CN107287225A - Gold ion is detected and adsorption system and its Host Strains, gold ion recovery method - Google Patents
Gold ion is detected 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 is detected 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 escherichia coli outer membrane protein OmpA DNA sequence dnas and gold ion associated proteins GolB DNA sequence dnas interconnected, and the escherichia coli outer membrane protein OmpA 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 be realized the detection to heavy metals in industrial wastewater gold and recyclable enrichment and be reclaimed by biological means, can be overcome the shortcoming of prior art, not resulted in 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 in heavy metal, physics, Electronic Performance are excellent
Metal, with higher stability, its application field is widely.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
Immune system can as the immunocytes such as leucocyte inhibitor.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, being produced in the production process such as irrational exploitation and the smelting of gold and secondary operation
Accessory substance, these problems have badly influenced 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
Reclaim 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 that 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 in human body with various enzymes occur combine to form compound, 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
The purpose for reclaiming heavy metal is reached into precipitation or suspension.But chemical method has three shortcomings：First is that chemical method has can
Can be because the amount of the chemical reagent of addition is improper and causes secondary pollution；Second is that chemical method is selectively not high enough, especially
It is that the extremely close each heavy metal species effect of handling properties is bad, has obtained certain purpose metal even if reclaiming, purity is not also high,
Also need to further processing；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 prior art it is above-mentioned it is not enough 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 existing gold ion and reclaim that purity is low, effect is undesirable, and pollution environment
Technical problem.
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 includes fluorescin DNA sequence dna, and the fluorescin DNA sequence dna and first
Promoter is connected；The escherichia coli outer membrane protein OmpA DNA sequence dnas and gold ion that the absorbing unit includes interconnecting are combined
Protein G olB DNA sequence dnas, and the escherichia coli outer membrane protein OmpA DNA sequence dnas are connected with the second promoter；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, escherichia coli outer membrane protein OmpA and the gold ion associated proteins GolB of adsorption system expression.
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 provides 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 provides the preparation method of a kind of above-mentioned gold ion detection and adsorption system, including following step
Suddenly：
Escherichia coli outer membrane protein OmpA DNA sequence dnas are amplified from genome of E.coli, from salmonella typhimurium
Gold ion associated proteins GolB DNA sequence dnas are amplified in genomic DNA；
Using the escherichia coli outer membrane protein OmpA DNA sequence dnas and the gold ion associated proteins GolB DNA sequence dnas as
Template, amplifies OmpA-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 OmpA-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 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.
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 OmpA-GolB absorbed portions, efficient absorption gold ion.After the system is connected in plasmid
In the Bacillus coli cells body for importing non-pathogenic, Visual retrieval and absorption gold ion under promoter regulation, therefore this kind
There is good effect to the detection of gold ion and absorption by improved engineering bacteria；Because of reporter fluorescence albumen, Escherichia coli
Outer membrane protein OmpA and gold ion associated proteins GolB high efficient expression, its engineering bacteria can express gold ion detection and absorption egg
White surface display system, therefore gold ion is detected and adsorption system is easily prepared, cost is relatively low, easy to operate.
The gold ion recovery method that the present invention is provided, can collect gold ion detection and absorption integration, 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 reclaims convenient.By the processing to thalline and it is secondary use, the present invention can finally pass through
Biological means are realized the detection to heavy metals in industrial wastewater gold and recyclable enrichment and reclaimed；Therefore, the present invention can overcome
The shortcoming of prior art, does not result in secondary pollution, 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 Lpp-OmpA-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 sensitivity Detection effect diagram in the water body of the embodiment of the present invention 5；
Fig. 7 (a) is gold ion selective enumeration method effect diagram in the water body of the embodiment of the present invention 6；
Fig. 7 (b) is gold ion selective enumeration method effect diagram in the water body of the embodiment of the present invention 5；
Fig. 8 is the protein expression proof diagram of the embodiment of the present invention 7；
Fig. 9 is the influence of the concentration and induction time of gold ion in the water body of the embodiment of the present invention 5 to detection；
Figure 10 is the gold ion absorption ability design sketch of the embodiment of the present invention 8.
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 includes fluorescin DNA sequence dna, and the fluorescin DNA sequence dna and first
Promoter is connected；Absorbing unit includes the escherichia coli outer membrane protein OmpA DNA sequence dnas and gold ion associated proteins interconnected
GolB DNA sequence dnas, and escherichia coli outer membrane protein OmpA DNA sequence dnas are connected with the second promoter；The detection unit, which is located at, inhales
The upstream of coupon member.
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 dnas such as SEQ ID
NO:Shown in 3, gold ion associated proteins GolB DNA sequence dnas 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 serves to gold ion Visual retrieval report, in a preferred embodiment of the invention in the present embodiment
In, fluorescin is red fluorescent protein RFP, its DNA sequence dna such as SEQ ID NO:Shown in 13.
In the gold ion detection of one embodiment of the present invention and adsorption system, the second promoter and Escherichia coli outer membrane
Lipoprotein Lpp DNA sequence dnas, lipoprotein Lpp DNA sequence dnas such as SEQ ID NO are also associated between albumen OmpA DNA sequence dnas:22
It is shown.Under this condition, the Lpp albumen of the system expression can strengthen escherichia coli outer membrane protein OmpA surface display energy
Power so that gold ion associated proteins GolB on cell membrane combine it is more firm closer.
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
Fluorescin, escherichia coli outer membrane protein OmpA and the gold ion of gold ion detection and the adsorption system expression of the embodiment of the present invention
Associated proteins GolB；Or the gold ion detection including another embodiment of the present invention and fluorescin, the fat egg of adsorption system expression
White Lpp, escherichia coli outer membrane protein OmpA and 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 present embodiment gold from
Son detection and adsorption system.Meanwhile, the embodiments of the invention provide a kind of Host Strains, the Host Strains 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 provides the preparation method of a kind of above-mentioned gold ion detection and adsorption system, and it is special
Levy and be, comprise the following steps：
S011：Escherichia coli outer membrane protein OmpA DNA sequence dnas are amplified from genome of E.coli, it is husky from mouse typhus
Gold ion associated proteins GolB DNA sequence dnas are amplified in door Salmonella genomic DNA；
S012：Using above-mentioned escherichia coli outer membrane protein OmpA DNA sequence dnas and gold ion associated proteins GolB DNA sequence dnas as
Template, amplifies the DNA sequence dna of OmpA-GolB fusion proteins；
S013：The amplification fluorescent protein DNA sequence from coli expression carrier genome；
S014：By the first promoter, the fluorescin DNA sequence dna, the second promoter, OmpA-GolB fusion eggs
White DNA sequence dna is sequentially connected.
In one embodiment, the primer such as SEQ ID NO of escherichia coli outer membrane protein OmpA DNA sequence dnas are expanded:1、SEQ
ID NO:Shown in 2；Expand the primer SEQ ID NO of gold ion associated proteins GolB DNA sequence dnas:4、SEQ ID NO: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 amplification is obtained, 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.
In another embodiment, by amplifying lipoprotein Lpp DNA sequence dna, connection from expression lipoprotein Lpp strain
In the second promoter PgolBBetween escherichia coli outer membrane protein OmpA DNA sequence dnas, make gold ion associated proteins GolB in cell
Combined on film more firm closer.
Finally, the embodiment of the present invention 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：Gold ion is obtained after the Host Strains of gold ion, separation are adsorbed with acid treatment.
In a preferred embodiment, in above-mentioned steps S021, the liquid containing gold ion can be various industrial wastewaters, ore deposit
Contain gold ion recovered liquid in industry waste water etc., the concentration range of gold ion is 0.01 μM~20 μM in the liquid；The present embodiment
There is provided Host Strains just has good Detection results when gold ion concentration is 0.01 μM, and is detected when gold ion concentration is 10 μM
Best results.In above-mentioned steps S022, the time range of stewing process is 10h~15h, and Host Strains are to golden in liquid in the range of this
The adsorption effect of ion reaches most preferably.
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 gold ion detecting system expression plasmid and identification
1. gold ion induced gene P in detecting systemgolS-GolS-PgolBThe amplification of fragment
Use special primer pGolts1 (SEQ ID NO:8) with pGolts2 (SEQ ID NO:9) from Salmonella typhimurium
Bacterium genomic DNA (the reference position in NCBI：NC_003197) in amplify DNA sequence dna (the SEQ ID of the golden induced gene of coding
NO:10)；The gold ion induced gene (Gold inductive gene) of the present embodiment is promoter PgolS-GolS-PgolB,
It can express GolS albumen, and GolS albumen usually plays the effect checked, just can be from promoter P in the presence of having gold iongolB
DNA 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:11) with rfp2 (SEQ ID NO: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 above-mentioned 1st and 2 points of amplified productions reclaimed as masterplate, RFP-terminator is amplified
The DNA sequence dna of fragment, 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.
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 T4 DNA 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) using the above-mentioned RFP-terminator of DNA restriction enzyme XbaI and PstI digestions amplification of DNA fragments, so
Using the insertion of T4DNA ligases, 1) step is built in the Escherichia coli biological brick expression vector pZH2 completed afterwards.
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. gold ion induced gene 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 P of the promoter of sub- controlling genegolBDNA sequence dna (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 escherichia coli outer membrane protein A (OmpA) fragment
According to required amplification target DNA sequence and base interworking principle, design special primer Ompa1 (SEQ ID NO:1)
With Ompa2 (SEQ ID NO:2), from genome of E.coli DNA (the reference positions in NCBI：NC_000913) in amplify
The 1st DNA sequence dna (the SEQ ID NO to the 159th amino acids of encoding E. coli outer membrane protein A (OmpA):3).
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) 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) middle expansion
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
Sequence, 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：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 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.
The structure of 3.Lpp-OmpA-GolB fusion protein expression plasmids and identification
1) design special primer L1 (SEQ ID NO:20) with L2 (SEQ ID NO:21), from the big of expression lipoprotein Lpp
Lipoprotein Lpp DNA (SEQ ID NO are amplified in enterobacteria:22) product, uses DNA restriction enzyme XbaI and PstI enzymes
Solve lipoprotein Lpp fragments and above-mentioned PgolBFragment, OmpA fragments, GolB fragments, the amplified production of terminator fragments,
Then inserted using T4DNA ligases in Escherichia coli biological brick expression vector pZH2, Lpp-OmpA-GolB expressing fusion proteins
Plasmid construction collection of illustrative plates please refer to Fig. 3；The DNA sequence dna of wherein OmpA-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 Lpp-OmpA-GolB fusion protein expression plasmids, as a result see Fig. 4.
Embodiment 3Gold ion detection 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- Lpp-OmpA-GolB-terminator enzymatic fragments, make it have cohesive end and then use
T4DNA ligases are inserted into Escherichia coli biological brick plasmid backbone 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.
2. gold ion detects and adsorbed the extraction result of integrated expression plasmid see Fig. 5.
Embodiment 4Gold ion is detected and adsorption system plasmid is transferred to Host Strains
1) the recombinant plasmid pZH2 for obtaining above-described embodiment 4 imports Bacillus coli cells DH5 α by chemical conversion process
In, by containing ampicillin (50 μ g mL-1) LB solid mediums in 37 DEG C cultivate 16 hours, therefrom screen
Go out positive colony and preserved.
The heat-shock transformed and dilution spread step of competent cell is as follows：By the product of coupled reaction in competent cell
In (DH5 α), finger flicks mixing；Ice bath 42 DEG C of heat shocks 95 seconds after 30 minutes, fast transfer is on ice；Ice bath is often managed after 2 minutes
The prior 37 DEG C preheated μ l of LB culture mediums 900 are added, 37 DEG C, 200rpm cultures make it recover in 1 hour；3000rpm centrifugations 30
Second, remove after 0.9ml supernatants, remove layer 0.1ml and be coated with corresponding resistant panel, 37 DEG C are cultivated 10 hours.
2) 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 by Shanghai biotechnology service company complete, gold ion detection and
The extraction and identification of adsorption system plasmid.
Embodiment 5Gold ion is detected and adsorption system detects the test of gold ion ability
1. influence of the concentration of gold ion to detection
1) ampicillin (50 μ g mL will be contained in right amount for detecting that the strain Escherichia coli of gold ion is accessed-1) LB
In overnight incubation in 37 DEG C in fluid nutrient medium；By incubated overnight bacterium solution 1:100 be diluted into right amount contain ampicillin (50 μ g
mL-1) in LB fluid nutrient mediums in being cultivated in 37 DEG C to nutrient solution OD600=0.6, final concentration of 0 is added into nutrient solution；
0.001；0.01；0.1；0.25；0.5；1；5；10；20 μM of gold ion, nutrient solution continues overnight incubation in 37 DEG C；
2) culture 10h inoculum cleans 1 by centrifuging after (8000 revs/min, 2min) collection with deionized water
It is secondary；
3) thalline after cleaning is resuspended using 1 × PBS；
4) bacterium solution after being resuspended is detected using ELIASA.
5) specific testing result is see Fig. 6, and engineered engineering bacteria just has well when gold ion concentration is 0.01 μM
Detection results, and Detection results are best when gold ion concentration is 10 μM.
2. the influence of the concentration and induction time of gold ion to detection
1) ampicillin (50 μ g mL will be contained in right amount for detecting that the strain Escherichia coli of gold ion is accessed-1) LB
In overnight incubation in 37 DEG C in fluid nutrient medium；By incubated overnight bacterium solution 1:100 be diluted into right amount contain ampicillin (50 μ g
mL-1) in LB fluid nutrient mediums in being cultivated in 37 DEG C to nutrient solution OD600=0.6, four different concentration gradients are set, at four
Different time gradients are set under different concentration gradients.Four different gold concentration gradients, respectively 0.1,1,5,20 μm of ol,
Time gradient is respectively 0.5,1,1.5,2,3,5,7.5,10h.Cultivate 10h.
2) culture 10h inoculum cleans 1 by centrifuging after (8000 revs/min, 2min) collection with deionized water
It is secondary；
3) thalline after cleaning is resuspended using 1 × PBS；
4) bacterium solution after being resuspended is detected using ELIASA.
Specific testing result just has good inspection see Fig. 9, engineered engineering bacteria when gold ion concentration is 0.01 μM
Effect is surveyed, and Detection results are best when gold ion concentration is 10 μM.To thalline carry out fluoroscopic examination result be shown in gold from
When sub- concentration is 0.1 μm of ol, fluorescence intensity increases successively, and maximum 16000 or so is reached in 10h.It is 1 in gold ion concentration
During μm ol, peak shape distribution is presented in fluorescence intensity, and maximum 45000 or so is reached in 5h.When gold ion concentration is 5 μm of ol,
Peak shape distribution is presented in fluorescence intensity, and maximum 65000 or so is reached in 5h.
So understand：This system can be equivalent to the coupling chemically reacted with a decomposition reaction of a concentration dependent,
The concentration action rule of kinetics equation is met, i.e. initial concentration is higher, the time needed for reaction reaches balance is shorter, and by
In the presence of decomposition reaction, green fluorescent protein is eventually degraded, therefore peak type can be presented in the function of fluorescence intensity relative time
Distribution.
3. hybrid metal GOLD FROM PLATING SOLUTION ion selectivity is detected
1) ampicillin (50 μ g mL will be contained in right amount for detecting that the strain Escherichia coli of gold ion is accessed-1) LB
In overnight incubation in 37 DEG C in fluid nutrient medium；By incubated overnight bacterium solution 1:100 be diluted into right amount contain ampicillin (50 μ g
mL-1) in LB fluid nutrient mediums in being cultivated in 37 DEG C to nutrient solution OD600=0.6, addition gold, gold, silver, copper, cadmium, nickel, zinc and silver,
Copper, cadmium, nickel, zinc, nutrient solution continue overnight incubation in 37 DEG C；
2) culture 10h inoculum cleans 1 by centrifuging after (8000 revs/min, 2min) collection with deionized water
It is secondary；
3) thalline after cleaning is resuspended using 1 × PBS；
4) bacterium solution after being resuspended is detected using ELIASA.
5) specific testing result is see Fig. 7 (b), and the result that fluoroscopic examination is carried out to thalline shows hybrid metal to gold ion
Fluoroscopic examination have minor way, golden selectivity is still very high.
Embodiment 6Gold ion is detected and adsorption system selects gold ion the detection of sexuality
1. ampicillin (50 μ g mL will be contained in right amount for detecting that the strain Escherichia coli of gold ion is accessed-1) LB
In overnight incubation in 37 DEG C in fluid nutrient medium；By incubated overnight bacterium solution 1:100 be diluted into right amount contain ampicillin (50 μ g
mL-1) in LB fluid nutrient mediums in being cultivated in 37 DEG C to nutrient solution OD600=0.6, final concentration of 10 μ is separately added into nutrient solution
M silver, nickel, zinc, copper, cadmium, chromium, mercury, lead ion, nutrient solution continues to cultivate 10h in 37 DEG C；
2. cultivating 10h 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.
Specific testing result is see Fig. 7 (a), and engineered engineering bacteria has preferable selectivity to gold ion.
Embodiment 7The plasmid of expression Lpp-OmpA, Lpp-OmpA-GolB fusion protein is transferred to Host Strains and Host Strains
Culture
1. the plasmid of expression Lpp-OmpA fusion proteins is transferred to Host Strains
1) chemical conversion process importing Bacillus coli cells DH5 will be passed through for the plasmid for expressing Lpp-OmpA fusion proteins
In α, by containing ampicillin (50 μ g mL-1) LB solid mediums in 37 DEG C cultivate 16 hours, therefrom screen
Go out positive colony and preserved.
The heat-shock transformed and dilution spread step of competent cell is as follows：By the product of coupled reaction in competent cell
In (DH5 α), finger flicks mixing；Ice bath 42 DEG C of heat shocks 95 seconds after 30 minutes, fast transfer is on ice；Ice bath is often managed after 2 minutes
The prior 37 DEG C preheated μ L of LB culture mediums 900 are added, 37 DEG C, 200rpm cultures make it recover in 1 hour；3000rpm centrifugations 30
Second, remove after 0.9ml supernatants, remove layer 0.1ml and be coated with corresponding resistant panel, 37 DEG C are cultivated 10 hours.
2) 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.Lpp-OmpA is merged
Protein expressing plasmid Sequencing chromatogram is see Fig. 2.
2. the plasmid of expression Lpp-OmpA-GolB fusion proteins is transferred to Host Strains
1) will to import Escherichia coli for expressing the plasmids of Lpp-OmpA-GolB fusion proteins by chemical conversion process thin
In born of the same parents DH5 α, by containing ampicillin (50 μ g mL-1) LB solid mediums in 37 DEG C cultivate 16 hours, from
In filter out positive colony and preserved.
The heat-shock transformed and dilution spread step of competent cell is as follows：By the product of coupled reaction in competent cell
In (DH5 α), finger flicks mixing；Ice bath 42 DEG C of heat shocks 95 seconds after 30 minutes, fast transfer is on ice；Ice bath is often managed after 2 minutes
The prior 37 DEG C preheated μ L of LB culture mediums 900 are added, 37 DEG C, 200rpm cultures make it recover in 1 hour；3000rpm centrifugations 30
Second, remove after 0.9ml supernatants, remove layer 0.1ml and be coated with corresponding resistant panel, 37 DEG C are cultivated 10 hours.
2) 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.
3. Fiber differentiation
Ammonia will be contained in right amount for expressing the strain Escherichia coli of Lpp-OmpA, Lpp-OmpA-GolB fusion protein and accessing
Parasiticin (50 μ g mL-1) LB fluid nutrient mediums in overnight incubation in 37 DEG C；By incubated overnight bacterium solution 1:100 are diluted into
Contain ampicillin (50 μ g mL in right amount-1) in LB fluid nutrient mediums in being cultivated in 37 DEG C to nutrient solution OD600=0.6, Xiang Pei
It is respectively 1 μM that final concentration is added in nutrient solution；5μM；20 μM are induced, and nutrient solution continues overnight incubation in 37 DEG C.
4. protein expression is verified
The bacterium (5000rpm, 5min) of overnight incubation in 37 DEG C is collected by centrifugation, supernatant is abandoned.Precipitation uses 1mL PBS
(PMSF of the 100mM containing 10uL) is resuspended.Use broken bacterium (10min, the ultrasonic 5s, interval collected of sonicator
5s), after 4 DEG C, 12000rpm centrifugation 10min, precipitation uses TDSET buffer (1%Triton X-100,0.2%
Sodium Deoxycholate, 0.1%SDS, 10mM tetrasodium EDTA, 10mM Tris/HCl) processing is twice.
12000rpm centrifuges 10min, and supernatant discarding, precipitation is resuspended using 500 μ L PBS.
1) SDS-PAGE is verified
1. 12%SDS-PAGE glue is prepared, specific formula see the table below 1.
Table 1
2. take 20 μ L to crush supernatant and precipitation re-suspension liquid, add 5 μ 5 × sample buffers of L, 95 DEG C of constant temperature processing
10min.12000rpm centrifuges 10min afterwards.
3. 1 × electrophoretic buffer, loading, first 75V constant-pressure operations 30min, rear 160V constant pressures are added in electrophoresis tank
50min。
4. coomassie brilliant blue staining liquid dyeing 20min is added after the completion of.
5. Coomassie brilliant blue destainer decolourizes 2 times, each 20min.
6. it is imaged, is taken pictures under white light using gel imager.
2) western blot are verified
1. 12%SDS-PAGE protein isolates.
2. the pvdf membrane close with glue size is cut, 20s, then the 2min that is soaked in water are soaked in methyl alcohol.
3. 1L electricity is added in electrophoresis tank and turns liquid, using wet type transferring film method method transferring film, the placement order of glue and film is：Sea
250mA Constant Electric Currents turn 2h under silk floss/filter paper/glue/film/filter paper/sponge, low temperature.
4. after the completion of transferring film, film is taken out, in certain orientation shear angle to distinguish positive and negative.Film is cleaned using TBST 3 times, every time
5min。
5. after the completion of cleaning, the skimmed milk powers of 50mL 5% closing 2h is added.
6. after the completion of closing, film is cleaned 4 times using TBST, each 5min.With 1：The anti-Flag tag antibodies of 1000 dilutions
Make primary antibody, 4 DEG C of overnight incubations.
7. TBST cleans film 4 times, each 5min.Use 1：Goat anti-mouse IgG-HRP the antibody of 1000 dilutions makees secondary antibody,
Normal temperature is incubated 2h,
8. TBST cleans film 4 times, each 5min.Add the 400 low background chemiluminescence detection agents of μ L cECL on film, use
Gel imager is imaged, taken pictures.
As a result as shown in figure 8, being respectively from left to right：
1:Under simple gold ion environment, GolB albumen is largely induced, because GolB albumen end carries the foregoing description
FLAG labels, therefore can be recognized by fluorescence antibody, finally development produces band on film.
2:On the premise of gold ion is mixed with other metal ions, GolS albumen still can be lured by gold ion specificity
Lead and lose the ability of checking, cause GolB expression.
3:When only existing other metal ions, GolS albumen is still incorporated on DNA the effect checked of playing, therefore GolB eggs
Do not express in vain.
Embodiment 8The detection of Lpp-OmpA, Lpp-OmpA-GolB gold ion absorption ability
Two kinds of engineering bacterias Lpp-OmpA, Lpp-OmpA-GolB carry out adsorption experiment.Experiment first two bacterium, which is rule, to be incubated at
In LB solid mediums (amicillin resistance).
Influence of the gold ion concentration to adsorption capacity
1. picking single bacterium colony is inoculated in 5mL LB (ampicillin containing 5uL) culture medium respectively from two culture dishes
In, 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, add Au3+It is respectively 1 μm of ol, 5 μm of ol, 20 μm of ol to final concentration.37℃、220rpm
Continue to cultivate 10h.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.
Specific testing result is see Figure 10.Result is shown in figure, and Lpp-OmpA-GolB engineering bacterias show strong to gold ion
Strong adsorption capacity.
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>Gold ion is detected and adsorption system and its Host Strains, gold ion recovery method
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 45
<212> DNA
<213>The primer of amplification coding escherichia coli outer membrane protein OmpA gene
<400> 1
ctttcttcga attcgcggcc gcttctagag atgaaaaaga cagct 45
<210> 2
<211> 46
<212> DNA
<213>The primer of amplification coding escherichia coli outer membrane protein OmpA gene
<400> 2
ctttcttcct gcagcggccg tactagtaca tacgggtagc gatttc 46
<210> 3
<211> 477
<212> DNA
<213>Escherichia coli outer membrane protein OmpA DNA sequence dna
<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
accgctaaac tgggttaccc aatcactgac gacctggaca tctacactcg tctgggtggc 360
atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga caccggcgtt 420
tctccggtct tcgctggcgg tgttgagtac gcgatcactc ctgaaatcgc tacccgt 477
<210> 4
<211> 45
<212> DNA
<213>The primer of amplification coding gold ion associated proteins GolB gene
<400> 4
ctttcttcga attcgcggcc cttctagaga atgcagttcc atatt 45
<210> 5
<211> 63
<212> DNA
<213>The primer of amplification coding gold ion associated proteins GolB gene
<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> 705
<212> DNA
<213>The DNA sequence dna of OmpA-GolB fusion proteins
<400> 7
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
accgctaaac tgggttaccc aatcactgac gacctggaca tctacactcg tctgggtggc 360
atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga caccggcgtt 420
tctccggtct tcgctggcgg tgttgagtac gcgatcactc ctgaaatcgc tacccgtatg 480
cagttccata ttgatgacat gacctgcggc ggctgcgcca gtacggtaaa aaagacgatt 540
ctgactctcg atgctaatgc gacggtgaga actgacccgg cgacgcgtct ggttgacgtt 600
gaaacgtcgc tatccgcgga gcagattgcc gccgccctgc aaaaggccgg tttcccgccg 660
cgcgagaggc tcgaggatta caaggatgac gacgataaga tctga 705
<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 sequence 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
<210> 20
<211> 25
<212> DNA
<213>The primer of amplification coding lipoprotein Lpp gene
<400> 20
atgaaagcta ctaaactggt actgg 25
<210> 21
<211> 25
<212> DNA
<213>The primer of amplification coding lipoprotein Lpp gene
<400> 21
ttacttgcgg tatttagtag ccatg 25
<210> 22
<211> 236
<212> DNA
<213>Lipoprotein Lpp DNA sequence dna
<400> 22
atgaaagcta ctaaactggt actgggcgcg gtaatcctgg gttctactct gctggcaggt 60
tgctccagca acgctaaaat cgatcagctg tcttctgacg ttcagactct gaacgctaaa 120
gttgaccagc tgagcaacga cgtgaacgca atgcgttccg acgttcggct gctaaagatg 180
acgcagctcg tgctaaccag cgtctggaca acatggctac taaataccgc aagtaa 236
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 escherichia coli outer membrane protein OmpA DNA sequence dnas and gold ion associated proteins GolB interconnected
DNA sequence dna, and the escherichia coli outer membrane protein OmpA DNA sequence dnas are connected with the second promoter；The detection unit is located at institute
State the upstream of 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；And/or
The escherichia coli outer membrane protein OmpA DNA sequence dnas such as SEQ ID NO:Shown in 3, the gold ion associated proteins GolB
DNA sequence dna such as SEQ ID NO:Shown in 6.
3. gold ion detection as claimed in claim 1 or 2 and adsorption system, it is characterised in that second promoter and institute
State and be also associated with lipoprotein Lpp DNA sequence dnas, the lipoprotein Lpp DNA between escherichia coli outer membrane protein OmpA DNA sequence dnas
Sequence such as SEQ ID NO:Shown in 22.
4. a kind of gold ion detection and adhesion protein surface display system, it is characterised in that including as described in claim 1 or 2
Gold ion detection and adsorption system expression fluorescin, escherichia coli outer membrane protein OmpA and gold ion associated proteins
GolB；Or
Including fluorescin, lipoprotein Lpp, the large intestine bar expressed as the gold ion detection described in claim 3 and adsorption system
Bacterial outer membrane protein OmpA and gold ion associated proteins GolB.
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：
Escherichia coli outer membrane protein OmpA DNA sequence dnas are amplified from genome of E.coli, from salmonella typhimurium gene
Gold ion associated proteins GolB DNA sequence dnas are amplified in group DNA；
Using the escherichia coli outer membrane protein OmpA DNA sequence dnas and the gold ion associated proteins GolB DNA sequence dnas as template,
Amplify OmpA-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 OmpA-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 the amplification large intestine
The primer of bacillus outer membrane protein OmpA DNA sequence dnas such as SEQ ID NO:1、SEQ ID NO:Shown in 2；Expand the gold ion knot
The primer SEQ ID NO of hop protein GolB DNA sequence dnas:4、SEQ ID NO: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 0.01 μM~20 μM to spend scope；And/or
The time range of the stewing process is 10h~15h.
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Application publication date: 20171024 |