CN107338259A - Gold ion detects and adsorption system and its Host Strains, gold ion recovery method - Google Patents
Gold ion detects and adsorption system and its Host Strains, gold ion recovery method Download PDFInfo
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Abstract
The invention belongs to gene engineering technology field, and in particular to a kind of gold ion detection and adsorption system and its Host Strains, gold ion recovery method.The gold ion detects and adsorption system includes detection unit and the absorbing unit being connected with the detection unit, and the detection unit includes fluorescin DNA sequence dna, and the fluorescin DNA sequence dna is connected with the first promoter；The absorbing unit includes the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and gold ion associated proteins GolB DNA sequence dnas interconnected, and the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas are connected with the second promoter；The detection unit is located at the upstream of the absorbing unit.The present invention can realize the detection and recyclable enrichment and recovery to heavy metals in industrial wastewater gold by biological means, the shortcomings that can overcoming prior art, will not cause secondary pollution, environmentally friendly.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of gold ion detection and adsorption system and its host
Bacterium, gold ion recovery method.
Background technology
Gold is the noble metal that the mankind more early have found and utilized, due to its resource scarcity and distinctive physicochemical property, from ancient times with
To be considered as first of hardware, there is the title of " king of metal ".Gold is that rare chemistry, physics, Electronic Performance are excellent in heavy metal
Metal, there is higher stability, its application field is very extensive.Because gold utensil is for highly stable physicochemical property, such as
Corrosion resistance, electric conductivity, thermal conductivity and stability, enable it to use extensively in the middle of many modern high technologies, gold ion
Can be as the inhibitor of the immunocytes such as leucocyte in immune system.In addition, gold is equally played the part of in biology and medicine
Drill important effect, such as bacterial resistance and cancer drug development.
Pollution problem of the today's society heavy metal to soil, water, environment is increasingly taken seriously.The mineral products money of heavy metal gold
While source is developed, some enters in environment, then by ground air, surface water, underground water and biological chain until big
The routes of transmission such as air ring, production and living and the ecosystem to the people produce pollution, destroy or even threaten the existence of people
Environment and life and health.In addition, produced in the production process such as irrational exploitation and the smelting of gold and secondary operation
Accessory substance, these problems have seriously affected the development of Gold Industry.It is well known that the content of gold is extremely limited,
Belong to rare Precious Metals Resources, if we can reasonably develop golden mineral resources, reduce the waste of resource, can be very big
The effective rate of utilization of golden mineral resources is improved in degree.Thus it is possible to the detection to the gold ion in environment, and can be reasonable
Recovery environment in gold ion it is particularly significant.With the continuous expansion of golden application, especially gold is in food security aspect
And the use of medical and health, so as to cause many gold ions to be entered by the enrichment of food chain in animal and plant body, energy
Protein denaturation is set to cause environment and the health hazard of people.Because gold ion can not be decomposed in the environment, additionally it is possible in water
In other noxious materials combine the bigger product of generation toxicity, these toxic products are absorbed by animals and plants, to Digestive
System, immune system do great damage.Gold ion can also combine to form compound in human body with what various enzymes occurred, make enzyme
Itself loses activity, and then is destroyed the physical function of people, the health of harmful to human, when serious even life-threatening.It is special
Other tervalence gold ion can cause organism toxicosis, influence the health of organism with being had an effect in organism.So heavy metal
The detection and recovery of gold ion are gradually of interest for society, find accurate, sensitive, quick golden detection method in current scientific circles
Have very important significance, how to detect and reclaim the gold ion in environment turns into the hot issue of current era.
Existing heavy metal removal method is mainly chemical method, by adding chemical reagent and heavy metal ion shape
Reach the purpose of recovery heavy metal into precipitation or suspension.But chemical method has three shortcomings：First is that chemical method has can
Secondary pollution can be caused because the amount of the chemical reagent of addition is improper；Second is that chemical method is selectively not high enough, especially
It is that the extremely similar each heavy metal species effect of handling properties is bad, even if recovery has obtained certain purpose metal, purity is not also high,
Also need to further handle；3rd is the heavy metal in chemical method recovery industrial wastewater, and produced sewage is not friendly for environment
Good, the chemical reagent such as precipitating reagent may cause bigger destruction for natural environment.Therefore, existing method is difficult to realize to work
The efficient selective detection and enrichment and recovery of gold ion in industry waste water.
The content of the invention
It is an object of the invention to overcome the above-mentioned deficiency of prior art, there is provided a kind of detection of gold ion and adsorption system and
Its Host Strains, gold ion recovery method, it is intended to solve that existing gold ion recovery selectivity is not strong, purity is low, effect is undesirable, with
And the technical problem of pollution 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 detection of gold ion and adsorption system, including detection unit and single with the detection
The absorbing unit of member connection, the detection unit include fluorescin DNA sequence dna, and the fluorescin DNA sequence dna and first
Promoter connects；The absorbing unit include interconnect bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and gold from
Sub- associated proteins GolB DNA sequence dnas, and the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and the second promoter connect
Connect；The detection unit is located at the upstream of the absorbing unit.
On the other hand, the present invention provides a kind of gold ion detection and adhesion protein surface display system, including by above-mentioned gold
Ion detection and fluorescin, bacillus subtilis spore clothing PROTEIN C otC and the gold ion associated proteins of adsorption system expression
GolB。
On the other hand, the present invention provides a kind of expression vector, including the detection of above-mentioned gold ion and adsorption system.
Another aspect, the present invention provide a kind of Host Strains, and the Host Strains include above-mentioned expression vector or the Host Strains
Express above-mentioned gold ion detection and adhesion protein surface display system.
Another further aspect, the present invention provide the preparation method of a kind of above-mentioned gold ion detection and adsorption system, including following step
Suddenly：
Bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas are amplified from Bacillus subtilis genes group, are hindered from mouse
Gold ion associated proteins GolB DNA sequence dnas are amplified in cold salmonella gene group；
With the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and the gold ion associated proteins GolB DNA
Sequence is template, amplifies CotC-GolB fusion protein DNA sequence dnas；
The amplification fluorescent protein DNA sequence from coli expression carrier genome；
By the first promoter, the fluorescin DNA sequence dna, the second promoter, the CotC-GolB fusion proteins DNA
Sequence is sequentially connected.
Finally, the present invention provides a kind of gold ion recovery method, comprises the following steps：
Liquid containing gold 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 gold ion are carried out；
The Host Strains of gold ion are adsorbed with acid treatment, gold ion is obtained after separation.
The regulatory mechanism design of gold ion detection and adsorption system based on gold ion in salmonella typhimurium of the present invention,
Detection unit and absorbing unit are wherein contained, detection unit includes reporter fluorescence protein DNA sequence, and it can be with Visual retrieval
Gold ion, and absorbing unit includes CotC-GolB absorbed portions, efficient absorption gold ion.After the system is connected in plasmid
Import in the B. subtilis cell body of non-pathogenic, Visual retrieval and absorption gold ion under promoter regulation, therefore
Detection and absorption of the improved engineering bacteria of this kind of process to gold ion have good effect；Because of reporter fluorescence albumen, withered grass
Bacillus spore clothing PROTEIN C otC and gold ion associated proteins GolB high efficient expression, its engineering bacteria can express gold ion inspection
Survey and adhesion protein surface display system, therefore gold ion detects and adsorption system is easily prepared, cost is relatively low, easy to operate.
Gold ion recovery method provided by the invention, gold ion detection and absorption integration can be collected, with the work of the present invention
Journey bacterium is to the processing containing golden industrial wastewater：Engineering bacterium solution is added in waste water, thalline can be being detected containing the same of golden waste water
When outside cell membrane great expression GolB albumen, by waste water gold ion combine.It is right after the combination absorption of gold ion is completed
Thalline is assembled and precipitated, and thalline recovery is convenient.By the processing to thalline and secondary use, the present invention can finally pass through
Biological means realize the detection and recyclable enrichment and recovery to heavy metals in industrial wastewater gold；Therefore, the present invention can overcome
The shortcomings that prior art, secondary pollution will not be caused, it is environmentally friendly.
Brief description of the drawings
Fig. 1 is the gold ion detecting system plasmid construction collection of illustrative plates of the embodiment of the present invention 1；
Fig. 2 is the electroresis appraisal figure of the gold ion detecting system plasmid of the embodiment of the present invention 1；Wherein M is DNA molecular amount mark
Standard, 1 is gold ion detecting system plasmid swimming lane；
Fig. 3 is the gold ion absorption system plasmid construction collection of illustrative plates of the embodiment of the present invention 2；
Fig. 4 is the electroresis appraisal figure of the gold ion absorption system plasmid of the embodiment of the present invention 2；Wherein M is DNA molecular amount mark
Standard, 1 is expressing fusion protein CotC-GolB plasmid swimming lanes；
Fig. 5 is the detection of the gold ion of the embodiment of the present invention 3 and adsorption system plasmid construction collection of illustrative plates；
Fig. 6 is gold ion selective enumeration method effect diagram in the water body of the embodiment of the present invention 5；
Fig. 7 is the detection of the gold ion of the embodiment of the present invention 6 and adsorption system to gold ion absorption effect contrast figure.
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 a kind of gold ion detection and adsorption system, including detection unit and with this
The absorbing unit of detection unit connection, detection unit include fluorescin DNA sequence dna, and the fluorescin DNA sequence dna and first
Promoter connects；Absorbing unit includes the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and gold ion knot interconnected
Hop protein GolB DNA sequence dnas, and bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas are connected with the second promoter；The inspection
Survey the upstream that unit is located at absorbing unit.
In one embodiment, the first promoter is PgolS-GolS-PgolB, its sequence such as SEQ ID NO:Shown in 10；Second
Promoter is PgolB, its sequence such as SEQ ID NO:Shown in 19.Escherichia coli outer membrane protein OmpA DNA sequence dna such as SEQ ID
NO:Shown in 3, gold ion associated proteins GolB DNA sequence dna such as SEQ ID NO:Shown in 6.
Specifically, fluorescin is a kind of luminous, visual albumen, can be red fluorescent protein RFP or green fluorescence
Albumen EGFP, mainly serve in the present embodiment to gold ion Visual retrieval report, in a preferred embodiment of the invention
In, fluorescin is red fluorescent protein RFP, its DNA sequence dna such as SEQ ID NO:Shown in 13.
On the other hand, the embodiment of the present invention provides a kind of gold ion detection and adhesion protein surface display system, and it includes
By fluorescin, the bacillus subtilis spore clothing PROTEIN C otC of gold ion detection and the adsorption system expression of the embodiment of the present invention
With gold ion associated proteins GolB.
On the other hand, the embodiments of the invention provide a kind of expression vector, the expression vector include the gold of the present embodiment from
Son detection and adsorption system.Meanwhile the embodiments of the invention provide a kind of Host Strains, the Host Strains to include the expression of the present embodiment
Carrier；Or the gold ion detection of expression the present embodiment and adhesion protein surface display system.
Another aspect, the embodiment of the present invention provide the preparation method of a kind of above-mentioned gold ion detection and adsorption system, and it is special
Sign is, comprises the following steps：
S011：Bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas are amplified from Bacillus subtilis genes group,
Gold ion associated proteins GolB DNA sequence dnas are amplified from salmonella typhimurium genome；
S012：With above-mentioned bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and gold ion associated proteins GolB DNA
Sequence is template, amplifies CotC-GolB fusion protein DNA sequence dnas；
S013：The amplification fluorescent protein DNA sequence from coli expression carrier genome；
S014：By the first promoter, fluorescin DNA sequence dna, the second promoter, CotC-GolB fusion protein DNA sequence dnas
It is sequentially connected.
In one embodiment, the primer such as SEQ ID of bacillus subtilis spore clothing PROTEIN C otC DNA sequence dna are expanded
NO:1、SEQ ID NO:Shown in 2；Expand the primer such as SEQ ID NO of gold ion associated proteins GolB DNA sequence dna:4、SEQ
ID NO:Thing shown in 5.
And it is different according to specific fluorescin, the amplimer of selection is also different, the fluorescin DNA sequence dna of the present embodiment
For red fluorescent protein RFP DNA sequence dnas, it can expand red from the coli expression carrier genome in this laboratory
Fluorescin RFP DNA sequence dnas, and amplimer such as SEQ ID NO:11、SEQ ID NO:Shown in 12.And in one embodiment,
First promoter is PgolS-GolS-PgolB, and its amplimer such as SEQ ID NO:8、SEQ ID NO:Shown in 9, second starts
Son is PgolB, and its amplimer such as SEQ ID NO:17、SEQ ID NO:Shown in 18, two kinds of promoters can hinder from from mouse
Cold salmonella gene group expands to obtain, and the P of the present embodimentgolBCarried it is preferred that being expressed from the Escherichia coli biological brick in this laboratory
Expanded in body pZH2, it is more efficient.
Finally, the embodiment of the present invention also provides a kind of gold ion recovery method, comprises the following steps：
S021：Liquid containing gold ion is provided；
S022：It will be added to 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 gold ion are carried out；
S024：The Host Strains of gold ion are adsorbed with acid treatment, gold ion is obtained after separation.
In one selects embodiment, in above-mentioned steps S021, the liquid containing gold ion can be various industrial wastewaters, mining industry
Contain gold ion recovered liquid in waste water etc., the concentration range of gold ion is preferably 0.1 μM~20 μM in the liquid；Above-mentioned steps
In S022, the time range of stewing process is 40h~48h, and Host Strains play good suction to gold ion in liquid in the range of this
Attached effect.
It is of the invention successively to carry out test of many times, it is further detailed as reference pair invention progress now to lift A partial experiment result
Thin description, is described in detail with reference to specific embodiment.
Embodiment 1The structure of gold ion detecting system expression plasmid and identification
1. gold ion induced gene in detecting system：Promoter PgolS-GolS-PgolBAmplification
Use special primer pGolts1 (SEQ ID NO:And pGolts2 (SEQ ID NO 8):9) from Salmonella typhimurium
Bacterium genomic DNA (the reference position in NCBI：NC_003197) in amplify DNA sequence dna (the SEQ ID for encoding golden induced gene
NO:10), reaction condition is as follows.The gold ion induced gene (Gold inductive gene) of the present embodiment is promoter
PgolS-GolS-PgolBGolS, it can express GolS albumen, and GolS albumen usually plays the effect checked, with the presence of gold ion
Shi Caihui is from promoter PgolBDNA sequence dna on come off, trigger downstream gene expression.
PCR reaction systems (50 μ l)：
PCR reaction 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.
2. the amplification of reporter gene RFP-terminator fragments in detecting system
1) amplification of red fluorescent protein RFP fragment genes
Use special primer rfp1 (SEQ ID NO:And rfp2 (SEQ ID NO 11):12) from the large intestine bar in this laboratory
Coding red fluorescent protein RFP DNA sequence dna (SEQ ID NO are amplified in bacterium expression vector genomic DNA:13) bar, is reacted
Part 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, 1 minute
5th step：Second step is repeated to the 4th step, 29 circulations
6th step：72℃.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:14)
With term2 (SEQ ID NO:15), terminator is amplified from all coli expression carrier genomic DNAs in this laboratory
Terminator DNA sequence dna (SEQ ID NO:16), 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 RFP-terminator fragments
According to required amplification target DNA sequence and base interworking principle, special primer rfp1 (SEQ ID NO are used:11)
With term2 (SEQ ID NO:15), using the amplified production of RFP fragments and terminator fragments as masterplate, OmpA- is amplified
The DNA sequence dna of GolB fragments, reaction condition are 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.
3. the structure and identification of gold ion induced gene and reporter gene fusion protein expressing plasmid in detecting system
1) the above-mentioned gold ion induced gene of DNA restriction enzyme EcoRI and PstI digestions is used：Promoter PgolS-
GolS-PgolBExpand obtained DNA fragmentation (SEQ ID NO:10), then it is inserted into this laboratory institute using T4DNA ligases
In some Escherichia coli biological brick expression vector pZH2.
Endonuclease reaction system (20 μ l)：
Reaction condition：37 DEG C, water-bath 10 hours.
DNA coupled reactions system (10 μ l, 100ng DNA)：
Reaction condition：16 DEG C, water-bath 16 hours.
2) the above-mentioned RFP-terminator of DNA restriction enzyme XbaI and PstI digestions amplification of DNA fragments is used, so
Afterwards using in the insertion of T4DNA ligases 1) the Escherichia coli biological brick expression vector pZH2 of step structure completion.
Endonuclease reaction system (20 μ l)：
Reaction condition：37 DEG C, water-bath 10 hours.
DNA coupled reactions system (10 μ l, 100ng DNA)：
Reaction condition：16 DEG C, water-bath 16 hours.
3) in detecting system the extraction result of gold ion induced gene and reporter gene fusion protein expressing plasmid see Fig. 1
(GolB promoters are promoter P in figuregolS-GolS-PgolB), electroresis appraisal result is see Fig. 2.
Embodiment 2The structure of gold ion absorption system expression plasmid and identification
1. promoter P in adsorption systemgolBThe amplification of fragment
According to required amplification target DNA sequence and base interworking principle, design special primer PgolB1(SEQ ID NO:17)
And PgolB2(SEQ ID NO:18), amplified from all Escherichia coli biological brick expression vector pZH2 in this laboratory gold from
The promoter P of sub- controlling genegolBSequence (SEQ ID NO:19), reaction condition is as follows.
PCR reaction systems (50 μ l)：
PCR reaction 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 gold ion absorption genetic fragment in adsorption system
1) gene magnification of bacillus subtilis spore clothing PROTEIN C otC fragments
According to required amplification target DNA sequence and base interworking principle, design special primer CotC1 (SEQ ID NO:1)
With CotC2 (SEQ ID NO:2), from Bacillus subtilis genes group DNA (the reference positions in NCBI：NC_000964 expand in)
Increase and the 1st DNA sequence dna (the SEQ ID NO to the 159th amino acids of encoding B. subtilis gemma clothing PROTEIN C otC:3),
Reaction condition is 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 gold ion associated proteins GolB fragments
According to required amplification target DNA sequence and base interworking principle, design special primer Golb1 (SEQ ID NO:4)
With Golb2 (SEQ ID NO:5) from salmonella typhimurium genomic DNA (the reference position in NCBI：NC_003197) in expand
Increase DNA sequence dna (the SEQ ID NO for coding gold ion associated proteins GolB:6), and in its C-terminal plus coding FLAG-tag's
(FLAG molecular weight is 1012Da to sequence, is one section of mark label being made up of 8 amino acid residues, can be identified by antibody, is used
Tested in follow-up Western protein blots, prove that albumen is expressed so as to produce band), reaction condition is 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：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：4 DEG C of insulations.
PCR primer is reclaimed, it is standby.
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:14)
With term2 (SEQ ID NO:15), terminator is amplified from all coli expression carrier genomic DNAs in this laboratory
Terminator DNA sequence dna (SEQ ID NO:16), 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.CotC-GolB fusion protein expression plasmids and identification
1) above-mentioned P is digested using DNA restriction enzymes XbaI and PstIgolBFragment, CotC fragments, GolB fragments and
The amplified production of terminator fragments, then inserted using T4DNA ligases in Escherichia coli biological brick expression vector pZH2,
CotC-GolB fusion protein expression plasmids structure collection of illustrative plates please refer to Fig. 3, and the DNA sequence dna of wherein CotC-GolB fusion proteins is：
SEQ ID NO:7。
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) extraction and identification of CotC-GolB fusion protein expression plasmids, as a result see Fig. 4.
Embodiment 3Gold ion detects and structure and the identification of adsorption system expression plasmid
1. the P obtained in above-described embodiment 1 is digested using DNA restriction enzymes PstI and SpeIgolS-GolS-PgolB-
RFP-terminator enzymatic fragments, make it have cohesive end, are digested using DNA restriction enzymes PstI and XbaI above-mentioned
The P obtained in embodiment 2golB- CotC-GolB-terminator enzymatic fragments, cohesive end is made it have, is then used
T4DNA ligases are inserted into Escherichia coli biological brick plasmid backbone pZH2, by Escherichia coli biological brick expression vector pZH2
Purpose fragment get off to be incorporated into PDG1730 bacillus subtilis tables using DNA restriction enzyme PstI and EcoRI enzyme digestions
Up to carrier, expressed on the genome of homologous recombination to bacillus subtilis, that is, obtain gold ion detection and absorption integration
System.
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. gold ion detects and adsorbs the extraction result of integrated expression plasmid see Fig. 5.
Embodiment 4Gold ion detects and adsorption system plasmid is transferred to Host Strains
1) by the expressive plasmid carrier obtained by above-mentioned homologous recombination, using CaCl2Conversion method converts bacillus coli DH 5 alpha,
Recombination and integration carrier is identified in digestion.
2) conversion of Bacillus subtillis, activation Bacillus subtillis, which is transferred in the culture mediums of SP I, 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, for details, reference can be made to《TAKARA is limited
Property restriction endonuclease buying catalogue processed and technical manual》.Sequencing by Shanghai biotechnology service company complete, gold ion detection and
The extraction and identification of adsorption system plasmid.
Embodiment 5Gold ion detects and adsorption system selects gold ion the detection of sexuality
1. spectinomycin (50 μ g mL will be contained in right amount for detecting the Bacillus subtilis strain of gold ion and accessing-1)
In LB fluid nutrient mediums in 37 DEG C overnight incubation；Bacterium solution 1 will be incubated overnight:100 are diluted into and contain spectinomycin (50 μ g in right amount
mL-1) in DSM fluid nutrient mediums in 37 DEG C culture to nutrient solution OD600=0.6, final concentration of 20 are separately added into nutrient solution
μM silver, nickel, zinc, copper, cadmium, chromium, lead ion, nutrient solution continue in 37 DEG C cultivate 48h；
After 2. culture 48h inoculum is by centrifuging (8000 revs/min, 2min) collections, 1 is cleaned with deionized water
It is secondary；
3. the thalline after cleaning is resuspended using 1 × PBS；
4. the bacterium solution after being resuspended using ELIASA detection.
Specific testing result is see Fig. 6, and it was found from figure, engineered engineering bacteria has preferable selectivity to gold ion.
Embodiment 6The detection of CotC-GolB, WT168 gold ion absorption ability
Engineering bacteria CotC-GolB, WT168 (bacillus subtilis often uses strain, control group) carry out adsorption experiment, detection
Adsorption capacities of the CotC-GolB to gold ion.Experiment first two bacterium, which is rule, is incubated in LB solid mediums that (spectinomycin resists
Property).
1. picking single bacterium colony is inoculated in 5mL LB (spectinomycin containing 5uL) culture medium respectively from two culture dishes, and 37
DEG C, 220rpm overnight incubations.
2. by inoculum with 1：100 are re-seeded into 100mL DSM (ampicillin containing 100uL) culture medium,
37 DEG C, 220rpm cultivated to OD600=0.6, add Au3+It is respectively 1 μm of ol, 5 μm of ol, 20 μm of ol to final concentration.37℃、
220rpm continues to cultivate 48h.Each experimental group sets 3 repetitions.
3. collecting bacterium, supernatant is abandoned.ddH2O is cleaned 3 times.- 80 DEG C freeze and are precipitated with freeze dryer lyophilised bacteria after 6h.
4. being cleared up after weighing with 1mL concentrated nitric acids to complete, 10mL is settled to distilled water, finally using inductive etc. from
Sub- emission spectrum (ICP-AES) detection, for specific testing result see Fig. 7, Fig. 7 is to enumerate suction when gold ion concentration is 20 μm of ol
Attached effect, understand that CotC-GolB engineering bacterias have good adsorption capacity to gold ion from figure.
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
All any modification, equivalent and improvement 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>Gold ion detects and adsorption system and its Host Strains, gold ion recovery method
<160> 19
<210> 1
<211> 46
<212> DNA
<213>The primer of amplification coding bacillus subtilis spore clothing PROTEIN C otC gene
<400> 1
ctttcttcga attcgcggcc gcttctagag atgggttatt acaaaa 46
<210> 2
<211> 41
<212> DNA
<213>The primer of amplification coding bacillus subtilis spore clothing PROTEIN C otC gene
<400> 2
ctttcttcct gcagcggccg tactagtaca taaatgtagt g 41
<210> 3
<211> 201
<212> DNA
<213>Bacillus subtilis spore clothing PROTEIN C otC DNA sequence dna
<400> 3
atgggttatt acaaaaaata caaagaagag tattatacgg tcaaaaaaac gtattataag 60
aagtattacg aatatgataa aaaagattat gactgtgatt acgacaaaaa atatgatgac 120
tatgataaaa aatattatga tcacgataaa aaagactatg attatgttgt agagtataaa 180
aagcataaaa aacactacta a 201
<210> 4
<211> 45
<212> DNA
<213>The primer of the gene of amplification coding gold ion associated proteins GolB fragments
<400> 4
ctttcttcga attcgcggcc cttctagaga atgcagttcc atatt 45
<210> 5
<211> 63
<212> DNA
<213>The primer of the gene of amplification coding gold ion associated proteins GolB fragments
<400> 5
gtttcttcct gcagcggccg tactagtata aaggatgacg acgataagat ctgaaagctt 60
ggg 63
<210> 6
<211> 228
<212> DNA
<213>Gold ion associated proteins GolB DNA sequence dna
<400> 6
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> 7
<211> 429
<212> DNA
<213>The DNA sequence dna of CotC-GolB fusion proteins
<400> 7
atgggttatt acaaaaaata caaagaagag tattatacgg tcaaaaaaac gtattataag 60
aagtattacg aatatgataa aaaagattat gactgtgatt acgacaaaaa atatgatgac 120
tatgataaaa aatattatga tcacgataaa aaagactatg attatgttgt agagtataaa 180
aagcataaaa aacactacta aatgcagttc catattgatg acatgacctg cggcggctgc 240
gccagtacgg taaaaaagac gattctgact ctcgatgcta atgcgacggt gagaactgac 300
ccggcgacgc gtctggttga cgttgaaacg tcgctatccg cggagcagat tgccgccgcc 360
ctgcaaaagg ccggtttccc gccgcgcgag aggctcgagg attacaagga tgacgacgat 420
aagatctga 429
<210> 8
<211> 43
<212> DNA
<213>Expand promoter PgolS-GolS-PgolB primer
<400> 8
ctttcttcga attcgcggcc gcttctagag aaggaaaggt caa 43
<210> 9
<211> 43
<212> DNA
<213>Expand promoter PgolS-GolS-PgolB primer
<400> 9
gtttcttcct gcagcggccg tactagtact tttgtgtggg aac 43
<210> 10
<211> 3231
<212> DNA
<213>Promoter PgolS-GolS-PgolB sequence
<400> 10
acaacgaagg aaaggtcaag cgttcctgac gggtttttac ggggcgtggg tcggcatcgt 60
ggcgtaaatg tctcgcatca tcctctttta tgagccatct cacattctcg ccgaaccgtg 120
cagcctgaat acgcttgacc ttcccacaat ggcaagcttt aggctttctg ataccgaata 180
gtcaggatgg ggaagtcgtc atgagtcagt cagaaaatcg tcacgacacg ataagcttac 240
ttattgaagg tatgacctgc gcgtcgtgcg tcgctcgcgt tgaaaaaggt attaaggctg 300
tgcctggcgt aacggacgct acggtgaatc tggcgacgga gcgcgccacc gtccgcggga 360
cggcgtcggc ggaggcggtg atcgcggcga ttgaaaaaac ggggtataag gcgcgaccga 420
tagagacggc ggggcagggc gaagacgact ctgaagagaa aaaagaggcc gagcgcgtca 480
ggctgaagcg cgatctgatt ctcgccagcg tgctggcgct ccccgttttt gtgcttgaaa 540
tgggctcgca ccttattcct ggtatgcacg agtgggtgat aaaaacgatt ggcctgcaac 600
aaagctggta ctggcaattt gcactgaccc tgttggtgct gacgatcccc ggtcgccgtt 660
tttaccttaa agggttcccg gcgctggcgc gtctggcgcc ggacatgaac tcgctggtcg 720
ccgtgggaac cgcagcggca ttcggctact cgctggtggc gacctttacg cccgacctgt 780
tgcctgaagg gacggtaaac gtctattacg aggccgccgc agtgattgtc gcgcttattc 840
tgctggggcg ctttctggag gcaagggcga aggggcggac ttccgaagcg attaaacgtc 900
tggtggggct acaggcgcgg gtcgcgcatg tgttacgcga gggccgcatc gtggatatcc 960
ctgtcgacga ggttgtgctg ggcgactgtg tggaggttcg gccaggcgag cggatcccgg 1020
ttgacggcga agtgaccgaa ggccgcagct tcgttgacga gtcgatgatt accggcgaac 1080
cgataccggt tgagaaatcc gcaggaagcg cggtggtggg cggtaccgta aaccaaaaag 1140
gcgcgctcac gctgcgggcg accgccgtcg gcggacagac catgctggcg caaatcattc 1200
gtctggtgga acaggcgcag ggttcaaaac tgccgattca ggccgtcgtg gataaagtga 1260
cgctgtggtt tgtcccgatg gtgatgctta ttgctgcgct gacctttgtg gtatggctgg 1320
cgtttgggcc gtcgccagcg ctgactttcg ctctgatcaa tggcgttgcg gttctgatta 1380
tcgcctgtcc ttgcgcgatg ggcctggcga cgccgacctc tattatggtg ggaaccggtc 1440
gtggggcgga aatgggcgtg ctgttccgta agggggaggc gttacagcta ctcaaagacg 1500
ctaaggtggt ggccgtagac aaaaccggga cgcttaccga aggccgcccg gtactgaccg 1560
atctcgacgt ggccagcggc tttgaacgcc gtgaggtgct ggcgaaagtc gcggcggtag 1620
aatcgcgttc agagcatccg attgcccgtg cgattgtcgt gtcggcagaa gaggaaggga 1680
tcgcgctacc aggcatgagc ggcttcgaat cggtgaccgg gatgggcgta tacgctaccg 1740
ttgacggtac gcgtgttgac gtgggggctg atcgctatat gcgcgaaatt ggcgtggata 1800
ttagcggctt cgccaccacc gccgaacggt tagggcagga agggaaatcg ccgctctatg 1860
cggctattga cggtcaactg gcggcgatta tcgccgtggc cgatccgatc aaacccagta 1920
cgcccgccgc aattaacgct ttacatcagc tcggcattaa ggtcgccatg atcactggcg 1980
ataatgcccg cacggcacag gctatcgcca gacagttagg aattgatgat gtggttgccg 2040
aggtattgcc agaagggaaa gtcgaggcga tacggcgcct gaaagcggcg tatgggcagg 2100
tggcgtttgt cggcgatggc atcaacgatg cgccagcgct ggcggagtcc gacgtggggc 2160
tggcgattgg caccggcacc gatgtggcgg tggaatccgc cgacgtcgta ctaatgtccg 2220
gcaacctgca aggcgtgccg aatgctatcg cgctgtctaa agcgaccatc cgcaatatcc 2280
accagaatct gttctgggcc tttgcttaca atacggcgct gattcctgtc gcggcaggcg 2340
cgctatttcc ggtctggggc atattattgt caccggtatt cgccgcaggg gcgatggcga 2400
tgtcgagcgt gttcgtgctg ggcaacgctt tgcggctgcg ccgtttccgg gcgccgatgg 2460
caaccccatc cgacacatcc acgacatgag gaggagcgtc atgaacatcg gtaaagcagc 2520
taaagcatcg aaagtctcgg ccaaaatgat tcgctactat gaacagattg gtctgattcc 2580
cgcggcaagt cggacggatt ccggctatcg ggcctatacc caggctgatg ttaatcaatt 2640
gcattttata cgccgcgcgc gcgacctcgg tttttcagtt gctgaaatca tgaacatcgg 2700
taaagcagct aaagcatcga aagtctcggc caaaatgatt cgctactatg aacagattgg 2760
tctgattccc gcggcaagtc ggacggattc cggctatcgg gcctataccc aggctgatgt 2820
taatcaattg cattttatac gccgcgcgcg cgacctcggt ttttcagttg ctgaaatcag 2880
cgacttactg aatctttgga ataaccagtc gcggcaaagc gctgacgtca aacgcctggc 2940
gcagacgcac attgatgaac tggacagacg tatccagaac atgcagcaca tggcgcaaac 3000
cctcaaagcg ctgattcact gctgcgccgg cgacgcgctg ccagattgcc ccattctgca 3060
tacgcttgga caacctgacg atagcgagcc ggaggcgcgt accggagcgg tattgcgacg 3120
tcctcgtcgc cacggactgg caaagcgtct gtaagtcctg agattacgct tgaccttcca 3180
acactggcaa ggtccagact ggcaacagtt cccacacaaa aggagttcac t 3231
<210> 11
<211> 50
<212> DNA
<213>The primer of amplification coding red fluorescent protein RFP gene
<400> 11
ctttcttcga attcgcggcc gcttctagag atggcttcct ccgaagacgt 50
<210> 12
<211> 41
<212> DNA
<213>The primer of amplification coding red fluorescent protein RFP gene
<400> 12
gtttcttcct gcagcggccg tactagtaat taagcaccgg t 41
<210> 13
<211> 681
<212> DNA
<213>Red fluorescent protein RFP DNA sequence dna
<400> 13
atggcttcct ccgaagacgt tatcaaagag ttcatgcgtt tcaaagttcg tatggaaggt 60
tccgttaacg gtcacgagtt cgaaatcgaa ggtgaaggtg aaggtcgtcc gtacgaaggt 120
acccagaccg ctaaactgaa agttaccaaa ggtggtccgc tgccgttcgc ttgggacatc 180
ctgtccccgc agttccagta cggttccaaa gcttacgtta aacacccggc tgacatcccg 240
gactacctga aactgtcctt cccggaaggt ttcaaatggg aacgtgttat gaacttcgaa 300
gacggtggtg ttgttaccgt tacccaggac tcctccctgc aagacggtga gttcatctac 360
aaagttaaac tgcgtggtac caacttcccg tccgacggtc cggttatgca gaaaaaaacc 420
atgggttggg aagcttccac cgaacgtatg tacccggaag acggtgctct gaaaggtgaa 480
atcaaaatgc gtctgaaact gaaagacggt ggtcactacg acgctgaagt taaaaccacc 540
tacatggcta aaaaaccggt tcagctgccg ggtgcttaca aaaccgacat caaactggac 600
atcacctccc acaacgaaga ctacaccatc gttgaacagt acgaacgtgc tgaaggtcgt 660
cactccaccg gtgcttaata a 681
<210> 14
<211> 43
<212> DNA
<213>Expand terminator terminator primer
<400> 14
ctttcttcga attcgcggcc gcttctagag cggcggcatg gac 43
<210> 15
<211> 37
<212> DNA
<213>Expand terminator terminator primer
<400> 15
gtttcttcct gcagcggccg tactagtaaa aggccca 37
<210> 16
<211> 171
<212> DNA
<213>Terminator terminator sequence
<400> 16
accggcggca tggacgagct gtacaagtaa taatactaga gccaggcatc aaataaaacg 60
aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 120
ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata t 171
<210> 17
<211> 50
<212> DNA
<213>Expand promoter PgolB primer
<400> 17
gtttcttcga attcgcggcc gcttctagag gacgcgctgc cagattgccc 50
<210> 18
<211> 49
<212> DNA
<213>Expand promoter PgolB primer
<400> 18
gtttcttcct gcagcggccg ctactagtaa gtgaactcct tttctgtgg 49
<210> 19
<211> 192
<212> DNA
<213>Promoter PgolB sequence
<400> 19
atgcagttcc atattgatga catgacctgc ggcggctgcg ccagtacggt aaaaaagacg 60
attctgactc tcgatgctaa tgcgacggtg agaactgacc cggcgacgcg tctggttgac 120
gttgaaacgt cgctatccgc ggagcagatt gccgccgccc tgcaaaaggc cggtttcccg 180
ccgcgcgaga gg 192
Claims (10)
1. a kind of gold ion detection and adsorption system, it is characterised in that be connected including detection unit and with the detection unit
Absorbing unit, the detection unit includes fluorescin DNA sequence dna, and the fluorescin DNA sequence dna and the first promoter connect
Connect；The absorbing unit includes the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and gold ion combination egg interconnected
White GolB DNA sequence dnas, and the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas are connected with the second promoter；It is described
Detection unit is located at the upstream of the absorbing unit.
2. gold ion detection as claimed in claim 1 and adsorption system, it is characterised in that the fluorescin is red fluorescence
Albumen RFP, its DNA sequence dna such as SEQ ID NO:Shown in 13；And/or
First promoter is PgolS-GolS-PgolB, its sequence such as SEQ ID NO:Shown in 10；Second promoter is
PgolB, its sequence such as SEQ ID NO:Shown in 19.
3. gold ion detection as claimed in claim 1 and adsorption system, it is characterised in that the bacillus subtilis spore clothing
PROTEIN C otC DNA sequence dnas such as SEQ ID NO:Shown in 3, the gold ion associated proteins GolB DNA sequence dnas such as SEQ ID NO:6
It is shown.
4. a kind of gold ion detection and adhesion protein surface display system, it is characterised in that including by any institutes of claim 1-3
The gold ion detection stated and fluorescin, bacillus subtilis spore clothing PROTEIN C otC and the gold ion of adsorption system expression combine
Protein G olB.
5. a kind of expression vector, it is characterised in that including the gold ion detection as described in claim 1-3 is any and absorption system
System.
6. a kind of Host Strains, it is characterised in that the Host Strains include expression vector as claimed in claim 5；Or
The Host Strains express gold ion detection as claimed in claim 4 and adhesion protein surface display system.
7. a kind of gold ion detection and the preparation method of adsorption system, it is characterised in that comprise the following steps：
Bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas are amplified from Bacillus subtilis genes group, it is husky from mouse typhus
Gold ion associated proteins GolB DNA sequence dnas are amplified in door Salmonella genome；
With the bacillus subtilis spore clothing PROTEIN C otC DNA sequence dnas and the gold ion associated proteins GolB DNA sequence dnas
For template, CotC-GolB fusion protein DNA sequence dnas are amplified；
The amplification fluorescent protein DNA sequence from coli expression carrier genome；
By the first promoter, the fluorescin DNA sequence dna, the second promoter, the CotC-GolB fusion proteins DNA sequence dna
It is sequentially connected.
8. gold ion detection as claimed in claim 7 and the preparation method of adsorption system, it is characterised in that expand the withered grass
The primer such as SEQ ID NO of bacillus spore clothing PROTEIN C otC DNA sequence dnas:1、SEQ ID NO:Shown in 2；Expand the gold
The primer such as SEQ ID NO of ions binding Protein G olB DNA sequence dnas:4、SEQ ID NO:Thing shown in 5；And/or
The fluorescin DNA sequence dna is red fluorescent protein RFP DNA sequence dnas, and its amplimer such as SEQ ID NO:11、
SEQ ID NO:Shown in 12；First promoter is PgolS-GolS-PgolB, and its amplimer such as SEQ ID NO:8、SEQ
ID NO:Shown in 9；Second promoter is PgolB, and its amplimer such as SEQ ID NO:17、SEQ ID NO:Shown in 18.
9. a kind of gold ion recovery method, it is characterised in that comprise the following steps：
Liquid containing gold ion is provided；
It will be added after Host Strains culture described in claim 6 in the liquid, stewing process；
After there is precipitation in the solution, the Host Strains that filtration treatment obtains being adsorbed with gold ion are carried out；
The Host Strains of gold ion are adsorbed with acid treatment, gold ion is obtained after separation.
10. gold ion recovery method as claimed in claim 9, it is characterised in that gold ion is dense in the liquid of offer
It is 0.1 μM~20 μM to spend scope；And/or
The time range of the stewing process is 40h~48h.
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| 宋天宇 等: ""枯草芽胞杆菌芽胞表面展示技术研究进展"", 《生命科学仪器》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958612A (en) * | 2022-02-23 | 2022-08-30 | 武汉科技大学 | Screening method of chlamydomonas reinhardtii for specifically identifying heavy metal cadmium |
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