb1a2
- rfp
- dimercurion
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
- G01N21/6404—Atomic fluorescence
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Abstract
The invention discloses a kind of for detecting the bioengineered strain of dimercurion, and the bioengineered strain contains expression vector merR op-ompA-rfp-pSB1A2, wherein merR op-ompA-rfp is recombination, and pSB1A2 is plasmid backbone.The invention also discloses a kind of for detecting the preparation method and purposes of the bioengineered strain of dimercurion.Bioengineered strain provided by the invention highly selective, high sensitivity identification and can detect the target metal ions mercury in water environment, to 0.025 μM of low-concentration metallic IONS OF H g2+There is apparent response, and other metal ions of high concentration have not been responded to.Moreover, 1uM Hg is added2+When, it analyzes engineering bacteria responsiveness in the differential responses time and obvious fluorescence intensity occurs in 15min relative short time, with the extension of reaction time, fluorescence intensity gradually increases.
Description
Technical field
The present invention relates to water pollution detection technique fields.It is more particularly related to a kind of for detecting bivalent mercury
Bioengineered strain of ion and its preparation method and application.
Background technique
In heavy metal pollution research, metal ion Hg of the density in 5.0g/mL or more2+It is that bio-toxicity significantly pollutes
One of object.Once polluting into water environment, soil, organism, it will cause the situations for being difficult to remove.Data is shown, extremely low
Concentration Hg2+Also human health can be caused to poison, a series of damage such as human body alimentary canals, kidney, dirty, even more harm maincenter mind occurs
Cause mercury poisoning through system.
In recent years, production technology largely discharges mercury raffinate, causes heavy metal Hg pollution in rising trend.Heavy metal Hg pollution
It is not degradable, and inhibit the bioactivity of the life macromolecules substances such as human body protein, enzyme to influence eubolism.In view of mercury dirt
The serious harm to environment and human body life and health is contaminated, to Hg in water environment2+Detection is even more important.Initial chemical-agent technique,
Detection sensitivity and discrimination have limitation, can not a certain heavy metal ion of specific recognition, and selected organic reagent discharge ring
Border causes secondary pollution.Since modern age, the continuous development and improvement of the technological means and research method of mercury detection.Has sensitivity
Fabulous analysis method just has atomic fluorescence spectrometry (Atomic Fluorescence Spectrometry, AFS), atom to inhale
Receive spectroscopic methodology (Atmotic Absorption Spectrometry, AAS), atomic emission spectrometry (Atomic Emission
Pectrometry, AES) and inductively coupled plasma mass spectrometry (Iductively Coupled Plasma-Mass
Spectrometry, ICP-MS) etc..But these dependent on large-scale instrument detection method it is with high costs, sample treatment is complicated, institute
Take time length, it is difficult to realize low cost, simple, quickly detection.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide one kind by utilizing MerR albumen to target metal ions Hg2+High selection
Property, the identification of high sensitivity and binding characteristic and the excellent fluorescent spectroscopic properties of red fluorescent protein mFRP1, realize to water dirt
Trace Hg in dye system2+The bioengineered strain of the Visual retrieval of specificity.
In order to realize these purposes and other advantages according to the present invention, provides a kind of bioengineered strain and contain table
Up to carrier merR op-ompA-rfp-pSB1A2, wherein merR op-ompA-rfp is recombination, and pSB1A2 is plasmid bone
Frame.
The present invention also provides a kind of for detecting the preparation method of the bioengineered strain of dimercurion, including following step
It is rapid:
Step 1: the amplification of merR op, ompA, rfp genetic fragment are realized respectively using pcr amplification reaction, after amplification
MerR op genetic fragment connected with ompA genetic fragment by overlap extension pcr, and pass through pcr amplification reaction realize
Amplification, obtains target gene fragment merR op-ompA；
Step 2: using restriction enzyme XbaI and SpeI to pSB1A2 plasmid backbone and target gene fragment merR
Op-ompA carries out double digestion, makes target gene fragment merR op-ompA and pSB1A2 plasmid backbone while exposing identical viscous
Property end, target gene fragment merR op-ompA is connected with pSB1A2 plasmid backbone by T4 DNA ligase, realize
The building of merR op-ompA-pSB1A2 plasmid；
Step 3: using restriction enzyme SpeI to the rfp gene after merR op-ompA-pSB1A2 plasmid and amplification
Segment carries out single endonuclease digestion, then the rfp genetic fragment with T4 DNA ligase by merR op-ompA-pSB1A2 plasmid and after expanding
The building of expression vector merR op-ompA-rfp-pSB1A2 is completed in connection；
Step 4: expression vector merR op-ompA-rfp-pSB1A2 converted by chemical transformation thin to competence
It is intracellular, obtain the bioengineered strain.
Preferably, the target gene fragment merR op-ompA in step 1 is obtained especially by following methods: respectively
Using fully synthetic merR op, ompA, rfp gene order as template, first time pcr amplification reaction is carried out, then will be after amplification
The complementary fragment of merR op and ompA genetic fragment end realizes the company of merR op and ompA by overlap extension pcr
It connects, obtains junction fragment merR op-ompA, carry out second of pcr amplification reaction, realize junction fragment merR op-ompA's
Amplification, obtains target gene fragment merR op-ompA.
Preferably, the competent cell in step 4 is E.coli DH5 α competent cell.
Preferably, when first time pcr amplification reaction, it includes ImeR-OP XF, OP- that merR op, which needs the primer being added,
OmpA UP, it includes OP-ompA DN, OmpA SR that ompA, which needs the primer being added, and it includes mRFP1 that rfp, which needs the primer being added,
SF,mRFP1 SR；The primer that second of pcr amplification reaction is added includes IMerR-OP XF and OmpA SR.
Preferably, reaction temperature when carrying out double digestion in step 2 is 37 DEG C, reaction time 1h.
Preferably, reaction temperature when carrying out single endonuclease digestion in step 3 is 37 DEG C, reaction time 1h.
Preferably, first time pcr amplification reaction includes 30 circulations, wherein a circulation successively includes 98 DEG C of denaturation
10s, 55 DEG C of annealing 10s, 72 DEG C of extension 5s；Second of pcr amplification reaction includes 30 circulations, wherein a circulation is successively wrapped
Include 98 DEG C of denaturation 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 10s.
The present invention also provides a kind of for detecting the purposes of the bioengineered strain of dimercurion, which is used for
Whether detection water environment contains Hg2+。
Preferably, which identifies Hg2+Reaction time be 15min or more.
The present invention is include at least the following beneficial effects:
Bioengineered strain provided by the invention can be highly selective identification and detect the target metal ions in water environment
Mercury, to 0.025 μM of metal ion Hg2+There is apparent response, and to other metal ion Ag of high concentration+、Au3+、Cd2 +、 Cr3+、Cu2+、Fe3+、Ni2+、Pb2+、Zn2+It does not respond to.Moreover, under a certain concentration (1 μM), when analyzing differential responses
Between middle engineering bacteria responsiveness there is obvious fluorescence intensity in 15min relative short time, with the reaction time (15min~
8h) change, fluorescence intensity increases.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the plasmid map of pSB1A2 plasmid backbone of the present invention；
Fig. 2 is the building flow chart of expression vector of the present invention；
Fig. 3 is the electrophoretogram after merR op of the present invention, ompA, rfp, merR op-ompA gene fragment amplification；
Fig. 4 is merR op-ompA-pSB1A2 plasmid of the present invention and the electrophoretogram that digestion is identified；
Fig. 5 is merR op-ompA-rfp-pSB1A2 expression vector of the present invention and the electrophoretogram that digestion is identified；
Fig. 6 is bioengineered strain of the present invention to various concentration Hg2+The fluorescence intensity figure of Visual retrieval；
Fig. 7 is for bioengineered strain of the present invention to same concentrations Hg in different incubation times2+Visual retrieval
Fluorescence intensity figure；
Fig. 8 is fluorescence intensity figure of the bioengineered strain of the present invention to 10 metal ion species Visual retrievals.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, to enable those skilled in the art's reference
Specification word can be implemented accordingly.
It should be noted that experimental method described in following embodiments is unless otherwise specified conventional method, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.
The present invention provides a kind of for detecting the bioengineered strain of dimercurion, and the bioengineered strain contains expression and carries
Body merR op-ompA-rfp-pSB1A2, wherein merR op-ompA-rfp is recombination, and pSB1A2 is plasmid backbone.
PSB1A2 plasmid backbone is used for construction recombination plasmid, and pSB1A2 plasmid map is as shown in Figure 1.
As shown in Fig. 2, the present invention also provides a kind of for detecting the preparation method of the bioengineered strain of dimercurion, wrap
Include following steps:
Step 1: the amplification of merR op, ompA, rfp genetic fragment are realized respectively using pcr amplification reaction, after amplification
MerR op genetic fragment connected with ompA genetic fragment by overlap extension pcr, and pass through pcr amplification reaction realize
Amplification, obtains target gene fragment merR op-ompA；
The gene order of merR as shown in SEQ ID NO.1, the gene order of op as shown in SEQ ID NO.2, ompA's
Gene order is as shown in SEQ ID NO.3, and the gene order of rfp is as shown in SEQ ID NO.4, recombination merR op-
The gene order of ompA-rfp is as shown in SEQ ID NO.5.
Target gene fragment merR op-ompA in step 1 is obtained especially by following methods: respectively with fully synthetic
MerR op, ompA, rfp gene order be template, carry out first time pcr amplification reaction, then by after amplification merR op and
The complementary fragment of ompA genetic fragment end realizes the connection of merR op and ompA by overlap extension pcr, is connected
Segment merR op-ompA carries out second of pcr amplification reaction, realizes the amplification of junction fragment merR op-ompA, obtains mesh
Genetic fragment merR op-ompA.If the following table 1 is first time pcr amplification reaction system (100ul).If the following table 2 is second
Pcr amplification reaction system (100ul).
Table 1
Table 2
When first time pcr amplification reaction, it includes upstream primer IMerR-OP XF under that merR op, which needs the primer being added,
Swimming primer OP-OmpA UP, it includes upstream primer OP-OmpA DN and downstream primer OmpA SR that ompA, which needs the primer that is added,
It includes upstream primer mRFP1 SF and downstream primer mRFP1 SR that rfp, which needs the primer being added,；Second of pcr amplification reaction adds
The primer entered includes IMerR-OP XF and OmpA SR, and the sequence of above-mentioned each primer is as shown in table 3 below.In first time PCR amplification
Archaeal dna polymerase is also added into when reaction and second of pcr amplification reaction.
Table 3
First time pcr amplification reaction includes 30 circulations, wherein a circulation is successively moved back including 98 DEG C of denaturation 10s, 55 DEG C
Fiery 10s, 72 DEG C of extension 5s；Second of pcr amplification reaction includes 30 circulations, wherein a circulation successively includes 98 DEG C of denaturation
10s, 55 DEG C of annealing 10s, 72 DEG C of extension 10s.Such as the response procedures that the following table 4 is first time pcr amplification reaction.If the following table 5 is the
The response procedures of secondary PCR amplified reaction.
Table 4
Table 5
Genetic fragment merR op, ompA, rfp, merR op-ompA use Ago-Gel after expanding by PCR program
Electrophoresis identified, electrophoresis result show swimming lane 1,2,3,4 near 500bp, between 500bp-750bp, near 750bp,
1000bp nearby has apparent single slice, with merR op (506bp), ompA (549bp), rfp (742bp) and merR op-
The size of ompA (1054bp) genetic fragment is consistent completely, as shown in figure 3, showing that PCR amplification result is in the main true.In Fig. 3,1
Represent merR op gene amplification product；2 represent ompA gene amplification product；3 represent rfp gene amplification product；4 represent merR
Op-ompA gene amplification product.
Step 2: using restriction enzyme XbaI and SpeI to PSB1A2 plasmid backbone and target gene fragment merR
Op-ompA carries out double digestion, makes target gene fragment merR op-ompA and pSB1A2 plasmid backbone while exposing identical viscous
Property end, target gene fragment merR op-ompA is connected with pSB1A2 plasmid backbone by T4DNA ligase, realize merR
The building of op-ompA-pSB1A2 plasmid；Reaction temperature when carrying out double digestion is 37 DEG C, reaction time 1h.
The target gene fragment connected by overlap extension pcr is handled using restriction enzyme XbaI and SpeI
MerR op-ompA and pSB1A2 plasmid backbone, reaction system digestion products after 37 DEG C of isothermal reaction 1h pass through 1% agarose
Gel electrophoresis experiment and gel extraction experiment carry out concentration determination after purification.Endonuclease bamhi merR op-ompA and pSB1A2 matter
Grain skeleton is after gel extraction, under the action of T4 ligase, realizes that the success of merR op-ompA segment and pSB1A2 connect
It connects, connection product is converted by chemical method into E.coli DH5 α competent cell.Picking monoclonal carries out the side by step 1
Method carries out PCR identification, and PCR identifies that correct monoclonal carries out plasmid extraction and digestion identification experiment, as shown in figure 4,1 in Fig. 4
The result for respectively representing merR op-ompA-pSB1A2 plasmid and the identification of XbaI, SpeI double digestion with 2.Meanwhile through the raw work in Shanghai
Biotech firm's sequencing proves that merR op-ompA segment is successfully inserted into the XbaI/SpeI digestion position of pSB1A2 plasmid backbone
Between point, and building process does not mutate.
The double enzyme digestion reaction system of target gene fragment merR op-ompA and pSB1A2 plasmid backbone is as shown in table 6 below.
Then, target gene fragment merR op-ompA is constructed to pSB1A2 plasmid backbone by connection reaction, realizes merR op-
The building of ompA-pSB1A2 carrier, specific reaction system is as shown in table 7, in 16 DEG C of constant temperature overnight after take a small amount of connection product into
Row conversion, picking PCR identification and the correct monoclonal of digestion identification send to the raw work in Shanghai after being incubated overnight in LB culture medium raw
Object company is sequenced, and the correct carrier merR op-ompA-pSB1A2 of sequencing result is carried out subsequent experimental.
Table 6
Table 7
Step 3: using restriction enzyme SpeI to the rfp gene after merR op-ompA-PSB1A2 plasmid and amplification
Segment carries out single endonuclease digestion, then the rfp genetic fragment with T4DNA ligase by merR op-ompA-PSB1A2 plasmid and after expanding
The building of expression vector merR op-ompA-rfp-pSB1A2 is completed in connection；Over-lap PCR is exactly Overlap extension PCR skill in Fig. 2
Art；Reaction temperature when carrying out single endonuclease digestion in step 3 is 37 DEG C, reaction time 1h.
Rfp genetic fragment and merR op-ompA-pSB1A2 after amplification, reaction are handled using restriction enzyme SpeI
System digestion products after 37 DEG C of isothermal reaction 1h are laggard by the experiment of 1% agarose gel electrophoresis and gel extraction experiment purifying
Row concentration determination.As shown in figure 5,1 and 2 representing expression vector in Fig. 5 as merR op-ompA-rfp-pSB1A2 and SpeI enzyme
Cut the result of identification, it was demonstrated that after merR op-ompA segment is successfully connected to plasmid pSB1A2, merR op-ompA-pSB1A2 with
Rfp segment use SpeI single endonuclease digestion after, reuse T4 ligase can be realized plasmid merR op-ompA-rfp-pSB1A2 at
Function building.It is transferred in competent cell E.coli DH5 α, is sequenced to prevent having base deletion or mutation again.It is raw through the raw work in Shanghai
The sequencing of object company proves that rfp segment is successfully inserted into the meta position of pSB1A2 carrier Yu merR op-ompA segment, and constructs
Cheng Wei mutates.
MerR op-ompA-pSB1A2 and the single endonuclease digestion reaction system of the rfp genetic fragment after amplification are as shown in table 8 below.
Then, the rfp genetic fragment after amplification is constructed to merR op-ompA-pSB1A2 by connection reaction, realizes that expression carries
The building of body merR op-ompA-rfp-pSB1A2, specific reaction system is as shown in table 9, then is converted, picking PCR identification
Shanghai Sheng Gong biotech firm is sent to after being incubated overnight in LB culture medium with the correct monoclonal of digestion identification to be sequenced, and will be surveyed
It is real for subsequent performance test after the correct expression vector merR op-ompA-rfp-pSB1A2 scribing line of sequence result and conservation
It tests.
Table 8
Table 9
Step 4: expression vector merR op-ompA-rfp-PSB1A2 converted by chemical transformation thin to competence
It is intracellular, obtain the bioengineered strain.Competent cell in step 4 is E.coli DH5 α competent cell, as host
Bacterium.Specific conversion process are as follows: (1) the competent cell E.coli DH5 α dispensed is taken to thaw from nitrogen gas tank on ice；(2)
1ul expression vector merR op-ompA-rfp-PSB1A2 is added, tube wall can be flicked to mix, in placing 30min on ice； (3)
42 DEG C of water-bath heat shock 90s, avoid shaking；(4) 5min on ice is put back to rapidly；(5) plus 500ul sterilizes LB (being free of resistance), in 37
DEG C 250rpm constant temperature incubation 60min；(6) take 50ul bacterium solution uniform in sprawling in resistant LB solid medium board；(7) plate
It is inverted in 37 DEG C of oven overnight cultures.
<bioengineered strain performance test>
One, engineering bacteria identifies Hg2+Sensitivity behaviour research
(1) from scribing line culture LB plate on the picking expression vector of op-ompA-rfp-pSB1A2 containing merR single colonie,
It is inoculated in the LB liquid medium of 3mL sterilizing (containing 150 μ g/mL carbenicillins), crosses liquid in 37 DEG C of 250rpm constant temperature incubations；
(2) bacterium solution being incubated overnight is incubated in the LB liquid medium of 250mL in the expansion of 1:100 ratio (containing 150
Ug/mL carbenicillin), in 37 DEG C of 250rpm constant temperature incubations until OD600=1；
(3) packing is above-mentioned grows to OD600=1 bacterium solution (5mL/ pipe), is added respectively into the bacterium solution dispensed corresponding
Hg2+Standard solution to final concentration is respectively 0 μM, 0.001 μM, 0.01 μM, 0.025 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5
μM, 10 μM, 15 μM, 20 μM, continue to cultivate 4h (setting 5 repetition test) in 37 DEG C of 250rpm；
(4) 6000rpm is centrifuged 3min and collects thallus, is washed with 500 μ L 10mM PBS buffer solution；
(5) add 1mL 10mM PBS buffer solution to be resuspended to the thallus after washing, use Shimadzu RF-6000 fluorescence spectrophotometry
Measurement resuspended bacterium solution fluorescence intensity is simultaneously taken pictures.
In experiment, give engineering bacteria certain density Hg respectively2+(0μM、0.001μM、0.01μM、0.025μM、 0.05μ
M, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM), according to the method described above, finally measure various concentration Hg2+Lower engineering
Bacterium shows fluorescence intensity, by Fig. 6, it is apparent that with Hg2+The increase of concentration, system fluorescence intensity first increases to be declined afterwards,
And Hg2+Concentration shows that fluorescence intensity is most strong when reaching 0.5 μM.Work as Hg2+Concentration is in 0.025 μM~15 μM of ranges, engineering bacteria
Show obvious fluorescence intensity.Show that red fluorescence is consistent with measurement fluorescence intensity in cell membrane surface under various concentration, it is red strong
Degree gradient weakens again after increasing.Experiment Result show bioengineered strain can efficiently, in Visual retrieval water environment Low Concentration Mercury from
Son.
Two, engineering bacteria identifies Hg2+Peak optimization reaction time study
(1) from the single colonie of the picking plasmid of op-ompA-rfp-pSB1A2 containing merR on the LB plate of scribing line culture, inoculation
(contain 150 μ g/mL carbenicillins) in the LB liquid medium of 3mL sterilizing, crosses liquid in 37 DEG C of 250rpm constant temperature incubations；
(2) bacterium solution being incubated overnight is incubated in the LB liquid medium of 250mL in the expansion of 1:100 ratio (containing 150
Ug/mL carbenicillin), in 37 DEG C of 250rpm constant temperature incubations until OD600=1；
(3) add 200 μ L, 500 μM of Hg in OD600=1 bacterium solution to above-mentioned grow to2+(5mL/ is dispensed after standard solution
Pipe), continue culture in 37 DEG C of 250rpm (5 repetitions of setting are tested)；
(4) bacterium solution is taken out respectively in 0min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 6000rpm is centrifuged 3min and collects
Thallus is washed with 500 μ L 10mM PBS buffer solution；
(5) add 1mL 10mM PBS buffer solution to be resuspended to the thallus after washing, use Shimadzu RF-6000 fluorescence spectrophotometry
Measurement resuspended bacterium solution fluorescence intensity is simultaneously taken pictures.
In certain Hg2+Under concentration (1 μM), mercury ion enters cell interior, the MerR protein binding with cell inner expression
Afterwards, the expression for activating downstream ompA-rfp gene expresses that red fluorescent protein mRFP1 in bacterium surface, thus in bacterium table
Face shows that fluorescence intensity, Fig. 7 result indicate, with mercury ion time change is added, system fluorescence intensity has significant difference.It is incubating
After educating 15min, engineering bacteria has shown that faint fluorescence intensity, and naked eyes are visible；After being incubated for 1h, engineering bacteria is shown glimmering
Luminous intensity rises rapid；Between 1h~4h, the fluorescence intensity ascendant trend of engineering bacteria is obvious；Between 4h~8h, engineering bacteria
It shows stronger fluorescence intensity, visually observes fairly obvious.Research achievement shows that this bioengineered strain can be realized in a short time
Trace Hg in water environment2+Visual retrieval.
Three, engineering bacteria identifies Hg2+Specific performance study
(1) from the single colonie of the picking plasmid of op-ompA-rfp-pSB1A2 containing merR on the LB plate of scribing line culture, inoculation
(contain 150 μ g/mL carbenicillins) in the LB liquid medium of 3mL sterilizing, crosses liquid in 37 DEG C of 250rpm constant temperature incubations；
(2) bacterium solution being incubated overnight is incubated in the liquid M9 culture medium of 250mL in the expansion of 1:100 ratio (containing 150
Ug/mL carbenicillin), in 37 DEG C of 250rpm constant temperature incubations until OD600=1；
(3) OD600=1 bacterium solution packing (5mL/ pipe) is grown to by above-mentioned, final concentration is added to the bacterium solution dispensed respectively
Respectively 0.05 μM and 0.1 μM of Hg2+、Ag+、Au3+、Cd2+、Cr3+、Cu2+、Fe3+、Ni2+、Pb2+、Zn2+, in 37 DEG C of 250rpm
Continue to cultivate 12h (5 repetitions of setting are tested)；
(4) 6000rpm is centrifuged 3min and collects thallus, is washed with 500 μ L 10mM PBS buffer solution；
(5) add 1mL 10mM PBS buffer solution to be resuspended to the thallus after washing, use Shimadzu RF-6000 fluorescence spectrophotometry
Measurement resuspended bacterium solution fluorescence intensity is simultaneously taken pictures.
10 metal ion species (Hg are separately added into the LB liquid medium of culturing engineering bacterium2+、Ag+、Au3+、Cd2+、 Cr3+、
Cu2+、Fe3+、Ni2+、Pb2+、Zn2+), compare the fluorescence intensity that engineering bacteria responds different metal ions to reflect engineering bacteria to Hg2 +Selectivity.As seen from Figure 8, Hg is added2+When, engineering bacteria specificity shows fluorescence intensity, to Hg2+There is preferable selectivity,
To effectively detect Hg in water environment2+Concentration.And other metal ion (Ag in water environment+、 Au3+、Cd2+、Cr3+、Cu2+、Fe3 +、Ni2+、Pb2+、Zn2+) although engineering bacteria can also be made to show fluorescence intensity, it is relatively very weak.Opposite result of study shows engineering bacteria
To Hg2+There is preferable selectivity, it can mercury ion content in specific detection water environment.
This experiment utilizes technique for gene engineering, passes through the success of the genetic engineering means such as PCR amplification, digestion, connection and conversion
MerR op-ompA-rfp-pSB1A2 expression vector is constructed, the bacterium containing expression vector can pass through metal-regulatory albumen
" transcriptional activation " mechanism regulating red fluorescent protein mRFP1 of MerR microbial cell surface expression, to realize trace
Hg2+Visual retrieval.Result of study shows Hg in water environment2+Concentration is different, the red egg that bioengineered strain surface is shown
White mRFP1 fluorescence intensity is different, and the fluorescence intensity of bioengineered strain is in the trend of growth at any time.Meanwhile above-mentioned biology
Engineering bacteria can only Hg in specific detection water environment2+, and other metal ion Ag+、Au3+、Cd2+、Cr3+、 Cu2+、Fe3+、Ni2+、
Pb2+、Zn2+It does not respond to.Therefore, in this research biological detection engineering bacteria successful exploitation, be ecological, environmental protective, selectivity it is good,
Important foundation has been established in high sensitivity, low in cost, reusable mercury pollution detection.
In addition, reagent used in the present invention is as shown in the following table 10.
Table 10
Instrument and equipment used in the present invention is as shown in table 11 below.
Table 11
Culture medium prescription used in the present invention is as shown in table 12 below.
Table 12
Electrophoresis liquid used in the present invention is 50 × TAE electrophoresis liquid (1L), specific the preparation method comprises the following steps: weighing 242g Tris
Alkali is settled to 1000 after 57.1ml glacial acetic acid and 100ml 0.5M NaOH (pH=8.0) is added after being completely dissolved with deionized water
Ml, working solution concentration be 1 ×.
4mL 50 is added the preparation method comprises the following steps: weighing 2g agar powder (Agarose G-10) in agarose used in the present invention
200mL is settled to after × TAE, moderate heat is heated to solid and is completely dissolved.
The formula of M9 culture medium used in the present invention is as shown in table 13, wherein the formula of 1 × M9 salting liquid and preparation side
Method such as table 14, MgSO4Glucose-CaCl2The formula of solution is as shown in Table 15, weighs corresponding weight by the formula of table 15
MgSO4, glucose, CaCl2Afterwards, then plus pure water constant volume to 20mL, dispensed after crossing 0.22 μm of sterilised membrane filter, place -20 DEG C of guarantors
It deposits.
Table 13
Table 14
Table 15
The formula of 6 × DNA sample-loading buffer (1mL) used in the present invention is as shown in table 16.
Table 16
0.1M phosphate buffer (PBS) used in the present invention the preparation method comprises the following steps: weigh 80g NaCl, 2g KCl,
36.3g Na2HPO4·12H2O and 2.4g KH2PO4Be dissolved in 800mL distilled water, with 4M dilute hydrochloric acid adjust solution pH value to
7.4, it has adjusted after pH value plus distilled water is settled to 1L, last 120 DEG C of high pressure sterilizations 30min, saved in 4 DEG C of refrigerators.
Agarose gel electrophoresis experiment has been used in the present invention.Agarose gel electrophoresis experiment is mainly used to separate and identify
DNA molecular.DNA molecular is mainly by ribose, phosphoric acid and base composition, and the structure of ribose-phosphate backbone is repeated, makes identical
The double chain DNA molecule of quantity possesses identical electrostatic charge, and the nucleic acid as amphiphatic molecule is in pH=8.0 or so, entirely
Charge is negatively charged, will move at a same speed to anode in electrophoretic action, therefore under identical electric field strength, DNA molecular
Migration rate depend on DNA molecular itself molecular weight and structure.DNA molecular identical for configuration, migration velocity with
The size of relative molecular weight is inversely proportional.And the DNA molecular different for molecular weight identical configuration, agarose gel electrophoresis experiment are same
Sample is available to be efficiently separated, the migration rate of the DNA molecular of various configuration in certain electric field are as follows: covalently closed circle supercoil
DNA > linear DNA > open loop double-stranded cyclic DNA.General agarose gel electrophoresis experiment be suitable for separation size 0.2kb~
DNA fragmentation within the scope of 50kb.By taking the experiment of 1% agarose gel electrophoresis as an example, primary operational process are as follows: 2g agarose is weighed,
It is added after 50 × TAE of 4ml electrophoretic buffer mixes and is settled to 200ml with deionized water, it is complete that agarose is heated in micro-wave oven
Room temperature is cooling after fully dissolved, pours into mold rapidly after temperature is down to 60 DEG C or so and 2-4ul ethidium bromide is added.It is uniformly mixed
After continue cool to colloid solidification after, the gel prepared is transferred in electrophoresis system by mold, it is slow that 1 × TAE electrophoresis is added
Fliud flushing was not to there be glue surface slightly.Backward Ago-Gel swimming lane in DNA sample is added, the gel slab after sample-adding be powered immediately into
Row electrophoresis, voltage 100V, sample are mobile from cathode (black) to positive (red) direction.When bromophenol blue is moved to distance in sample
When at offset plate lower edge about 1/3, stop electrophoresis.It is careful to take out gel, it observes in the UV lamp, DNA, which exists, then shows that red is glimmering
Striation band is taken pictures preservation using Gel Doc XR gel imaging system.
Gel extraction experiment has been used in the present invention.It is solidifying to agarose using Omega Bio-Tek gel extraction kit
The DNA fragmentation that gel electrophoresis experiment is cut is recycled, and key step is as follows: 1) gel being placed in the EP pipe completely to sterilize claims
Weight calculates the net weight of gel, and the ratio of 100ul Binding Buffer XP2 is added according to the gel of every 100mg weight
The Binding Buffer XP2 of appropriate volume is added in EP pipe.2) EP pipe is put into 55-60 DEG C of waters, until to solidifying
Glue melts completely, and the gel that during which can turn upside down accelerates the thawing of gel.3) it will be dissolved after gel melts completely with liquid-transfering gun
Careful being transferred to of liquid is equipped with collecting pipeIn DNA Mini column, 10000xg is centrifuged 1 minute at room temperature, is discarded
Centrifugate.4) again to300ul Binding Buffer XP2 is added in DNA Mini column, equally at room temperature
10000xg discards centrifugate after being centrifuged 1 minute.5) to700ul SPW Wash is added in DNA Mini column
Filtrate is discarded after Buffer, room temperature high speed centrifugation 1min.6) after repeating step 5), room temperature high speed centrifugation 3min is dry againDNA Mini column.7) sample collection tube is replaced into sterile EP to manage, the MilliQ of 20-30 ul sterilizing is added
H2O infiltrationDNA Mini column is stored at room temperature high speed centrifugation eluted dna sample after 2min, uses
NanoDrop1000 trace of albumin nucleic acid analyzer saves after measuring concentration to -4 DEG C.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
The sequence of merR is as follows in the present invention:
TTACGGCATAGCAGAACCAGCCAGTGAAGCACCACCTTGCAGCGAAGCGATCA
GCGGACACGACACGTTACCGCGACGAGCATGACATGCGCAAACCAGTTCTGACAGC
ACTGCTTCCATACGTGCCAGGTCTGCCATCTTTTCACGGACATCCTTCAGTTTGTGTT
CGGCCAGAGAGCTCGCTTCTTCGCAATGCGTACCGTCTTCCAGGCGCAGCAGTTCTG
CGATTTCATCCAGGGAGAAGCCCAGACGCTGAGCACTTTTGACAAAGCGAACACGG
GTCACGTCTGCTTCACCGTAGCGACGAATAGAGCCATACGGCTTATCCGGTTCCAGC
AGCAGGCCTTTGCGTTGATAGAAGCGGATCGTTTCCACATTCACACCGGCAGCCTTA
GCGAAGACACCAATCGTCAGGTTTTCCAGGTTATTTTCCAT
The sequence of op (operator) is as follows in the present invention:
ATCGCTTGACTCCGTACATGAGTACGGAAGTAAGGTTACGCTATCCAATTTCAAT
TCGAAAGGACAAGCGC
The sequence of ompA is as follows in the present invention:
ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTA
GCGCAGGCCGCTCCGAAAGATAACACCTGGTACACTGGTGCTAAACTGGGCTGGTC
CCAGTACCATGACACTGGTTTCATCAACAACAATGGCCCGACCCATGAAAACCAACT
GGGCGCTGGTGCTTTTGGTGGTTACCAGGTTAACCCGTATGTTGGCTTTGAAATGGGT
TACGACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAA
GCTCAGGGCGTTCAACTGACCGCTAAGCTGGGTTACCCAATCACTGACGACCTGGAC
ATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATG
GTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGA
TCACTCCTGAAATCGCTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACG
CACACACCATCGGCACTCGTCCGGACAACGGAGGATCC
The sequence of rfp is as follows in the present invention:
ATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATG
GAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCC
CCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCC
TTCGCCTGGGACATCCTGTCCCCTCAGTTCCAGTACGGCTCCAAGGCCTACGTGAAG
CACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGG
GAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTC
CCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCT
CCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGG
ATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGATGAGGCTGAAGCTGAA
GGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACATGGCCAAGAAGCCCG
TGCAGCTGCCCGGCGCCTACAAGACCGACATCAAGCTGGACATCACCTCCCACAAC
GAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGG CGCCTAA
The sequence of recombination merR op-ompA-rfp is as follows in the present invention:
TCTAGATTACGGCATAGCAGAACCAGCCAGTGAAGCACCACCTTGCAGCGAAG
CGATCAGCGGACACGACACGTTACCGCGACGAGCATGACATGCGCAAACCAGTTCT
GACAGCACTGCTTCCATACGTGCCAGGTCTGCCATCTTTTCACGGACATCCTTCAGTT
TGTGTTCGGCCAGAGAGCTCGCTTCTTCGCAATGCGTACCGTCTTCCAGGCGCAGCA
GTTCTGCGATTTCATCCAGGGAGAAGCCCAGACGCTGAGCACTTTTGACAAAGCGA
ACACGGGTCACGTCTGCTTCACCGTAGCGACGAATAGAGCCATACGGCTTATCCGGT
TCCAGCAGCAGGCCTTTGCGTTGATAGAAGCGGATCGTTTCCACATTCACACCGGCA
GCCTTAGCGAAGACACCAATCGTCAGGTTTTCCAGGTTATTTTCCATATCGCTTGACT
CCGTACATGAGTACGGAAGTAAGGTTACGCTATCCAATTTCAATTCGAAAGGACAAG
CGCATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTA
GCGCAGGCCGCTCCGAAAGATAACACCTGGTACACTGGTGCTAAACTGGGCTGGTC
CCAGTACCATGACACTGGTTTCATCAACAACAATGGCCCGACCCATGAAAACCAACT
GGGCGCTGGTGCTTTTGGTGGTTACCAGGTTAACCCGTATGTTGGCTTTGAAATGGGT
TACGACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAA
GCTCAGGGCGTTCAACTGACCGCTAAGCTGGGTTACCCAATCACTGACGACCTGGAC
ATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATG
GTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGA
TCACTCCTGAAATCGCTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACG
CACACACCATCGGCACTCGTCCGGACAACGGAGGATCCACTAGTATGGCCTCCTCCG
AGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAAC
GGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCC
AGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATC
CTGTCCCCTCAGTTCCAGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATC
CCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAAC
TTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGA
GTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAAT
GCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGGATGTACCCCGAGGACG
GCGCCCTGAAGGGCGAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTAC
GACGCCGAGGTCAAGACCACCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCGC
CTACAAGACCGACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGT
GGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGCCTAAACTAGT。
SEQUENCE LISTING
<110>Guangxi Teachers College
<120>for detecting bioengineered strain of dimercurion and its preparation method and application
<130> CN18NN9855I
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 435
<212> DNA
<213>artificial sequence
<400> 1
ttacggcata gcagaaccag ccagtgaagc accaccttgc agcgaagcga tcagcggaca 60
cgacacgtta ccgcgacgag catgacatgc gcaaaccagt tctgacagca ctgcttccat 120
acgtgccagg tctgccatct tttcacggac atccttcagt ttgtgttcgg ccagagagct 180
cgcttcttcg caatgcgtac cgtcttccag gcgcagcagt tctgcgattt catccaggga 240
gaagcccaga cgctgagcac ttttgacaaa gcgaacacgg gtcacgtctg cttcaccgta 300
gcgacgaata gagccatacg gcttatccgg ttccagcagc aggcctttgc gttgatagaa 360
gcggatcgtt tccacattca caccggcagc cttagcgaag acaccaatcg tcaggttttc 420
caggttattt tccat 435
<210> 2
<211> 71
<212> DNA
<213>artificial sequence
<400> 2
atcgcttgac tccgtacatg agtacggaag taaggttacg ctatccaatt tcaattcgaa 60
aggacaagcg c 71
<210> 3
<211> 549
<212> DNA
<213>artificial sequence
<400> 3
atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60
gccgctccga aagataacac ctggtacact ggtgctaaac tgggctggtc ccagtaccat 120
gacactggtt tcatcaacaa caatggcccg acccatgaaa accaactggg cgctggtgct 180
tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggttacga ctggttaggt 240
cgtatgccgt acaaaggcag cgttgaaaac ggtgcataca aagctcaggg cgttcaactg 300
accgctaagc tgggttaccc aatcactgac gacctggaca tctacactcg tctgggtggc 360
atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga caccggcgtt 420
tctccggtct tcgctggcgg tgttgagtac gcgatcactc ctgaaatcgc tacccgtctg 480
gaataccagt ggaccaacaa catcggtgac gcacacacca tcggcactcg tccggacaac 540
ggaggatcc 549
<210> 4
<211> 678
<212> DNA
<213>artificial sequence
<400> 4
atggcctcct ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc 60
tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120
acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180
ctgtcccctc agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc 240
gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300
gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac 360
aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc 420
atgggctggg aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag 480
atcaagatga ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc 540
tacatggcca agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac 600
atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc 660
cactccaccg gcgcctaa 678
<210> 5
<211> 1751
<212> DNA
<213>artificial sequence
<400> 5
tctagattac ggcatagcag aaccagccag tgaagcacca ccttgcagcg aagcgatcag 60
cggacacgac acgttaccgc gacgagcatg acatgcgcaa accagttctg acagcactgc 120
ttccatacgt gccaggtctg ccatcttttc acggacatcc ttcagtttgt gttcggccag 180
agagctcgct tcttcgcaat gcgtaccgtc ttccaggcgc agcagttctg cgatttcatc 240
cagggagaag cccagacgct gagcactttt gacaaagcga acacgggtca cgtctgcttc 300
accgtagcga cgaatagagc catacggctt atccggttcc agcagcaggc ctttgcgttg 360
atagaagcgg atcgtttcca cattcacacc ggcagcctta gcgaagacac caatcgtcag 420
gttttccagg ttattttcca tatcgcttga ctccgtacat gagtacggaa gtaaggttac 480
gctatccaat ttcaattcga aaggacaagc gcatgaaaaa gacagctatc gcgattgcag 540
tggcactggc tggtttcgct accgtagcgc aggccgctcc gaaagataac acctggtaca 600
ctggtgctaa actgggctgg tcccagtacc atgacactgg tttcatcaac aacaatggcc 660
cgacccatga aaaccaactg ggcgctggtg cttttggtgg ttaccaggtt aacccgtatg 720
ttggctttga aatgggttac gactggttag gtcgtatgcc gtacaaaggc agcgttgaaa 780
acggtgcata caaagctcag ggcgttcaac tgaccgctaa gctgggttac ccaatcactg 840
acgacctgga catctacact cgtctgggtg gcatggtatg gcgtgcagac actaaatcca 900
acgtttatgg taaaaaccac gacaccggcg tttctccggt cttcgctggc ggtgttgagt 960
acgcgatcac tcctgaaatc gctacccgtc tggaatacca gtggaccaac aacatcggtg 1020
acgcacacac catcggcact cgtccggaca acggaggatc cactagtatg gcctcctccg 1080
aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc 1140
acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca 1200
agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt 1260
tccagtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc 1320
tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg 1380
tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc 1440
gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg 1500
cctccaccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc 1560
tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga 1620
agcccgtgca gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca 1680
acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg 1740
cctaaactag t 1751
Claims (10)
1. the bioengineered strain for detecting dimercurion, which is characterized in that the bioengineered strain contains expression vector
MerR op-ompA-rfp-pSB1A2, wherein merR op-ompA-rfp is recombination, and pSB1A2 is plasmid backbone.
2. as described in claim 1 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that packet
Include following steps:
Step 1: the amplification of merR op, ompA, rfp genetic fragment are realized respectively using pcr amplification reaction, after amplification
MerR op genetic fragment is connected with ompA genetic fragment by overlap extension pcr, and is realized and expanded by pcr amplification reaction
Increase, obtains target gene fragment merR op-ompA；
Step 2: using restriction enzyme XbaI and SpeI to pSB1A2 plasmid backbone and target gene fragment merR op-
OmpA carries out double digestion, makes target gene fragment merR op-ompA and pSB1A2 plasmid backbone while exposing identical viscosity end
End, is connected target gene fragment merR op-ompA with pSB1A2 plasmid backbone by T4 DNA ligase, realizes merR
The building of op-ompA-pSB1A2 plasmid；
Step 3: using restriction enzyme SpeI to the rfp genetic fragment after merR op-ompA-pSB1A2 plasmid and amplification
Single endonuclease digestion is carried out, then is connected the rfp genetic fragment after merR op-ompA-pSB1A2 plasmid and amplification with T4 DNA ligase
It connects, completes the building of expression vector merR op-ompA-rfp-pSB1A2；
Step 4: converted expression vector merR op-ompA-rfp-pSB1A2 to competent cell by chemical transformation,
Obtain the bioengineered strain.
3. as claimed in claim 2 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that step
Target gene fragment merR op-ompA in rapid one is obtained especially by following methods: respectively with fully synthetic merR op,
OmpA, rfp gene order are template, carry out first time pcr amplification reaction, then by merR op and ompA the gene piece after amplification
The complementary fragment of section end realizes the connection of merR op and ompA by overlap extension pcr, obtains junction fragment merR
Op-ompA carries out second of pcr amplification reaction, realizes the amplification of junction fragment merR op-ompA, obtains target gene fragment
merR op-ompA。
4. as claimed in claim 2 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that step
Competent cell in rapid four is E.coli DH5 α competent cell.
5. as claimed in claim 3 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that the
When pcr amplification reaction, it includes ImeR-OP XF, OP-ompA UP that merR op, which needs the primer being added, and ompA needs to be added
Primer include OP-ompADN, OmpASR, it includes mRFP1SF, mRFP1SR that rfp, which needs the primer being added,；Second of PCR amplification
The primer that reaction is added includes IMerR-OP XF and OmpASR.
6. as claimed in claim 2 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that step
Reaction temperature when carrying out double digestion in rapid two is 37 DEG C, reaction time 1h.
7. as claimed in claim 2 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that step
Reaction temperature when carrying out single endonuclease digestion in rapid three is 37 DEG C, reaction time 1h.
8. as claimed in claim 3 for detecting the preparation method of the bioengineered strain of dimercurion, which is characterized in that the
Pcr amplification reaction includes 30 circulations, wherein a circulation successively includes 98 DEG C of denaturation 10s, 55 DEG C of annealing 10s, 72 DEG C
Extend 5s；Second of pcr amplification reaction includes 30 circulations, wherein a circulation is successively moved back including 98 DEG C of denaturation 10s, 55 DEG C
Fiery 10s, 72 DEG C of extension 10s.
9. as described in claim 1 for detecting the purposes of the bioengineered strain of dimercurion, which is characterized in that the biology
Engineering bacteria is for detecting whether water environment contains Hg2+。
10. as claimed in claim 9 for detecting the purposes of the bioengineered strain of dimercurion, which is characterized in that the life
Object engineering bacteria identifies Hg2+Reaction time be 15min or more.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129248A (en) * | 2019-05-24 | 2019-08-16 | 南宁师范大学 | Bioengineered strain and its preparation method and application for detecting dimercurion |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924602A (en) * | 2011-08-08 | 2013-02-13 | 深圳市艾派格生物技术有限公司 | Gold ion adsorption protein and method for enriching and recovering gold ion |
| WO2017079872A1 (en) * | 2015-11-09 | 2017-05-18 | Cathay R & D Center Co., Ltd. | Modified membrane permeability |
| CN107916243A (en) * | 2016-10-10 | 2018-04-17 | 中国科学院微生物研究所 | For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution |
-
2018
- 2018-07-05 CN CN201810732044.1A patent/CN108949659B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924602A (en) * | 2011-08-08 | 2013-02-13 | 深圳市艾派格生物技术有限公司 | Gold ion adsorption protein and method for enriching and recovering gold ion |
| WO2017079872A1 (en) * | 2015-11-09 | 2017-05-18 | Cathay R & D Center Co., Ltd. | Modified membrane permeability |
| CN107916243A (en) * | 2016-10-10 | 2018-04-17 | 中国科学院微生物研究所 | For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution |
Non-Patent Citations (5)
| Title |
|---|
| SHAILENDRA SINGH 等: "Bioremediation: environmental clean-up through pathway engineering", 《CURRENT OPINION IN BIOTECHNOLOGY》 * |
| TAPAN K.MISRA: "Bacterial Resistances to Inorganic Mercury Salts and Organomercurials", 《PLASMID》 * |
| 中国环境科学学会编: "《中国环境科学学会学术年会论文集 2012 第1卷》", 30 June 2012, 北京：中国农业大学出版社 * |
| 林稚兰 等: "微生物对重金属的抗性及解毒机理", 《微生物学通报》 * |
| 覃朝科 等: "《矿业开发中的重金属污染防治》", 30 September 2015, 北京：冶金工业出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129248A (en) * | 2019-05-24 | 2019-08-16 | 南宁师范大学 | Bioengineered strain and its preparation method and application for detecting dimercurion |
| CN110129248B (en) * | 2019-05-24 | 2022-03-01 | 南宁师范大学 | Bioengineering bacterium for detecting bivalent mercury ions and preparation 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 |
| 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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