CN107217067A - Heavy metal ion adsorbed system and its Host Strains, heavy metal removal method - Google Patents
Heavy metal ion adsorbed system and its Host Strains, heavy metal removal method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
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Abstract
The invention belongs to gene engineering technology field, and in particular to a kind of heavy metal ion adsorbed system and its Host Strains, heavy metal removal method.The heavy metal ion adsorbed system includes the bacillus subtilis membrane protein TasA DNA sequence dnas and heavy metal ion associated proteins DNA sequence dna being connected with each other, and the bacillus subtilis membrane protein TasA DNA sequence dnas are connected with promoter.Heavy metal ion adsorbed protein surface display system includes the bacillus subtilis membrane protein TasA and heavy metal ion associated proteins of the heavy metal ion adsorbed system expression by the present invention.The present invention can be realized to the recyclable enrichment of heavy metals in industrial wastewater ion and reclaimed by biological means, and can overcome the shortcoming of prior art, not result in secondary pollution, environmentally friendly.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of heavy metal ion adsorbed system and its Host Strains,
Heavy metal removal method.
Background technology
In recent years, with the fast development of China's economic construction, exploitation, smelting, processing and the business of heavy metal are manufactured
Activity is increasing, causes many heavy metals (such as lead, mercury, cadmium) to enter in big gas and water, soil, has triggered a series of serious
Contamination accident, great harm is caused to ecological environment.Acute poisoning and each anthropoid chronic disease that heavy metal is caused
Such as senile dementia, also the moment threatens the health of the mankind.And on the other hand, with scientific and technological level make rapid progress, that
A little chemical property is stable, physical attribute is excellent heavy metal such as gold, silver, copper, platinum, palladium etc., have been widely used in electronics, have led to
In the fields such as news, Aero-Space, chemical industry, medical treatment, and all kinds of small stores related to people's daily life.It is how reasonable
Heavy metal in nature is applied in the production and living of the mankind by ground, is allowed to and ecological environmental protection sustainability harmonious development
It is the significant challenge that current scientific worker faces.On the one hand, some heavy metal ion will be to organism in very low concentrations
With extremely strong toxicity, and it is different from organic compound, heavy metal has enriching, is difficult to be degraded in natural environment.By
In the limitation of production technology, a series of discarded object produced by production activities contains a substantial amounts of huge sum of money in such as industrial wastewater
Belong to ion, this can both be damaged to ecological environment, also production cost is remained high.At present for a huge sum of money in industrial wastewater
Belong to that the detection and enrichment of ion mainly use is all physical absorption and chemical-agent technique, the general detection sensitivity of these methods compared with
Low, the discrimination of the especially heavy metal ion close to various properties is poor, it is impossible to realize to certain specific heavy metal ion
Specific recognition and removal.And use large-scale analytical equipment such as ICP-MS (inductivity coupled plasma mass spectrometry), although its counterweight
The detection sensitivity of metal ion is higher, but operational means is cumbersome, take it is longer, it is necessary to by sample be sent to specific office by
Professional is detected, it is difficult to realize the quick inspection and monitoring in real time at scene, and recycles this on the basis of detection
A little large-scale instruments realize that the Specific adsorption of heavy metal is just more difficult.
To sum up, the chemical method of existing heavy metal removal has three shortcomings：First be chemical method be possible to by
It is improper and cause secondary pollution in the amount of the chemical reagent of addition；Second is that chemical method is selectively not high enough, is especially located
The extremely close various heavy metal ion effects of rationality matter are bad, obtained certain purpose metal even if reclaiming, purity is not also high,
Also need to further processing；3rd is that chemical method reclaims sewage produced during the heavy metal ion in industrial wastewater for ring
Border is unfriendly, and the chemical reagent such as precipitating reagent may cause bigger destruction for natural environment.During lead, gold are heavy metal with uranyl
The excellent heavy metal of rare chemistry, physics, Electronic Performance, application field is very wide, but difficult to realize pair of existing method
The efficient selective enrichment and recovery of heavy metals in industrial wastewater ion.
The content of the invention
It is an object of the invention to overcome prior art it is above-mentioned it is not enough there is provided a kind of heavy metal ion adsorbed system and its
Host Strains, heavy metal removal method, it is intended to solve existing heavy metal removal selectivity strong, purity be not low, effect not
Ideal, and pollute the technical problem of environment.
For achieving the above object, the technical solution adopted by the present invention is as follows：
On the one hand, the present invention provides a kind of heavy metal ion adsorbed system, includes the bacillus subtilis life of interconnection
Thing memebrane protein TasA DNA sequence dnas and heavy metal ion associated proteins DNA sequence dna, and the bacillus subtilis bud biomembrane egg
White TasA DNA sequence dnas are connected with promoter..
On the other hand, the present invention provides a kind of heavy metal ion adsorbed protein surface display system, including by an above-mentioned huge sum of money
Belong to the bacillus subtilis membrane protein TasA and heavy metal ion associated proteins of ionic adsorption system expression.
On the other hand, the present invention provides a kind of expression vector, including above-mentioned heavy metal ion adsorbed system.
Another aspect, the present invention provides a kind of Host Strains, and the Host Strains include above-mentioned expression vector；Or the Host Strains
Express above-mentioned heavy metal ion adsorbed protein surface display system.
Another further aspect, the present invention provides a kind of preparation method of above-mentioned heavy metal ion adsorbed system, comprises the following steps：
Bacillus subtilis membrane protein TasA DNA sequence dnas are amplified from Bacillus subtilis genes group；
The heavy metal ion associated proteins DNA is amplified from the expression protein-bonded strain genome of heavy metal ion
Sequence；
By promoter, the bacillus subtilis membrane protein TasA DNA sequence dnas and the heavy metal ion combination egg
White DNA sequence dna is sequentially connected.
Finally, the present invention provides a kind of heavy metal removal method, comprises the following steps：
Liquid containing heavy metal ion is provided；
It will be added after the Host Strains culture of the present invention in the liquid, stewing process；
After there is precipitation in the solution, the Host Strains that filtration treatment obtains being adsorbed with heavy metal ion are carried out；
The Host Strains of heavy metal ion are adsorbed with acid treatment, heavy metal ion is obtained after separation.
The heavy metal ion adsorbed system high-selectivity adsorption heavy metal ion of the present invention, it is according to salmonella typhimurium
The regulatory mechanism design of middle heavy metal ion, wherein containing key component in the bacterium regulatory mechanism.The present invention heavy metal from
Sub- adsorption system includes bacillus subtilis membrane protein TasA and the protein-bonded DNA sequence dna of heavy metal ion and regulation and control
Its promoter expressed, and it is easily prepared, cost is relatively low, easy to operate.Imported after the system is connected in plasmid non-
In pathogenic B. subtilis cell body, the bacillus subtilis biomembrane egg of absorbed portion is caused under promoter regulation
White TasA and heavy metal ion associated proteins great expression, can efficient selective Adsorption of Heavy Metals ion.Heavy metal can be expressed
The engineering bacteria of ionic adsorption protein surface display system, the absorption and recovery of heavy metal ion have good effect.
The heavy metal collection method that the present invention is provided, utilization can express heavy metal ion adsorbed protein surface display system
Engineering bacteria efficient absorption reclaims heavy metal ion, and engineering bacterium solution is added in the waste water containing heavy metal ion, and thalline can be
The outer great expression heavy metal ion associated proteins of cell membrane, the heavy metal ion in waste water is combined, and its selectivity is strong, acquisition
Purity is high；After heavy metal ion combination absorption is completed, thalline is assembled and precipitated, thalline reclaims convenient, secondary can make
With.The present invention can finally realize the recyclable enrichment and recovery to heavy metals in industrial wastewater ion by biological means；Cause
This, it enough overcomes the shortcoming of prior art, does not result in secondary pollution, environmentally friendly.
Brief description of the drawings
Fig. 1 is the detection of heavy metal ion system plasmid construction collection of illustrative plates of the embodiment of the present invention 1；
Fig. 2 is the electroresis appraisal figure of the detection of heavy metal ion system plasmid of the embodiment of the present invention 1；Wherein, M is DNA molecular
Amount standard, 1 is detection of heavy metal ion system plasmid swimming lane；
Fig. 3 is the heavy metal ion adsorbed system plasmid construction collection of illustrative plates of the embodiment of the present invention 2；
Fig. 4 is the electroresis appraisal figure of the heavy metal ion adsorbed system plasmid of the embodiment of the present invention 2；Wherein, M is DNA molecular
Amount standard, 1 is expressing fusion protein TasA-GolB/PbrR/SUP plasmid swimming lanes；
The pDG1730 plasmid vector collection of illustrative plates that Fig. 5 is used when being 3 heavy metal ion adsorbed system expression of the embodiment of the present invention；
Fig. 6 is the heavy metal ion adsorbed system of the embodiment of the present invention 6 to gold ion absorption effect contrast figure；
Fig. 7 is the heavy metal ion adsorbed system of the embodiment of the present invention 6 to uranyl ion adsorption effect comparison diagram；
Fig. 8 is the heavy metal ion adsorbed system of the embodiment of the present invention 6 to lead ion adsorption effect comparison diagram.
Embodiment
In order that technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Drawings and examples, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
On the one hand, the embodiments of the invention provide the withered grass bud of a kind of heavy metal ion adsorbed system, including interconnection
Spore bacillus membrane protein TasA DNA sequence dnas and heavy metal ion associated proteins DNA sequence dna, and the bacillus subtilis is biological
Memebrane protein TasA DNA sequence dnas are connected with promoter.
In one embodiment, the promoter is Pcot, its sequence such as SEQ ID NO:Shown in 18；Or the promoter also may be used
Think Pveg, its sequence such as SEQ ID NO:Shown in 21.And bacillus subtilis membrane protein TasA DNA sequence dnas such as SEQ
ID NO:Shown in 3.
Further, the downstream connection of heavy metal ion associated proteins DNA sequence dna has green fluorescent protein EGFP DNA sequences
Row, such as SEQ ID NO:Shown in 15.In this way, the expression of heavy metal ion adsorbed system can be detected further.
In another embodiment, the heavy metal ion associated proteins DNA sequence dna is lead ion associated proteins PbrR DNA sequence dnas,
It is such as SEQ ID NO:Shown in 6；Or heavy metal ion associated proteins DNA sequence dna is gold ion associated proteins GolB DNA sequence dnas,
It is such as SEQ ID NO:Shown in 9；Or heavy metal ion associated proteins DNA sequence dna is uranyl ion associated proteins SUP DNA sequence dnas,
It is such as SEQ ID NO:Shown in 12.Every functional protein DNA sequence dna that can be combined with heavy metal ion, is all implementation of the present invention
Metal ion associated proteins DNA sequence dna in example, is not enumerated here.
On the other hand, the embodiment of the present invention provides a kind of heavy metal ion adsorbed protein surface display system, it include by
The bacillus subtilis membrane protein TasA and gold ion of the heavy metal ion adsorbed system expression of the embodiment of the present invention are combined
Protein G olB.
On the other hand, the embodiments of the invention provide a kind of expression vector, the expression vector includes the huge sum of money of the present embodiment
Belong to ionic adsorption system.Meanwhile, the embodiments of the invention provide a kind of Host Strains, the expression that the Host Strains include the present embodiment is carried
Body；Or the heavy metal ion adsorbed protein surface display system of expression the present embodiment.
Another aspect, the embodiment of the present invention provides a kind of preparation method of above-mentioned heavy metal ion adsorbed system, including such as
Lower step：
S011：Bacillus subtilis membrane protein TasA DNA sequence dnas are amplified from Bacillus subtilis genes group；
S012：The heavy metal ion associated proteins are amplified from the expression protein-bonded strain genome of heavy metal ion
DNA sequence dna；
S013：By promoter, bacillus subtilis membrane protein TasA DNA sequence dnas and heavy metal ion associated proteins
DNA sequence dna is sequentially connected.
In one embodiment, the primer such as SEQ ID of the bacillus subtilis membrane protein TasA DNA sequence dnas are expanded
NO:1、SEQ ID NO:Shown in 2；In another embodiment, the promoter can be Pcot or Pveg, and expand Pcot primer such as
SEQ ID NO:16、SEQ ID NO:Shown in 17, Pveg primer such as SEQ ID NO are expanded:19、SEQ ID NO:Shown in 20,
Two promoter can all be amplified from this laboratory Bacillus subtilis genes group；In another embodiment, heavy metal ion knot
Hop protein DNA sequence dna be lead ion associated proteins PbrR DNA sequence dnas or gold ion associated proteins GolB DNA sequence dnas or uranyl from
Sub- associated proteins SUP DNA sequence dnas, and expand the primer such as DNA SEQ ID of the lead ion associated proteins PbrR DNA sequence dnas
NO:4、SEQ ID NO:Shown in 5, the primer such as DNA SEQ ID NO of the gold ion associated proteins GolB DNA sequence dnas are expanded:7、
SEQ ID NO:Shown in 8, the primer for expanding the uranyl ion associated proteins SUP DNA sequence dnas draws such as SEQ ID NO:10、SEQ
ID NO:Shown in 11., can be from salmonella typhimurium genome (the reference position in NCBI in the present embodiment：NC_003197)
With thermophilic copper bacillus gene group (the reference position in NCBI：NC_007973 gold ion associated proteins GolB is amplified in) respectively
DNA sequence dna and lead ion associated proteins PbrR DNA sequence dnas；And uranyl ion associated proteins SUP DNA sequence dnas can be artificial synthesized,
Effect is more preferable.
Finally, the embodiment of the present invention additionally provides a kind of heavy metal removal method, and it comprises the following steps：
S021：Liquid containing heavy metal ion is provided；
S022：It will be added after the Host Strains culture of the embodiment of the present invention in aforesaid liquid, stewing process；
S023：After there is precipitation in solution, the Host Strains that filtration treatment obtains being adsorbed with heavy metal ion are carried out；
S024：Heavy metal ion is obtained after the Host Strains of heavy metal ion, separation are adsorbed with acid treatment.
In selecting embodiment one, in above-mentioned steps S021, the liquid containing heavy metal ion can be various industrial wastewaters,
Recovered liquid containing heavy metal ion in mining industry waste water etc., the concentration range of heavy metal ion is preferably 0.1 μM in the liquid
~20 μM；In above-mentioned steps S022, the time range of stewing process is 40h~48h, and Host Strains are to a huge sum of money in liquid in the range of this
Category ion plays good adsorption effect.
It is of the invention successively to carry out test of many times, now lift A partial experiment result further detailed as reference pair invention progress
Thin description, is described in detail with reference to specific embodiment.
Embodiment 1The structure of detection of heavy metal ion system expression plasmid and identification
1. the amplification of heavy metal ion binding-protein gene fragment in detecting system
Use special primer (SEQ ID NO:4)(SEQ ID NO:5)(SEQ ID NO:7)(SEQ ID NO:8) from thermophilic
Copper vaccae genomic dna (the reference position in NCBI：NC_007973) with salmonella typhimurium genomic DNA (in NCBI
Quote position：NC_003197) in amplify coding heavy metal ion binding-protein gene DNA sequence dna (SEQ ID NO:6)
(SEQ ID NO:9), DNA sequence dna (the SEQ ID NO of artificial synthesized SUP genes:12), and corresponding amplimer is designed
(SEQ ID NO:10)(SEQ ID NO:11), reaction condition is as follows.
PCR reaction systems (50 μ l)：
PCR conditions：
The first step：95 DEG C, 3 minutes
Second step：95 DEG C, 1 minute
3rd step：60 DEG C, 2 minutes
4th step：72 DEG C, 4 minutes
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：4 DEG C of insulations.
PCR primer is reclaimed, it is standby.
The gene magnification of bacillus subtilis membrane protein 2. (TasA) fragment
According to required amplification target DNA sequence and base interworking principle, design special primer TasA1 (SEQ ID NO:1)
With TasA2 (SEQ ID NO:2), from Bacillus subtilis genes group DNA (the reference positions in NCBI：NC_000964 expand in)
Increase base sequence (the SEQ ID NO for encoding B. subtilis membrane protein TasA the 1st to the 314th DNA:3), instead
Answer condition as follows.
PCR reaction systems (50 μ l)：
PCR conditions：
The first step：95 DEG C, 2 minutes
Second step：95 DEG C, 0.5 minute
3rd step：63 DEG C, 0.5 minute
4th step：72 DEG C, 0.5 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：4 DEG C of insulations.
PCR primer is reclaimed, it is standby.
3. the amplification of reporter gene EGFP-terminator fragments in detecting system
1) amplification of green fluorescent protein EGFP fragment genes
Use special primer (SEQ ID NO:13)(SEQ ID NO:14) from all Bacillus coli expressions in this laboratory
Encoding green fluorescent protein EGFP DNA sequence dna (SEQ ID NO are amplified in vector gene group DNA:15), reaction condition is such as
Under.
PCR reaction systems (50 μ l)：
PCR conditions：
The first step：94 DEG C, 2 minutes
Second step：94 DEG C, 0.5 minute
3rd step：60 DEG C, 0.5 minute
4th step：72 DEG C, 1 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：10 DEG C of insulations.
PCR primer is reclaimed, it is standby.
2) gene magnification of terminator terminator fragments
According to required amplification target DNA sequence and base interworking principle, design special primer term1 (SEQ ID NO:22)
With term2 (SEQ ID NO:23), terminator is amplified from all coli expression carrier genomic DNAs in this laboratory
Terminator DNA sequence dna (SEQ ID NO:24), reaction condition is as follows.
PCR reaction systems (50 μ l)：
PCR reaction conditions：
The first step：94 DEG C, 2 minutes
Second step：94 DEG C, 0.5 minute
3rd step：60 DEG C, 0.5 minute
4th step：72 DEG C, 0.5 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：10 DEG C of insulations.
PCR primer is reclaimed, it is standby.
3) gene magnification of EGFP-terminator fragments
According to required amplification target DNA sequence and base interworking principle, special primer (SEQ ID NO are used:13)(SEQ
ID NO:23), using the above-mentioned 1st and 2 points of amplified productions reclaimed as masterplate, the DNA sequences of EGFP-terminator fragments are amplified
Row, reaction condition is as follows.
PCR reaction systems (50 μ l)：
PCR reaction conditions：
The first step：94 DEG C, 2 minutes
Second step：94 DEG C, 0.5 minute
3rd step：65 DEG C, 0.5 minute
4th step：72 DEG C, 1 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：10 DEG C of insulations.
PCR primer is reclaimed, it is standby.
4. the structure and identification of heavy metal ion surface display and reporter gene fusion protein expressing plasmid in detecting system
1) using the DNA fragmentation of the above-mentioned TasA genes of DNA restriction enzyme EcoRI and PstI digestions, then use
T4DNA ligases are inserted into all Escherichia coli biological brick expression vector pZH2 in this laboratory.
Endonuclease reaction system (20 μ l)：
Reaction condition：37 DEG C of water-baths 10 hours.
DNA coupled reactions system (10 μ l, 100ng DNA)：
Reaction condition：16 DEG C of water-baths 16 hours.
2) using DNA restriction enzyme XbaI and PstI digestions three heavy metal ion adsorbed albumen obtained above
DNA fragmentation (PbrR, GolB and SUP) and then the Escherichia coli biological brick that 1) step structure completion is inserted using T4DNA ligases
In expression vector pZH2.
Endonuclease reaction system (20 μ l)：
Reaction condition：37 DEG C of water-baths 10 hours.
DNA coupled reactions system (10 μ l, 100ng DNA)
Reaction condition：16 DEG C of water-baths 16 hours.
3) the above-mentioned EGFP-terminator fragments of DNA restriction enzyme XbaI and PstI digestions are finally used, are then made
2) step is inserted with T4DNA ligases to build in the Escherichia coli biological brick expression vector pZH2 completed, obtains heavy metal ion
Detecting system expression plasmid is shown in Fig. 1, and electroresis appraisal result is see Fig. 2.
Embodiment 2The structure of heavy metal ion adsorbed system expression plasmid and identification
1. special promoter fragment Ptas and Pveg amplification in adsorption system
According to required amplification target DNA sequence and base interworking principle, design special primer (SEQ ID NO:16)(SEQ
ID NO:Or (SEQ ID NO 17):19)(SEQ ID NO:20), amplified from this laboratory Bacillus subtilis genes group
Ptas sequences (the SEQ ID NO of the promoter of heavy metal ion controlling gene:Or Pveg sequences (SEQ ID NO 18):21), instead
Answer condition as follows.
PCR reaction systems (50 μ l)：
PCR conditions：
The first step：95 DEG C, 2 minutes
Second step：95 DEG C, 0.5 minute
3rd step：63 DEG C, 0.5 minute
4th step：72 DEG C, 0.5 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C 10 minutes
7th step：4 DEG C of insulations.
PCR primer is reclaimed, it is standby.
2. the amplification of heavy metal ion adsorbed genetic fragment in adsorption system
1) gene magnification of bacillus subtilis membrane protein (TasA) fragment
According to required amplification target DNA sequence and base interworking principle, design special primer TasA1 (SEQ ID NO:1)
With TasA2 (SEQ ID NO:2), from Bacillus subtilis genes group DNA (the reference positions in NCBI：NC_000964 expand in)
Increase base sequence (the SEQ ID NO for encoding B. subtilis membrane protein TasA the 1st to the 314th DNA:3), instead
Answer condition as follows.
PCR reaction systems (50 μ l)：
PCR conditions：
The first step：95 DEG C, 2 minutes
Second step：95 DEG C, 0.5 minute
3rd step：63 DEG C, 0.5 minute
4th step：72 DEG C, 0.5 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：4 DEG C of insulations.
PCR primer is reclaimed, it is standby.
2) gene magnification of heavy metal ion associated proteins fragment
See embodiment 1.
3) gene magnification of terminator terminator fragments
According to required amplification target DNA sequence and base interworking principle, design special primer term1 (SEQ ID NO:22)
With term2 (SEQ ID NO:23), terminator is amplified from all coli expression carrier genomic DNAs in this laboratory
Terminator DNA sequence dna (SEQ ID NO:24), reaction condition is as follows.
PCR reaction systems (50 μ l)：
PCR conditions：
The first step：94 DEG C, 2 minutes
Second step：94 DEG C, 0.5 minute
3rd step：60 DEG C, 0.5 minute
4th step：72 DEG C, 0.5 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72 DEG C, 10 minutes
7th step：10 DEG C of insulations.
PCR primer is reclaimed, it is standby.
The structure of 3.TasA-GolB fusion protein expression plasmids and identification
1) above-mentioned TasA and three heavy metal species ions binding albumen are digested using DNA restriction enzymes XbaI and PstI
DNA fragmentation, is then inserted in Escherichia coli biological brick expression vector pZH2, TasA-PbrR/GolB/ using T4DNA ligases
SUP fusion protein expression plasmids, which build collection of illustrative plates, please refer to Fig. 3, and qualification result is shown in Fig. 4.
Endonuclease reaction system (20 μ l)
Reaction condition：37 DEG C of water-baths 10 hours.
DNA coupled reactions system (10 μ l (100ng DNA)：
Reaction condition：16 DEG C of water-baths 16 hours.
Embodiment 3The structure of heavy metal ion adsorbed system homologous recombination expression plasmid and identification
1. digesting promoter Pcot/Pveg fragments obtained above using DNA restriction enzymes PstI and SpeI, use
DNA restriction enzymes PstI and XbaI digest above-mentioned TasA-PbrR/GolB/SUP DNA fragmentation, are then connected using T4DNA
Connect enzyme to be inserted into Escherichia coli biological brick plasmid backbone pZH2, by the purpose piece in Escherichia coli biological brick expression vector pZH2
Section using DNA restriction enzyme PstI and EcoRI digestions get off to be incorporated into PDG1730 bacillus subtilises expression vector (see
Fig. 5), homologous recombination obtains described heavy metal ion adsorbed system to being expressed on the genome of bacillus subtilis.
Endonuclease reaction system (20 μ l)：
Reaction condition：37 DEG C of water-baths 10 hours.
DNA coupled reactions system (10 μ l, 100ng DNA)：
Reaction condition：16 DEG C of water-baths 16 hours.
Embodiment 4Heavy metal ion adsorbed system plasmid is transferred to Host Strains
1) by the expressive plasmid carrier CaCl obtained by above-mentioned homologous recombination2Conversion method converts bacillus coli DH 5 alpha, enzyme
Cut identification recombination and integration carrier.
2) conversion of Bacillus subtillis, activation Bacillus subtillis, which is transferred in SPI culture mediums, cultivates a period of time, so
After go in the culture mediums of SP II culture and form competence, restructuring pDG1730 plasmids are cut into wire conversion Bacillus subtillis, tool
Body method is with reference to Zeigler (2002).
3) screening and identification of recombinant bacterium
Using endonuclease cutting after the plasmid of regular colony PCR methods and extracting positive colony, it for details, reference can be made to《TAKARA is limited
Property restriction endonuclease buying catalogue processed and technical manual》.Sequencing is completed by Shanghai biotechnology service company.Heavy metal ion is inhaled
The extraction and identification of attached and recovery system plasmid.
Embodiment 5The detection of heavy metal ion adsorbed system heavy metal ion selectivity ability
1. spectinomycin (50 μ g mL will be contained in right amount for detecting that the Bacillus subtilis strain of heavy metal ion is accessed-1) LB fluid nutrient mediums in overnight incubation in 37 DEG C；By incubated overnight bacterium solution 1:100 are diluted into and contain spectinomycin in right amount
(50μg mL-1) in DSM fluid nutrient mediums in being cultivated in 37 DEG C to nutrient solution OD600=0.6, it is separately added into nutrient solution dense eventually
The silver for 20 μM, nickel, zinc, copper, cadmium, chromium, lead ion are spent, nutrient solution continues to cultivate 48h in 37 DEG C；
2. cultivating 48h inoculum by centrifuging after (8000 revs/min, 2min) collection, 1 is cleaned with deionized water
It is secondary；
3. the thalline after cleaning is resuspended using 1 × PBS；
4. detect the bacterium solution after being resuspended using ELIASA.
Testing result is shown：Engineered engineering bacteria has preferable selectivity to corresponding heavy metal ion.
Embodiment 6The detection of the heavy metal ion adsorbed ability of TasA-PbrR/GolB/SUP, WT168
To engineering bacteria TasA-PbrR/GolB/SUP (correspondence two kinds of different promoters of connection：Pcot or Pveg) and
WT168 (bacillus subtilis often uses strain, is herein control group) carries out adsorption experiment, detects heavy metal ion adsorbed ability.
Experiment first two bacterium, which is rule, to be incubated in LB solid mediums (Spectinomycin resistance).
1. picking single bacterium colony is inoculated in 5mL LB (spectinomycin containing 5uL) culture medium respectively from two culture dishes,
37 DEG C, 220rpm overnight incubations.
2. by inoculum with 1：100 are re-seeded into 100mL LB (ampicillin containing 100uL) culture medium, and 37
DEG C, 220rpm cultivated to OD600=0.6, it is respectively 1 μm of ol, 5 μm of ol, 20 μm of ol to add concentration of heavy metal ion final concentration.37
DEG C, 220rpm continue cultivate 48h.Each experimental group sets 3 repetitions.
3. bacterium is collected, supernatant is abandoned.ddH2O is cleaned 3 times.- 80 DEG C freeze after 6h with freeze dryer lyophilised bacteria precipitation.
4. cleared up after weighing with 1mL concentrated nitric acids to complete, 10mL be settled to distilled water, finally using inductive etc. from
Sub- emission spectrum (ICP-AES) detection.
When concentration of heavy metal ion is 20 μm of ol, it adsorbs testing result as shown in Fig. 6, Fig. 7 and Fig. 8：Fig. 6 is two kinds
Engineering bacteria (Pcot/Pveg-TasA-GolB) relative comparison group is to gold ion absorption design sketch, and Fig. 7 is two kinds of engineering bacteria (Pcot/
Pveg-TasA-SUP) relative comparison group is to uranyl ion the adsorption effect figure, and Fig. 8 is two kinds of engineering bacteria (Pcot/Pveg-TasA-
PbrR) the adsorption effect figure of the relative comparison group to lead ion.Testing result is shown：Engineered engineering bacteria is to a corresponding huge sum of money
Category ion has more preferable adsorptivity.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>Shenzhen Jing Yu bio tech ltd
<120>Heavy metal ion adsorbed system and its Host Strains, heavy metal removal method
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 46
<212> DNA
<213>The primer of amplification coding bacillus subtilis membrane protein TasA gene
<400> 1
ctttcttcga attcgcggcc gcttctagag atggggggaa ttttgt 46
<210> 2
<211> 47
<212> DNA
<213>The primer of amplification coding bacillus subtilis membrane protein TasA gene
<400> 2
ctttcttcct gcagcggccg tactagtaca tgttcaatgt gattctt 47
<210> 3
<211> 314
<212> DNA
<213>Bacillus subtilis membrane protein TasA DNA sequence dna
<400> 3
atggggggaa ttttgttatg aaacgcaaat tattatcttc tttggcaatt agtgcattaa 60
gtctcgggtt actcgtttct gcacctacag cttctttcgc ggctgaatct acatcaacta 120
aagctcatac tgaatccact atgagaacac agtctacagc ttcattgttc gcaacaatca 180
ctggcgccag caaaacggaa tggtctttct cagatatcga attgacttac cgtccaaaca 240
cgcttctcag ccttggcgtt atggagttta cattgccaag cggatttact gcaaacacga 300
aagacacatt gaac 314
<210> 4
<211> 46
<212> DNA
<213>The primer of amplification coding lead ion associated proteins PbrR gene
<400> 4
ctttcttcga attcgcggcc cttctagaga atgaatatcc agatcg 46
<210> 5
<211> 64
<212> DNA
<213>The primer of amplification coding lead ion associated proteins PbrR gene
<400> 5
gtttcttcct gcagcggccg tactagtata aaggatgacg acgatactag tcgcttggat 60
gggc 64
<210> 6
<211> 438
<212> DNA
<213>Lead ion associated proteins PbrR DNA sequence dna
<400> 6
atgaatatcc agatcggcga gcttgccaag cgcaccgcat gcccggtggt gaccattcgc 60
ttctacgaac aagaagggct gttgccgccg ccgggccgca gccgggggaa ttttcgcctg 120
tatggcgagg agcacgtgga gcgcttgcag ttcattcgtc actgccggtc tctggatatg 180
ccgttgagcg acgtacggac cttattgagt taccggaagc ggcccgacca ggattgcggt 240
gaagtcaata tgctcttaga tgagcacatc cgtcaggtcg aatctcggat cggagccttg 300
ctcgaactga agcaccattt ggtggaactg cgcgaagcct gttctggtgc caggcccgcc 360
caatcgtgcg ggattctgca aggactgtcg gactgcgtgt gtgatacgcg ggggaccacc 420
gcccatccaa gcgactag 438
<210> 7
<211> 45
<212> DNA
<213>The primer of amplification coding gold ion associated proteins GolB gene
<400> 7
ctttcttcga attcgcggcc cttctagaga atgcagttcc atatt 45
<210> 8
<211> 63
<212> DNA
<213>The primer of amplification coding gold ion associated proteins GolB gene
<400> 8
gtttcttcct gcagcggccg tactagtata aaggatgacg acgataagat ctgaaagctt 60
ggg 63
<210> 9
<211> 228
<212> DNA
<213>Gold ion associated proteins GolB DNA sequence dna
<400> 9
atgcagttcc atattgatga catgacctgc ggcggctgcg ccagtacggt aaaaaagacg 60
attctgactc tcgatgctaa tgcgacggtg agaactgacc cggcgacgcg tctggttgac 120
gttgaaacgt cgctatccgc ggagcagatt gccgccgccc tgcaaaaggc cggtttcccg 180
ccgcgcgaga ggctcgagga ttacaaggat gacgacgata agatctga 228
<210> 10
<211> 46
<212> DNA
<213>The primer of amplification coding uranyl ion associated proteins SUP gene
<400> 10
ctttcttcga attcgcggcc cttctagaga gtttcttcct gcagcg 46
<210> 11
<211> 64
<212> DNA
<213>The primer of amplification coding uranyl ion associated proteins SUP gene
<400> 11
gtttcttcct gcagcggccg tactagtata aaggatgacc ttcgataatg cggtgcagca tacg 64
<210> 12
<211> 267
<212> DNA
<213>Uranyl ion associated proteins SUP DNA sequence dna
<400> 12
ggcggaggtt ctggaggagg gagcaatgcc ctggattgcc gtgaacgcat tgaaaaagac 60
ctggaaaacc tggaaaaaga actgatggaa atgaaaagca tcaaactgtc tgatgacgaa 120
gaagcggtgg ttgaacgtgc cctgaattat cgcgatgaca gtgtctatta cctggaaaaa 180
ggcgatcata ttacctcctt tggttgtatc acgtacgcgg agggcctgac ggatagcctg 240
cgtatgctgc accgcattat cgaaggt 267
<210> 13
<211> 50
<212> DNA
<213>The primer of amplification coding green fluorescent protein EGFP gene
<400> 13
ctttcttcga attcgcggcc atatacatat gagtaaagga gaagaacttt 50
<210> 14
<211> 42
<212> DNA
<213>The primer of amplification coding green fluorescent protein EGFP gene
<400> 14
ttagtggccg tatgttggcc gtactagtaa ttaagcaccg gt 42
<210> 15
<211> 797
<212> DNA
<213>Green fluorescent protein EGFP DNA sequence dna
<400> 15
atatacatat gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat 60
tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa 120
catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc 180
caacacttgt cactactttg acttatggtg ttcaatgctt ttcccgttat ccggatcata 240
tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaacgcacta 300
tatttttcaa agatgacggg aactacaaga cgcgtgctga agtcaagttt gaaggtgata 360
cccttgttaa tcgtatcgag ttaaaaggta ttgattttaa agaagatgga aacattctcg 420
gacacaaact cgagtacaac tataactcac acaatgtata catcatggca gacaaacaaa 480
agaatggaat caaagttaac ttcaaaattc gccacaacat tgaagatgga tccgttcaac 540
tagcagacca ttatcaacaa aatactccaa accattacct gtcgacacaa tctgcccttt 600
cgaaagatcc caacgaaaag cgtgaccaca tggcatggat gagctctaca aatatcgata 660
tgaattccaa ctgagcgccg ctagcaagct tgcggccgca ctcgagcacc accaccacca 720
accattacct gtcgacacaa tctgcccttt cgaaagatcc caacgaaaag cgtgaccaca 780
caacatacgg ccactaa 797
<210> 16
<211> 43
<212> DNA
<213>Expand promoter Ptas primer
<400> 16
ctttcttcga attcgcggcc gcttctagag cggcttcagt tgt 43
<210> 17
<211> 37
<212> DNA
<213>Expand promoter Ptas primer
<400> 17
gtttcttcct gcagcggccg tactagtaaa ggtaagc 37
<210> 18
<211> 63
<212> DNA
<213>Promoter Ptas sequence
<400> 18
cttcagttgt aaacctggca acaggtttcg atataaaatc attcaataaa aggggagctt 60
acc 63
<210> 19
<211> 43
<212> DNA
<213>Expand promoter Pveg primer
<400> 19
ctttcttcga attcgcggcc gcttctagag aattttgtca aaa 43
<210> 20
<211> 36
<212> DNA
<213>Expand promoter Pveg primer
<400> 20
gtttcttcct gcagcggcca catttattgt acaaca 36
<210> 21
<211> 97
<212> DNA
<213>Promoter Pveg sequence
<400> 21
aattttgtca aaataatttt attgacaacg tcttattaac gttgatataa tttaaatttt 60
atttgacaaa aatgggctcg tgttgtacaa taaatgt 97
<210> 22
<211> 43
<212> DNA
<213>Expand terminator terminator primer
<400> 22
ctttcttcga attcgcggcc gcttctagag cggcggcatg gac 43
<210> 23
<211> 37
<212> DNA
<213>Expand terminator terminator primer
<400> 23
gtttcttcct gcagcggccg tactagtaaa aggccca 37
<210> 24
<211> 171
<212> DNA
<213>Terminator terminator sequence
<400> 24
accggcggca tggacgagct gtacaagtaa taatactaga gccaggcatc aaataaaacg 60
aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 120
ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata t 171
Claims (10)
1. a kind of heavy metal ion adsorbed system, it is characterised in that the bacillus subtilis membrane protein including interconnection
TasA DNA sequence dnas and heavy metal ion associated proteins DNA sequence dna, and the bacillus subtilis membrane protein TasA DNA
Sequence is connected with promoter.
2. heavy metal ion adsorbed system as claimed in claim 1, it is characterised in that the promoter is Pcot or Pveg,
The sequence of the Pcot such as SEQ ID NO:Shown in 18, the sequence such as SEQ ID NO of the Pveg:Shown in 21；And
The bacillus subtilis membrane protein TasA DNA sequence dnas such as SEQ ID NO:Shown in 3.
3. heavy metal ion adsorbed system as claimed in claim 1, it is characterised in that the heavy metal ion associated proteins
DNA sequence dna is lead ion associated proteins PbrR DNA sequence dnas, such as SEQ ID NO:Shown in 6；Or
The heavy metal ion associated proteins DNA sequence dna is gold ion associated proteins GolB DNA sequence dnas, such as SEQ ID NO:9 institutes
Show；Or
The heavy metal ion associated proteins DNA sequence dna is uranyl ion associated proteins SUP DNA sequence dnas, such as SEQ ID NO:12
It is shown.
4. heavy metal ion adsorbed system as claimed in claim 1, it is characterised in that the heavy metal ion associated proteins
The downstream connection of DNA sequence dna has green fluorescent protein EGFP DNA sequence dnas, such as SEQ ID NO:Shown in 15.
5. a kind of heavy metal ion adsorbed protein surface display system, it is characterised in that including any described by claim 1-3
Heavy metal ion adsorbed system expression bacillus subtilis membrane protein TasA and heavy metal ion associated proteins.
6. a kind of expression vector, it is characterised in that including the heavy metal ion adsorbed system as described in claim 1-3 is any.
7. a kind of Host Strains, it is characterised in that the Host Strains include expression vector as claimed in claim 6；Or
The Host Strains expression heavy metal ion adsorbed protein surface display system as claimed in claim 5.
8. a kind of preparation method of heavy metal ion adsorbed system, it is characterised in that comprise the following steps：
Bacillus subtilis membrane protein TasA DNA sequence dnas are amplified from Bacillus subtilis genes group；
The heavy metal ion associated proteins DNA sequences are amplified from the expression protein-bonded strain genome of heavy metal ion
Row；
By promoter, the bacillus subtilis membrane protein TasA DNA sequence dnas and the heavy metal ion associated proteins
DNA sequence dna is sequentially connected.
9. the preparation method of heavy metal ion adsorbed system as claimed in claim 8, it is characterised in that the amplification withered grass bud
The primer such as SEQ ID NO of spore bacillus membrane protein TasA DNA sequence dnas:1、SEQ ID NO:Shown in 2；And/or
The promoter is Pcot or Pveg；And amplification Pcot primer such as SEQ ID NO:16、SEQ ID NO:17 institutes
Show, expand the primer such as SEQ ID NO of the Pveg:19、SEQ ID NO:Shown in 20；And/or
The heavy metal ion associated proteins DNA sequence dna is lead ion associated proteins PbrR DNA sequence dnas or gold ion associated proteins
GolB DNA sequence dnas or uranyl ion associated proteins SUP DNA sequence dnas；And expand the lead ion associated proteins PbrR DNA sequences
The primer of row such as DNA SEQ ID NO:4、SEQ ID NO:Shown in 5；Expand the gold ion associated proteins GolB DNA sequence dnas
Primer such as DNA SEQ ID NO:7、SEQ ID NO:Shown in 8；Expand the uranyl ion associated proteins SUP DNA sequence dnas
Primer such as SEQ ID NO:10、SEQ ID NO:Shown in 11.
10. a kind of heavy metal removal method, it is characterised in that comprise the following steps：
Liquid containing heavy metal ion is provided；
It will be added after Host Strains culture described in claim 7 in the liquid, stewing process；
After there is precipitation in the solution, the Host Strains that filtration treatment obtains being adsorbed with heavy metal ion are carried out；
The Host Strains of heavy metal ion are adsorbed with acid treatment, heavy metal ion is obtained after separation.
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| CN110283769A (en) * | 2019-07-05 | 2019-09-27 | 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 lead ion detection |
| CN114438007A (en) * | 2022-03-25 | 2022-05-06 | 两山生态科技(山东)有限公司 | Recombinant bacillus subtilis capable of efficiently adsorbing multiple heavy metals and preparation method and application thereof |
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| CN114438007A (en) * | 2022-03-25 | 2022-05-06 | 两山生态科技(山东)有限公司 | Recombinant bacillus subtilis capable of efficiently adsorbing multiple heavy metals and preparation method and application thereof |
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