CN101509000A - Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof - Google Patents
Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof Download PDFInfo
- Publication number
- CN101509000A CN101509000A CNA2008102336257A CN200810233625A CN101509000A CN 101509000 A CN101509000 A CN 101509000A CN A2008102336257 A CNA2008102336257 A CN A2008102336257A CN 200810233625 A CN200810233625 A CN 200810233625A CN 101509000 A CN101509000 A CN 101509000A
- Authority
- CN
- China
- Prior art keywords
- gene
- copper
- dna
- pet28a
- resistant gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 68
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 229910052802 copper Inorganic materials 0.000 title claims abstract description 63
- 239000010949 copper Substances 0.000 title claims abstract description 63
- 239000013598 vector Substances 0.000 title claims description 9
- 238000002360 preparation method Methods 0.000 title claims description 5
- 241000894006 Bacteria Species 0.000 title abstract description 12
- 239000013612 plasmid Substances 0.000 claims abstract description 17
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 11
- 150000001413 amino acids Chemical class 0.000 claims abstract description 11
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 11
- 241000607714 Serratia sp. Species 0.000 claims abstract description 9
- 241000607715 Serratia marcescens Species 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 241000588724 Escherichia coli Species 0.000 claims description 23
- 108020004414 DNA Proteins 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 235000001014 amino acid Nutrition 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 9
- 108091008146 restriction endonucleases Proteins 0.000 claims description 9
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 8
- 101710112057 Lipoprotein NlpE Proteins 0.000 claims description 7
- 230000029087 digestion Effects 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 241000607734 Yersinia <bacteria> Species 0.000 claims description 6
- 241001464926 Yersinia mollaretii Species 0.000 claims description 6
- 239000003292 glue Substances 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- 238000005215 recombination Methods 0.000 claims description 5
- 230000006798 recombination Effects 0.000 claims description 5
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 4
- 238000009795 derivation Methods 0.000 claims description 4
- 238000013461 design Methods 0.000 claims description 4
- 239000013613 expression plasmid Substances 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 238000013467 fragmentation Methods 0.000 claims description 3
- 238000006062 fragmentation reaction Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 claims description 3
- 241000588807 Bordetella Species 0.000 claims description 2
- 239000013611 chromosomal DNA Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 6
- 102000004895 Lipoproteins Human genes 0.000 abstract description 4
- 108090001030 Lipoproteins Proteins 0.000 abstract description 4
- 230000008261 resistance mechanism Effects 0.000 abstract description 4
- 241000779673 Yersinia mollaretii ATCC 43969 Species 0.000 abstract 1
- 238000010367 cloning Methods 0.000 abstract 1
- 229910001431 copper ion Inorganic materials 0.000 abstract 1
- 239000000047 product Substances 0.000 description 10
- 241000607720 Serratia Species 0.000 description 9
- 229910001385 heavy metal Inorganic materials 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 210000002808 connective tissue Anatomy 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000010865 sewage Substances 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 101710105045 Lipoprotein E Proteins 0.000 description 1
- 102000015094 Paraproteins Human genes 0.000 description 1
- 108010064255 Paraproteins Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 241000405965 Scomberomorus brasiliensis Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a gene encoding copper resistance of Serratia marcescens. The gene is generated by a Serratia sp. KMR-3 strain; the whole length of the gene sequence is 1,117bp; derived copper resistance protein is encoded by 194 amino acids, is CutF protein with molecular weight of about 22KDa and theoretical pI value of 5.30; and the gene has highest homology with Yersinia mollaretii ATCC 43969 copper resistance lipoprotein N1pE, and reaches 70 percent. The invention also provides a recombinant plasmid containing the gene and a recombinant strain expressing the resistance gene. The method for expressing resistance protein heterology of the gene comprises the following steps: copper resistance related gene can be cloned through constructing a genome DNA library; according to comparison of amino acid sequences, the cloned protein is found to be CutF protein, and the protein is further expressed in colibacillus successfully. The technology of cloning the copper resistance gene establishes foundation for bacterium copper resistance mechanism and biological restoration technology of copper ion polluted region.
Description
Technical field
The invention belongs to the genetically engineered field, particularly, the gene that relates to Serratia coding copper resistant, dna sequence dna and deduced amino acid, and the heterogenous expression of resistance protein, relate to recombinant plasmid that contains this gene and the recombinant bacterial strain of expressing this resistant gene, and their preparation and expression method.
Background technology
Though heavy metal appears in some ecosystem naturally, but its industrial application causes many serious environmental problem, therefore heavy metal comes into one's own day by day to the pollution and the improvement of environment, and effective utilization of metal resistance bacterium then can be removed metal from the environment that pollutes.Therefore for the understanding of heavy metal resistance regulation and control, will help to handle bio-waste and assess the influence of industrial activity natural ecosystems.
Copper is a kind of important trace metal, plays the part of important role in cells physiological, and it also is the important prothetic group of a series of enzymes in the respiration.But, when surpassing finite concentration then pair cell have toxicity, the cupric ion of high density is mainly by producing highly active radical damage DNA, destroy membrane structure and making enzyme deactivation.Therefore; avoid the high density heavy metallic poison in order to protect cell; bacterium utilizes a series of resistance mechanisms: self avoid the murder by poisoning of copper as reaching the outer sequester of born of the same parents, outflow pump, enzyme detoxification and restoring system protection in permeability barrier, the born of the same parents, and guarantee to satisfy its nutritional requirement.
Clone's resistant gene is the prerequisite to the research of heavy metal resistance mechanism, simultaneously, the resistant gene of being cloned into is transformed, not only can provide certain theoretical foundation, can also provide theoretical investigation and application foundation for the development and application of the bioremediation technology of heavy metal contamination region for inquiring into bacterium heavy metal resistance mechanism.
Summary of the invention
The present invention aims to provide the dna sequence dna of a kind of copper resistant gene and this gene and the amino acid (Fig. 1) of derivation, and the proteic heterogenous expression of copper resistant (Fig. 2).
Another object of the present invention is to provide a kind of recombinant expression plasmid and recombinant bacterial strain of copper resistant gene.
Contain the recombinant plasmid pUC118-Cu1 and the pET28a-Cu1 of above-mentioned dna sequence dna, and recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 that utilizes carrier pET28a-Cu1 to transform.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The copper resistant gene, its dna sequence dna as shown in Figure 1.
The copper resistant gene, its aminoacid sequence as shown in Figure 1.
Described copper resistant gene, it is produced by thunder Bordetella (Serratia sp.) KMR-3 bacterial strain, gene order total length 1,117bp, the anti-cuproprotein of derivation is encoded by 194 amino acid, is CutF albumen, and molecular weight is about 22KDa, and theoretical pI value is 5.30; The highest with Mohs Yersinia (Yersinia mollaretii) ATCC 43969 copper resistant lipoprotein NlpE homologys, reach 70%.
The recombinant vectors pUC118-Cu1 that comprises the dna sequence dna of claim 1.
The recombinant vectors pET28a-Cu1 that comprises the dna sequence dna of claim 1.
Recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 that utilizes recombinant vectors pET28a-Cu1 to transform.
The preparation method of above-mentioned copper resistant gene, recombinant vectors, recombinant bacterial strain, extract the chromosomal DNA of serratia marcescens KMR-3 bacterial strain, carry out partially digested with restriction enzyme Sau3A I, glue reclaims enzyme and cuts product, and be connected with pUC118 BamH I/BAP carrier, connect product through ethanol sedimentation, electric Transformed E .coliDH5, coating contains the LB flat board of penbritin, successfully makes up genome dna library; Screen recon containing on the selectivity flat board of penbritin and cupric ion, from recombinant plasmid pUC118-Cu1, isolate the dna fragmentation of cupric resistant gene,, obtain DNA as shown in Figure 1 through digestion with restriction enzyme; Design primer amplification copper resistant gene is connected and transformed into escherichia coli BL21 (DE3) bacterial strain with the pET28a carrier, obtains recombinant plasmid pET28a-Cu1 and recombinant bacterial strain e. coli bl21 (the DE3)-pET28a-Cu1 that contains recombinant expression plasmid.
Serratia (Serratia sp.) the KMR-3 bacterial strain that the strain that utilization of the present invention is separated to the waste water around the smeltery, Kunming has high resistance to cupric ion, its preserving number is: CGMCC No.2636.By in bacillus coli DH 5, making up the genome dna library of this bacterium, be cloned into the gene relevant with copper resistant.The albumen of the copper resistant coded by said gene of being cloned into belongs to CutF albumen, and is the highest with Mohs Yersinia (Yersinia mollaretii) ATCC 43969 copper resistant lipoprotein NlpE homologys by 194 amino acid codings, reaches 70%.Obtain to contain the recombinant plasmid of copper resistant gene by ordinary method, transformed into escherichia coli bacterial strain successful expression the albumen of coding copper resistant gene.
In addition,, the copper resistant gene clone that the present invention relates to can be applied in the bacterial strain of sewage disposal to other, thereby improve it to the tolerance of copper and then reach the purpose that heavy metal ion are removed in enrichment, conversion etc. by molecular biology method.
The serratia that the present invention relates to (Serratia sp.) KMR-3 bacterial strain on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.2636.
Concrete technological step of the present invention is as follows: extract serratia KMR-3 strain chromosome DNA, carry out partially digested with restriction enzyme Sau3A I.Glue reclaims enzyme and cuts product, and is connected with pUC118 BamH I/BAP carrier.Connect product through ethanol sedimentation, electric Transformed E .coli DH5, coating contains the LB flat board of penbritin, successfully makes up genome dna library.Screen recon containing on the selectivity flat board of penbritin and cupric ion.From recombinant plasmid pUC118-Cu1, isolate the dna fragmentation of cupric resistant gene,, obtain DNA as shown in Figure 1 through digestion with restriction enzyme.Screen the gene relevant with copper resistant, behind the extraction plasmid Transformed E .coli, its tolerance to copper has been brought up to 300mg/L by 160mg/L, and therefore the copper resistant gene of being cloned into has the potentiality of the sewage disposal of being applied to.Order-checking shows: the copper resistant gene of being cloned into is encoded by 194 amino acid, and molecular weight is about 22KDa.Design primer amplification copper resistant gene is connected and transformed into escherichia coli BL21 (DE3) bacterial strain with the pET28a carrier, obtains recombinant plasmid pET28a-Cu1 and recombinant bacterial strain e. coli bl21 (the DE3)-pET28a-Cu1 that contains recombinant expression plasmid.Induce down at IPTG, in intestinal bacteria, carried out efficiently expressing (Fig. 2).
The copper resistant gene that the present invention is cloned into, the highest with Mohs Yersinia (Yersinia mollaretii) ATCC43969 copper resistant lipoprotein NlpE homology, reach 70%.Utilize software to carry out positioning analysis in this proteic cell.Find that by analyzing this anti-cuproprotein is evenly distributed in outside kytoplasm, plasma membrane, pericentral siphon, adventitia and the born of the same parents.And known bibliographical information shows, the CutF albumen of E.coli is outer membrane lipoprotein, participates in the outflow of cupric ion and transports cupric ion to the dependent enzyme of copper.Studies show that NlpE (CutF) is a kind of new outer membrane lipoprotein E, and NlpE is first example that relevant outer membrane lipoprotein participates in signal transduction and activation lysophospholipids.
At present, much studies show that: NlpE participation competent cell combines with abiotic surface, and transmits signal by activating the double-deck component system of Cpx; The adventitia loss that NlpE took place in the time of may participating in competent cell and contact with hydrophobic surface, and transmit signal and give the Cpx approach; NlpE also monitors the integrity of coating; Eliminate paraprotein in the pericentral siphon, repair the amino acid in the N hunger; The pressure of induction adventitia, the transmitter kinase c pxA of mediation signal to the cytoplasmic membrane.Though the CutF albumen that the present invention is cloned into has very high homology with NlpE, but much still different to some extent with NlpE in nature, especially from distributing, NlpE is distributed in outer after birth, the effect of mainly taking on the signal conduction, and CutF do not have and significantly strides the film district, and it more resembles a cytoplasmic protein, but how it has participated in the copper resistant of bacterium and still need do further research.
As mentioned above, the copper resistant gene and the encoded protein principal character thereof that the present invention relates to are as follows:
(1) serratia (Serratia sp.) KMR-3 bacterial strain produces.
(2) by 194 amino acid codings, molecular weight is about 22KDa.The highest with Mohs Yersinia (Yersiniamollare tii) ATCC43969 copper resistant lipoprotein NlpE homology, reach 70%.
(3) IPTG induces down, efficiently expresses in intestinal bacteria.
Description of drawings:
Fig. 1 clone's copper resistant gene order and deduced amino acid;
Fig. 2 resistance protein is at expression in escherichia coli figure.
M---albumen Marker wherein; Before 1---BL21 (DE3)-pET28a control strain IPTG induces; After 2---BL21 (DE3)-pET28a control strain IPTG induces; Before 3---BL21 (DE3)-pET28a-Cu1 recombinant bacterial strain IPTG induces; After 4---BL21 (DE3)-pET28a-Cu1 recombinant bacterial strain IPTG induces.
Embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiment, but do not limit the present invention with this.
Embodiment 1:
Serratia (Serra tiasp.) KMR-3 bacterial strain copper resistant gene clone and evaluation:
A strain that is separated to the waste water around the smeltery, Kunming has high resistance to cupric ion serratia (Serratia sp.) KMR-3 bacterial strain.This serratia (Serratia sp.) KMR-3 bacterial strain on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.2636.
The structure of genome dna library: the extraction (F. Ao Sibai etc., fine works molecular biology experiment guide, Beijing: Science Press, 1999) of the total DNA of serratia (Serratia sp.) KMR-3 strain gene group.With the partially digested genome DNA of restriction enzyme Sau3A I, glue reclaims enzyme and cuts product, is connected with pUC118 BamHI/BAP carrier, connects product Transformed E .coli DH5.Coating contains the LB flat board of penbritin, and the fraction of coverage of this genome dna library reaches 99.9%, is effective library, has successfully made up the genome dna library of this bacterium.Screen recon containing on the selectivity flat board of penbritin and cupric ion.
The evaluation of copper resistant gene: utilize pcr amplification and double digestion method to identify recon.To CaCl
2Cultivate in the LB liquid nutrient medium at the bacterium colony of growing on the selectivity flat board behind the method Transformed E .coli DH5, with vast Tyke test kit extracting plasmid.With M
13(RV/M4) be primer, detect converted product by pcr amplification, primer sequence is as follows: M
13(M
4): 5 '-GTTTTCCCAGTCACGACGTTGTAAA-3 ', M
13(RV): 5 '-ATTTCACACAGGAAACAGCTATGAC-3 '.Further detect and insert pulsating size by double digestion.The pcr amplification result is consistent with the double digestion result, inserts pulsating size and is about 1.2kb.
Screen the gene relevant with copper resistant, behind the extraction plasmid Transformed E .coli, its tolerance to copper has been brought up to 300mg/L by 160mg/L, and therefore the copper resistant gene of being cloned into has the potential of the sewage disposal of being applied to; Also the copper resistant gene clone that the present invention relates to can be applied in the bacterial strain of sewage disposal to other, thereby improve it to the tolerance of copper and then reach the purpose that heavy metal ion are removed in enrichment, conversion etc.
Embodiment 2:
The proteic heterogenous expression of copper resistant:
The copper resistant genetic analysis: order-checking shows copper resistant gene order total length 1,117bp, and the anti-cuproprotein of derivation is encoded by 194 amino acid, is CutF albumen.This proteic reckoning molecular weight is about 22KDa, and theoretical pI value is 5.30.Find that by analyzing this anti-cuproprotein is evenly distributed in outside kytoplasm, plasma membrane, pericentral siphon, adventitia and the born of the same parents.And known bibliographical information shows, the CutF albumen of E.coli is outer membrane lipoprotein, participates in the outflow of copper and transports cupric ion to the dependent enzyme of copper.Utilize Blast (Ver2.2.14) sequence homology comparison to show: itself and Mohs Yersinia (Yersinia mollare tii) ATCC 43969 copper resistant lipoprotein NlpE homologys are the highest, are 70%.
The subclone of copper resistant gene: according to sequencing result, design primer (restriction enzyme site that contains Nde I and Xho I respectively) is by pcr amplification copper resistant gene.The purpose clip size is about 600bp, detects segmental size by 1% agarose gel electrophoresis, and glue reclaims the purpose fragment.The purpose fragment of the copper resistant gene that pET28a plasmid and glue reclaim is used two kinds of restriction enzymes (Nde I and Xho I) to carry out enzyme simultaneously and is cut.Glue reclaims enzyme and cuts product, and uses T
4Dna ligase connects.Connect product transformed into escherichia coli BL21 (DE3) competent cell, correspondingly obtain a series of recombinant escherichia coli strains.Identify recon by pcr amplification and double digestion.Consistent as a result showing: the successful subclone of copper resistant gene is to the pET28a plasmid.The recombination bacillus coli that contains recombinant plasmid pET28a-Cu1 is called e. coli bl21 (DE3)-Cu1 bacterial strain.
The proteic heterogenous expression of copper resistant: will cut recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 bacterial strain of evaluation and e. coli bl21 (DE3)-pET28a that control strain promptly contains the pET28a plasmid by pcr amplification and enzyme, be inoculated in the LB liquid nutrient medium that contains kantlex, 37 ℃, 150rpm cultivates 12h.Bottle is shaken in inoculum size switching with 1%, is cultured to thalline OD
600Reach 0.6-0.8, take out the thalline of 1ml, the centrifugal 5min of 5000rpm abandons supernatant.Adding final concentration in remaining liquid nutrient medium is the IPTG of 1mM, induces 4h for 37 ℃.Carry out centrifugal to the inductive thalline equally.With sterilized water suspend induce preceding and induce after thalline, and add isopyknic 2 * sample-loading buffer simultaneously, mixing, 95 ℃ of thermo-cracking 15min in the centrifugal 5min of 12000rpm, draw the last all product of 101 supernatants as SDS-PAGE.The resolving gel concentration of SDS-PAGE is 12.5%, and concentrated gum concentration is 5%.SDS-PAGE result shows: the albumen of the copper resistant genes encoding of subclone is great expression (Fig. 2) in e. coli bl21 (DE3).
Claims (7)
1, copper resistant gene, its dna sequence dna as shown in Figure 1.
2, copper resistant gene, its encoded protein matter have the described aminoacid sequence of accompanying drawing 1.
3, copper resistant gene as claimed in claim 1 produces gene order total length 1 by husky Bordetella (Serratia sp.) the KMR-3 bacterial strain of thunder, 117bp, the anti-cuproprotein of derivation is encoded by 194 amino acid, is CutF albumen, molecular weight is about 22KDa, and theoretical pI value is 5.30; The highest with Mohs Yersinia (Yersiniamollare tii) ATCC 43969 copper resistant lipoprotein NlpE homologys, reach 70%.
4, the recombinant vectors pUC118-Cu1 that comprises the dna sequence dna of claim 1.
5, the recombinant vectors pET28a-Cu1 that comprises the dna sequence dna of claim 1.
6, recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 that utilizes recombinant vectors pET28a-Cu1 to transform.
7, the preparation method of the described material of claim 1 to 6, extract the chromosomal DNA of serratia marcescens KMR-3 bacterial strain, carry out partially digested with restriction enzyme Sau3A I, glue reclaims enzyme and cuts product, and be connected with pUC118 BamH I/BAP carrier, connect product through ethanol sedimentation, electric Transformed E .coli DH5, coating contains the LB flat board of penbritin, makes up genome dna library; Screen recon containing on the selectivity flat board of penbritin and cupric ion, from recombinant plasmid pUC118-Cu1, isolate the dna fragmentation of cupric resistant gene,, obtain DNA as shown in Figure 1 through digestion with restriction enzyme; Design primer amplification copper resistant gene is connected and transformed into escherichia coli BL21 (DE3) bacterial strain with the pET28a carrier, obtains recombinant plasmid pET28a-Cu1 and recombinant bacterial strain e. coli bl21 (the DE3)-pET28a-Cu1 that contains recombinant expression plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102336257A CN101509000A (en) | 2008-11-24 | 2008-11-24 | Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102336257A CN101509000A (en) | 2008-11-24 | 2008-11-24 | Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101509000A true CN101509000A (en) | 2009-08-19 |
Family
ID=41001532
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008102336257A Pending CN101509000A (en) | 2008-11-24 | 2008-11-24 | Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101509000A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717789B (en) * | 2009-12-14 | 2012-06-20 | 昆明理工大学 | Method for preparing culture medium for efficiently producing haematochrome |
| CN103937703A (en) * | 2014-03-10 | 2014-07-23 | 赵晗 | Serratia marcescens M7a and application of Serratia marcescens M7a in heavy metal ion removing |
-
2008
- 2008-11-24 CN CNA2008102336257A patent/CN101509000A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717789B (en) * | 2009-12-14 | 2012-06-20 | 昆明理工大学 | Method for preparing culture medium for efficiently producing haematochrome |
| CN103937703A (en) * | 2014-03-10 | 2014-07-23 | 赵晗 | Serratia marcescens M7a and application of Serratia marcescens M7a in heavy metal ion removing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Curson et al. | DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria | |
| Barabote et al. | Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations | |
| Elkins et al. | Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species | |
| Garcia et al. | Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton | |
| Chen et al. | Unique origin and lateral transfer of prokaryotic chlorophyll-b and chlorophyll-d light-harvesting systems | |
| Kunath et al. | From proteins to polysaccharides: lifestyle and genetic evolution of Coprothermobacter proteolyticus | |
| Fonseca et al. | Type IV-like pili facilitate transformation in naturally competent archaea | |
| Siezen et al. | Evolution of prokaryotic subtilases: genome‐wide analysis reveals novel subfamilies with different catalytic residues | |
| Xu et al. | Degradation and utilization of alginate by marine Pseudoalteromonas: a review | |
| Ivanova et al. | Complete genome sequence of the extremely Halophilic Halanaerobium praevalens type strain (GSL T) | |
| Bray et al. | Phylogenetic and structural diversity of aromatically dense pili from environmental metagenomes | |
| Wilmes et al. | Cytoplasmic and periplasmic proteomic signatures of exponentially growing cells of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 | |
| Cheng et al. | Isolation, identification, and whole genome sequence analysis of the alginate-degrading bacterium Cobetia sp. cqz5-12 | |
| CN101509000A (en) | Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof | |
| CN108018276A (en) | A kind of deep-sea bacterium keratinase and its encoding gene, zymoprotein production engineering bacterium and application | |
| Riedel et al. | Genome sequence of the orange-pigmented seawater bacterium Owenweeksia hongkongensis type strain (UST20020801 T) | |
| Lylloff et al. | Genomic and exoproteomic analyses of cold‐and alkaline‐adapted bacteria reveal an abundance of secreted subtilisin‐like proteases | |
| CN101712961B (en) | Fe hydrogenase gene and coded amino acid sequence by same and heterologous expression system | |
| Mavromatis et al. | Complete genome sequence of the moderate thermophile Anaerobaculum mobile type strain (NGA T) | |
| Nishio et al. | Analysis of strain-specific genes in glutamic acid-producing Corynebacterium glutamicum ssp. lactofermentum AJ 1511 | |
| Djao et al. | Complete genome sequence of Syntrophothermus lipocalidus type strain (TGB-C1T) | |
| Breider et al. | Genome sequence and emended description of Leisingera nanhaiensis strain DSM 24252 T isolated from marine sediment | |
| Baby et al. | Complete genome sequence of Vibrio gazogenes PB1: an estuarine bacterium capable of producing prodigiosin from starch or cellulose | |
| Pold et al. | Genome sequence of Verrucomicrobium sp. strain GAS474, a novel bacterium isolated from soil. Genome Announc 6: e01451-17 | |
| CN1177038C (en) | Halophilic and alkalophilic bacillus strain and its special plasmid and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20090819 |