CN102876702B - Shuttle expression vector with broad hosts - Google Patents
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- CN102876702B CN102876702B CN201210390124.6A CN201210390124A CN102876702B CN 102876702 B CN102876702 B CN 102876702B CN 201210390124 A CN201210390124 A CN 201210390124A CN 102876702 B CN102876702 B CN 102876702B
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- 239000013604 expression vector Substances 0.000 title abstract 3
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- 230000014509 gene expression Effects 0.000 claims abstract description 14
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a shuttle expression vector with broad hosts and provides a plasmid. The plasmid comprises the following components: a replication origin oriV, a gene for encoding replication protein Rep, a conjugation origin oriT, a gene for encoding conjugated protein Mob, an antibiotic selective marker gene and a foreign gene expression cassette, wherein the foreign gene expression cassette sequentially comprises a promoter, a multiple cloning endonuclease site in which a foreign gene is inserted, and a transcription termination sequence from upstream to downstream. The plasmid has a broad host ability, can be stably replicated in hosts and has high transconjugative frequency. The shuttle expression vector has great value for the genetic transformation of Acidithiobacillus caldus.
Description
Technical field
The present invention relates to a kind of shuttle expression carrier with extensive host.
Background technology
Biological metallurgy is that recent two decades carrys out one of very active subject of field of metallurgy, aspect the extraction of the metals such as copper, gold, uranium, is realizing industrial application.Biological metallurgy is that metalliferous mineral oxidation, reduction or complexing are decomposed in effect by utilizing microorganism, makes metal ion enter solution, further separation, enrichment, purifying and extract the technology of metal.Biological metallurgy, compared with conventional physics, chemical flotation and metallurgy method, has the outstanding advantages such as process is simple, cost is low, energy consumption is low, environmental pollution is little, be particularly suitable for lean ore, abandoned mine, unbalanced-ore and difficultly adopt, dump leaching and the Stope leaching in difficult choosing, difficult smelting ore deposit.Current resources requirement is large, easy exploiting resource is more and more in short supply, environmental requirement is improve constantly in the situation that, the application of microbial metallurgy technology has represented distinctive superiority and wide development space.
Acidithiobacillus caldus (Acidithiobacillus caldus) is distributed widely in Sulphide Ore, acid mine water and soil, plays an important role at the aspect such as processing, natural sulphur cycle of bacteriosmelt, coal desulfurization and sulfur-containing waste water.In the time that it and iron are oxidized metallurgical microorganism mixed culture, can effectively promote the stripping of metal ion, thereby improve metallurgical efficiency.Therefore, Acidithiobacillus caldus is a kind of important biological metallurgy microorganism.But in actual application, this bacterium exists poor growth, cell yield low and to shortcomings such as metallurgy environment bad adaptability, has seriously limited its range of application.Therefore, give full play to the function of Acidithiobacillus caldus at biological metallurgy, it be carried out to genetic modification imperative.
For realizing further investigation and the function modifications to Acidithiobacillus caldus, build a platform stable and effectively genetic operating system and be very important.Genetic operating system engaging frequency used in prior art is mostly below 10-4, affect the genetic manipulation efficiency (particularly knocking out the efficiency of plasmid) of Acidithiobacillus caldus, therefore, a kind of genetic operating system efficiently of exploitation is relevant scholar's problem demanding prompt solution.
Summary of the invention
The object of this invention is to provide a kind of shuttle expression carrier with extensive host.
The invention provides a kind of plasmid, comprise following element: the encoding gene of replication origin oriV, replication protein Rep, encoding gene and the exogenous gene expression box of joint starting point oriT, adaptor protein Mob; Described exogenous gene expression box comprises successively promotor, inserts for foreign gene to downstream polyclone restriction enzyme site (MCS) and transcription termination sequence from upstream.
Described replication origin oriV is if the sequence 1 of sequence table is from as shown in the 2041st to 2530 Nucleotide of 5 ' end.
Described joint starting point oriT is if the sequence 1 of sequence table is from as shown in the 4234th to 4235 Nucleotide of 5 ' end.
Described replication protein Rep is made up of replication protein RepA, replication protein RepB and replication protein RepC.The reverse complementary sequence of the encoding gene of described replication protein RepC is if the sequence 1 of sequence table is from as shown in the 81st to 755 Nucleotide of 5 ' end.The encoding gene of described replication protein RepB is if the sequence 1 of sequence table is from as shown in the 6337th to 7425 Nucleotide of 5 ' end.The reverse complementary sequence of the encoding gene of described replication protein RepA is if the sequence 1 of sequence table is from as shown in the 946th to 1803 Nucleotide of 5 ' end.
Described adaptor protein Mob is by adaptor protein MobE, adaptor protein MobD, in conjunction with albumen MobC, form in conjunction with albumen MobB with in conjunction with albumen MobA.The reverse complementary sequence of the encoding gene of described adaptor protein MobE is if the sequence 1 of sequence table is from as shown in the 2461st to 3105 Nucleotide of 5 ' end.The reverse complementary sequence of the encoding gene of described adaptor protein MobD is if the sequence 1 of sequence table is from as shown in the 3065th to 3748 Nucleotide of 5 ' end.The reverse complementary sequence of the encoding gene of described combination albumen MobC is if the sequence 1 of sequence table is from as shown in the 3761st to 4159 Nucleotide of 5 ' end.The encoding gene of described combination albumen MobB is if the sequence 1 of sequence table is from as shown in the 4227th to 4739 Nucleotide of 5 ' end.The encoding gene of described combination albumen MobA is if the sequence 1 of sequence table is from as shown in the 4897th to 7425 Nucleotide of 5 ' end.
Described promotor specifically can be the promotor of tetrathionate hydrolase gene tetH.The promotor of described tetrathionate hydrolase gene tetH is if the sequence 3 of sequence table is from as shown in the 76th to 156 Nucleotide of 5 ' end.
Described transcription termination sequence is if the sequence 5 of sequence table is from as shown in the 51st to 100 Nucleotide of 5 ' end.
Described multienzyme is cut cloning site (MCS) specifically can be as shown in the sequence of sequence table 4.
Described plasmid also can comprise more than one screening-gene.Described screening-gene can be kalamycin resistance gene and/or Streptomycin sulphate screening-gene.Described kalamycin resistance gene specifically can be if the sequence of sequence table 2 be from as shown in the 469th to 1263 Nucleotide of 5 ' end.Described Streptomycin sulphate screening-gene (aadA gene) specifically can be if the sequence of sequence table 6 be from as shown in the 132nd to 950 Nucleotide of 5 ' end.
Described plasmid specifically can be as shown in the sequence of sequence table 7.
Described foreign gene specifically can be as shown in the sequence of sequence table 8.
Replication origin oriV and replication protein Rep make plasmid have extensive host's ability.Engaging starting point oriT and adaptor protein Mob makes plasmid have the ability of efficient conjugal transfer.The promotor of tetrathionate hydrolase gene tetH can be by the transcription factor identification in Acidithiobacillus caldus (Acidithiobacillus caldus) SM-1, and making to insert gene can start and transcribe in Acidithiobacillus caldus (Acidithiobacillus caldus) SM-1.
The present invention also protects a kind of method (method first) of expression alien gene, comprises the steps:
(1) foreign gene is inserted to arbitrary multiple clone site of the above plasmid, obtain recombinant plasmid;
(2) by described recombinant plasmid transformed Host Strains and express described foreign gene in Host Strains.
In described method first, described Host Strains can be Acidithiobacillus caldus (Acidithiobacillus caldus), specifically can be Acidithiobacillus caldus (Acidithiobacillus caldus) SM-1.In described method first, described Host Strains can be intestinal bacteria, as intestinal bacteria SM10.
The present invention also protects the method (method second) of another kind of expression alien gene, comprises the steps:
(1) foreign gene is inserted to arbitrary multiple clone site of the above plasmid, obtain recombinant plasmid;
(2) by described recombinant plasmid transformed Host Strains, obtain donor bacterium;
(3) by cultivating altogether, the described recombinant plasmid in described donor bacterium is imported to described recipient bacterium, obtain recombinant bacterium;
(4) cultivate described recombinant bacterium, express described foreign protein.
In described method second, described Host Strains can be intestinal bacteria, as intestinal bacteria SM10.In described method second, described recipient bacterium can be Acidithiobacillus caldus (Acidithiobacillus caldus), specifically can be Acidithiobacillus caldus (Acidithiobacillus caldus) SM-1.
The present invention also protects the application of above arbitrary described plasmid in the genetic transformation of Acidithiobacillus caldus.
Described Acidithiobacillus caldus specifically can be Acidithiobacillus caldus (Acidithiobacillus caldus) SM-1.
Plasmid provided by the invention has extensive host's ability, can in host, stablize and copy, and have high conjugal transfer frequency.The present invention has great value for the genetic transformation of Acidithiobacillus caldus.
Brief description of the drawings
Fig. 1 is the fluoroscopic examination result of pLAtcE-eGFP after expression in escherichia coli
Fig. 2 is the fluoroscopic examination result after pLAtcE-eGFP expresses in Acidithiobacillus caldus.
Fig. 3 is the result of pcr amplification gene identification plasmid.
Fig. 4 is the structural representation of recombinant plasmid pLAtcE.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.PET-28a-c (+) carrier: Zhengzhou Weirui Biotechnology Co., Ltd., 69864-3.Intestinal bacteria SM10:Biomedal, BS3303.Acidithiobacillus caldus (Acidithiobacillus caldus) SM-1: reference: You XY, Guo X, Zheng HJ, Zhang MJ, Liu LJ, Zhu YQ, Zhu B, Wang SY, Zhao GP, Poetsch A, Jiang CY, Liu SJ.Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation.J Genet Genomics.2011,38 (6): 243-52.
The structure of embodiment 1, shuttle expression carrier (recombinant plasmid pLAtcE)
One, build plasmid pLAtc-Km
r
1, the double chain DNA molecule (8144bp) shown in the sequence 1 of composition sequence table.In the sequence 1 of sequence table, be the reverse complementary sequence of the encoding gene of replication protein RepC from the 81st to 755 Nucleotide of 5 ' end, the 946th to 1803 Nucleotide are the reverse complementary sequence of the encoding gene of replication protein RepA, the 2041st to 2530 Nucleotide are replication origin oriV, the 2461st to 3105 Nucleotide are the reverse complementary sequence of the encoding gene of adaptor protein MobE, the 3065th to 3748 Nucleotide are the reverse complementary sequence of the encoding gene of adaptor protein MobD, the 3761st to 4159 Nucleotide are the reverse complementary sequence in conjunction with the encoding gene of albumen MobC, the 4234th to 4235 Nucleotide are for engaging starting point oriT, the 4227th to 4739 Nucleotide are the encoding gene in conjunction with albumen MobB, the 4897th to 7425 Nucleotide are the encoding gene in conjunction with albumen MobA, the 6337th to 7425 Nucleotide are the encoding gene of replication protein RepB.
2, the double chain DNA molecule (1333bp) shown in the sequence 2 of composition sequence table.In the sequence 2 of sequence table, the 5th ' the 469th to 1263 Nucleotide of end are kalamycin resistance gene.
3, be template by double chain DNA molecule synthetic step 1 and the synthetic double chain DNA molecule of step 2 simultaneously, adopt the primer pair of F1 and R1 composition to carry out pcr amplification, obtain double-stranded circular DNA molecule (9426bp), called after recombinant plasmid pLAtc-Km
r.
F1：5’-CTTTCTCCAGCAGCGATTCCGCAGTAACCCTATCTCGGTCTATTCTTTTG-3’；
R1：5’-GGGTGAGTGGCATTCGGTTGTTATCTGGCGACACGGAAATGTTGAATAC-3’。
Two, build APMT fragment
1, the double chain DNA molecule (168bp) shown in the sequence 3 of composition sequence table.In the sequence 3 of sequence table, be the promotor of the tetrathionate hydrolase gene tetH in Acidithiobacillus caldus SM-1 genome from the 76th to 156 Nucleotide of 5 ' end.
2, the double chain DNA molecule (218bp) shown in the sequence 4 of composition sequence table.In the sequence 4 of sequence table, be EcoRI restriction endonuclease recognition sequence from the 25th to 30 Nucleotide of 5 ' end, the 59th to 64 Nucleotide are HindIII restriction endonuclease recognition sequence, 65-70 position Nucleotide is KpnI, Acc65I or Asp718I restriction endonuclease recognition sequence, the 77th to 82 Nucleotide are BamHI restriction endonuclease recognition sequence, the 118th to 123 Nucleotide are EcoRV restriction endonuclease recognition sequence, the 134th to 141 Nucleotide are NotI restriction endonuclease recognition sequence, the 141st to 146 Nucleotide are XhoI or SciI restriction endonuclease recognition sequence, the 193rd to 198 Nucleotide are MluI restriction endonuclease recognition sequence, the 197th to 202 Nucleotide are HpaI restriction endonuclease recognition sequence.
3, the double chain DNA molecule (130bp) shown in the sequence 5 of composition sequence table.In the sequence 5 of sequence table, be transcription termination sequence from the 51st to 100 Nucleotide of 5 ' end.
4, the double chain DNA molecule (1011bp) shown in the sequence 6 of composition sequence table.In the sequence 6 of sequence table, be Streptomycin sulphate screening-gene (aadA gene) from the 132nd to 950 Nucleotide of 5 ' end.
5, simultaneously using double chain DNA molecule synthetic to synthetic to double chain DNA molecule synthetic step 1, step 2 double chain DNA molecule, step 3 and the synthetic double chain DNA molecule of step 4 as template, carry out pcr amplification with the primer pair of F2 and R2 composition, obtain pcr amplification product (1471bp, called after APMT fragment).
F2：5’-CGTG
CCTAGGTCTAGATGAAAATGCCG-3’；
R2：5’-AAG
GACGTCTCGCCAATCCGGATATAGTTC-3’。
Three, construction recombination plasmid pLAtcE
1, the recombinant plasmid pLAtc-Km building with step 1
rfor template, carry out pcr amplification with the primer pair of F3 and R3 composition, obtain pcr amplification product (about 9412bp).
F3：5’-ATC
GACGTCCAACCGAATGCCACTCACCC-3’；
R3：5’-TCA
CCTAGGCAGCGACACGGAAATGTTGAATACTC-3’。
2, the pcr amplification product obtaining by restriction enzyme A vrII and AatII double digestion step 1, reclaims enzyme and cuts product.
3, the APMT fragment obtaining by restriction enzyme A vrII and AatII double digestion step 2, reclaims enzyme and cuts product.
4, the enzyme of the enzyme of step 2 being cut to product and step 3 is cut product and is connected, and obtains recombinant plasmid.Recombinant plasmid pLAtcE.The sequencing result of recombinant plasmid pLAtcE is as shown in the sequence 7 of sequence table (10885bp).The structural representation of recombinant plasmid pLAtcE is shown in Fig. 4.
The application of embodiment 2, shuttle expression carrier (recombinant plasmid pLAtcE)
One, construction recombination plasmid pLAtcE-eGFP
1, the double chain DNA molecule shown in the sequence 8 of composition sequence table (eGFP gene).
2, taking the synthetic double chain DNA molecule of step 1 as template, carry out pcr amplification with the primer pair of F4 and R4 composition, obtain pcr amplification product.
F4：5’-CCC
GAATTCATGGTGAGCAAGGGCGCCGAGCTGTTC-3’；
R4：5’-CCC
AAGCTTTCACTTGTACAGCTCATCCATGCCGTG-3’。
3, with the pcr amplification product of restriction enzyme EcoRI and HindIII double digestion step 2, reclaim enzyme and cut product.
4, with restriction enzyme EcoRI and HindIII double digestion recombinant plasmid pLAtcE, reclaim carrier framework.
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid pLAtcE-eGFP.According to sequencing result, recombinant plasmid pLAtcE-eGFP is carried out to structrual description as follows: between the EcoRI of recombinant plasmid pLAtcE and HindIII restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 8 of sequence table.
Two, build recombinant bacterium
Recombinant plasmid pLAtcE-eGFP is imported to intestinal bacteria SM10, obtain recombinant bacterium.
Three, the fluoroscopic examination of recombinant bacterium
1, recombinant bacterium step 2 being built is seeded in the LB liquid nutrient medium containing 50 μ g/ml kantlex and 50 μ g/ml Streptomycin sulphates, and under 37 DEG C of conditions, 200rpm cultivates 3h, obtains OD
600nm=0.4 culture system.
2, in the culture system of step 1, add potassium tetrathionate (making its concentration is 3mg/ml), 37 DEG C, 200rpm shaking culture 3h.
A situation arises for the eGFP fluorescence of the culture system 3, obtaining with fluorescent microscope detecting step 2.Fig. 1 is shown in by photo, can observe green fluorescence.
Four, the conjugal transfer ability of recombinant bacterium to Acidithiobacillus caldus
Starky liquid nutrient medium: containing 3g/L (NH
4)
2sO
4, 3g/L KH
2pO
4, 0.5g/L MgSO
47H
2o, 0.25g/LCaCl
22H
2o and 10g/L sulphur, other is water.
Washings: containing 3g/L (NH
4)
2sO
4, 3g/L KH
2pO
4, 0.5g/L MgSO
47H
2o and 0.25g/LCaCl
22H
2o, other is water.
The preparation method of Starky solid medium: solution A: (NH
4)
2sO
40.6g, KH
2pO
40.6g, MgSO
47H
2o0.1g, CaCl
22H
2o 0.05g, adding distil water is to 100ml, natural pH4.8,15 pounds/cm
2sterilizing 20min; Solution B: agar powder 2g, distilled water 100mL, 15 pounds/cm
2sterilizing 20min; Solution C: K
2s
4o
60.15g/mL, all the other are water, filtration sterilization; Solution A and solution B are cooled to 80 DEG C of mixing after boiling respectively, then add 4mL solution C to mix, and prepare solid plate.
Intestinal bacteria-Acidithiobacillus caldus engages substratum: at Starky-K
2s
4o
6in solid medium, add yeast powder, making its concentration is 0.05g/100ml.
The recombinant bacterium obtaining taking step 2 is donor bacterium, taking Acidithiobacillus caldus SM-1 as recipient bacterium, on the filter membrane (filter membrane is placed on above substratum) of intestinal bacteria-Acidithiobacillus caldus joint substratum, engages, and concrete steps are as follows:
1, under 37 DEG C of conditions, cultivate donor bacterium to the logarithm later stage, centrifugal collection donor bacterium, with washings washing 2 times.
2, under 40 DEG C of conditions, cultivate recipient bacterium to stationary phase, centrifugal collection donor bacterium, with washings washing 2 times.
3, with the resuspended donor bacterium of washings, obtaining donor bacteria concentration is the donor bacterium bacterium liquid of 100 μ g/mL, with the resuspended recipient bacterium of washings, obtaining recipient bacterium concentration is the recipient bacterium bacterium liquid of 100 μ g/mL, donor bacterium bacterium liquid and recipient bacterium bacterium liquid are mixed according to the volume ratio of 1:2, then coat on the nitrocellulose filter of intestinal bacteria-Acidithiobacillus caldus joint culture medium flat plate, cultivate 72h for 37 DEG C, then wash flat board and collect thalline with washings, with being coated with and being contained in the Starky solid medium flat board that contains 50 μ g/ml kantlex and 50 μ g/ml Streptomycin sulphates after the dilution of Starky liquid nutrient medium, the bacterium colony growing is the positive bacterium colony of microbiotic.
The quantity of the positive bacterium colony of microbiotic of engaging frequency=obtain/for carrying out quantity=1.50 × 10 of donor bacterium of this experiment
-3.
4, microbiotic positive bacteria is dropped into performing PCR qualification.
Primer pair for PCR qualification eGFP gene is as follows:
CCCGAATTCATGGTGAGCAAGGGCGCCGAGCTGTTC；
CCCAAGCTTTCACTTGTACAGCTCATCCATGCCGTG；
Target sequence fragment is 719bp.
For the identification of Km
rthe primer pair of gene is as follows:
AACCCTATCTCGGTCTATTCTTTTG；GCGACACGGAAATGTTGAATAC；
Target sequence clip size is 1280bp.
For the identification of Sm
rthe primer pair of gene is as follows:
GCCTAGGTCTAGATGAAAAT，AACTGCAGGAGCTCAGCCAATCGACTG；
Target sequence clip size is 985bp.
Result all obtains the positive band of suitable size.
Reclaim each object band order-checking, turn out to be eGFP gene, Km
rgene Sm
rgene order.
5, positive microbiotic bacterium colony is coated with after flat board at fluorescence microscopy Microscopic observation, result has green fluorescence, as shown in Figure 2.
6, from the positive bacterium colony of microbiotic, extract plasmid and transform intestinal bacteria SM10, on the LB solid medium flat board containing 50 μ g/ml kantlex and 50 μ g/ml Streptomycin sulphates, cultivate, picking positive colony enlarged culturing is also extracted plasmid and is carried out the PCR qualification that step 4 is identical.
The results are shown in Figure 3, all obtain the positive band of suitable size.
Reclaiming each object band order-checking, is eGFP gene, Km really
rgene Sm
rgene order.
Claims (4)
1. a plasmid, as shown in the sequence 7 of sequence table.
2. a method for expression alien gene, comprises the steps:
(1) foreign gene is inserted to the multiple clone site of plasmid described in claim 1, obtain recombinant plasmid;
(2) by described recombinant plasmid transformed Host Strains and express described foreign gene in Host Strains.
3. a method for expression alien gene, comprises the steps:
(1) foreign gene is inserted to the multiple clone site of plasmid described in claim 1, obtain recombinant plasmid;
(2) by described recombinant plasmid transformed Host Strains, obtain donor bacterium;
(3) by cultivating altogether, the described recombinant plasmid in described donor bacterium is imported to recipient bacterium, obtain recombinant bacterium;
(4) cultivate described recombinant bacterium, express described foreign gene.
4. the application of plasmid claimed in claim 1 in the genetic transformation of Acidithiobacillus caldus.
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| Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation;Xiao-Yan You 等;《JOURNAL OF GENETICS AND GENOMICS》;20111231;第38卷;第243-252页 * |
| Xiao-Yan You 等.Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation.《JOURNAL OF GENETICS AND GENOMICS》.2011,第38卷第243-252页. |
| 嗜酸性硫杆菌高效接合转移质粒载体及抗砷工程菌的构建;蒙建州;《中国优秀硕士学位论文全文数据库 基础科学辑》;20100430;第A006-29页 * |
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