CN107760703A - A kind of flat end cloning vector pUB857 of zero background and its construction method and application - Google Patents
A kind of flat end cloning vector pUB857 of zero background and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of flat end cloning vector pUB857 of zero background structures and its application in gene cloning and construction of gene library.The present invention is by using Gibson Assembly technologies, by the cI857 P that PCR amplifications obtain respectivelyRPromoter and ccdB genetic fragments are connected with the pUC19 plasmids handled through PvuII digestions, obtain flat end cloning vector pUB857.Flat end cloning vector pUB857 can clone any PCR primer, and clone's positive rate can reach 100% during using ccdB as anti-selection markers.In addition, the cloning vector can also be used for the efficient structure of genomic library, while effectively empty carrier can be avoided to disturb, be genome times afterwards comprehensively high flux genescreen and a kind of effective tool of functional verification.
Description
Technical field
The present invention relates to a kind of flat end cloning vector pUB857 of zero background and its construction method and application, and in particular to
One kind uses temperature sensitive type promoter cI857-PRToxin gene ccdB expression is controlled in this, as pUB857 grams of anti-selection markers
Grand carrier and its application in gene cloning and construction of gene library, belong to biological technical field.
Technical background
With the development of modern biotechnology, various molecule clone technologies arise at the historic moment.Molecule clone technology, i.e., in molecule
Specific dna sequence is cloned on level to express the method for target protein, target DNA is obtained frequently with digestion or PCR mode
Fragment, target DNA sequence is inserted into cloning vector with the method for vitro recombination after purification, matter then will be recombinated by transform mode
Grain is imported in suitable host bacterium, such as DH5 α, DH10B etc., then weight of the extraction needed for digestion identification from the Host Strains of screening
Group plasmid, finally obtains target gene.The clone of gene typically uses following technology：Digestion-enzyme connects, fusion DNA vaccine and homologous
Restructuring.Traditional recombinant clone screening depends on cumbersome time-consuming bacterium colony PCR or digestion verification, it is difficult to meets current high flux base
Because screening the requirement with functional verification.Use toxin gene as anti-selection markers to avoid the interference of empty carrier, can significantly subtract
Lack workload and shorten working hours.
DNA gyrase is the type topoisomerase of one kind 2 in bacterium, is existed in the form of the tetramer, respectively by gyrA and
The 2 A subunits and 2 B subunits of gyrB coded by said gene are formed.The enzyme is in DNA molecular by supercoil state change into relaxation
Played a decisive role during type state, be to maintain DNA molecular to be stabilized a kind of necessary enzyme.Wherein, ccdB is by 101
A kind of gene toxin albumen coded by individual amino acid, can with e. coli dna gyrase directly in conjunction with, it is lost activity,
So as to cause DNA molecular not produce supercoil, so as to which the DNA of oneself cutting can not be repaired, Apoptosis is ultimately resulted in
(Bernard P,Couturier M.Cell killing by the F plasmid CcdB protein involves
poisoning of DNA-topoisomerase II complexes[J].Journal of molecular biology,
1992,226(3):735-745.).Temperature sensitive type promoter PR is regulated and controled by aporepressor cI857, when temperature is less than 30 DEG C, resistance
Hold back the expression that albumen cI857 can stably suppress ccdB genes, and when temperature is at 37 DEG C, aporepressor cI857 is because of heat inactivation
And cause toxin gene ccdB expression (Villaverde A, et al.Fine regulation of cI857-
controlled gene expression in continuous culture of recombinant Escherichia
coli by temperature[J].Applied and environmental microbiology,1993,59(10):
3485-3487.)。
The content of the invention
For the practical problem and demand in gene cloning, the invention provides a kind of flat end cloning vector of zero background
PUB857, the flat end of zero background quickly connect carrier temperature sensitive type promoter cI857-PRControl toxin gene ccdB expression
In this, as the pUB857 cloning vectors of anti-selection markers, with the flat end PCR primer of high-efficient cloning and any there can be flat end
DNA fragmentation.
Technical scheme is as follows：
The flat end cloning vector pUB857 of a kind of zero background, to form linear DNA using PvuII digestion pUC19 plasmids
Chain, while connect ccdB and cI857-P in restriction enzyme siteRThe full length sequence of formation is SEQ ID NO.9 cloning vector, described
CcdB ORFs on be designed with SmaI restriction enzyme sites.
Further, the present invention also provides the flat end cloning vector pUB857 of above-mentioned zero background construction method, including
Design of primers, gene cloning and vector construction, cI857-P is expanded by synthetic primer PCRRThe piece of purpose fragment and ccdB mesh
Section, and pUC19 DNAs are subjected to digestion with restriction enzyme PvuII, linearized fragment pUC19 is reclaimed, finally will
ccdB、cI857-PR, linearisation pUC19 fragments be attached, obtain cloning vector pUB857.
The present invention utilizes Thermo-sensitive promoter element cI857-PRBacteriotoxin gene ccdB expression is controlled, and in ccdB
SmaI restriction enzyme sites are designed on ORFs, for cloning any DNA fragment.When target DNA fragment insertion ccdB's
The expression of original toxin gene can be destroyed after SmaI restriction enzyme sites, thus can make transformant in 37 DEG C of normal growths, and from connect product
Raw empty carrier, its transformant meeting great expression toxin gene ccdB when cultivating for 37 DEG C, causes Apoptosis, so as to realize
Clone's effect of " zero background ".The flat end cloning vector pUB857 of zero background of the present invention can be used for cloning any PCR primer, and
And clone's positive rate can reach 100% during using ccdB as anti-selection markers.In addition, the cloning vector can also be used for genome
The efficient structure in library, while effectively empty carrier can be avoided to disturb, it is that genome times afterwards comprehensively high flux genescreen is tested with function
A kind of effective tool of card.
Brief description of the drawings
Fig. 1 is the digestion verification figure of pUB857 carriers, and M represents that marker, a represent pUB857 carriers through NdeI single endonuclease digestions
Band, b represent band of the pUB857 carriers through NdeI and PciI double digestions.
Fig. 2 is the pUB857 plasmid maps that structure is completed.
Fig. 3 is DH5 α/pUB857 respectively in 30 DEG C (a) and the growth curve of 37 DEG C (b).
Fig. 4 is DH5 α/pUB857 upgrowth situations on 30 DEG C (a) and 37 DEG C of (b) culture plates respectively.
Fig. 5 be pUB857-ble transformants bleomycin resistance the result, Amp100 (a), Ble50 (b).
Fig. 6 is the bacterium colony PCR the result figures using pUB857 carrier cloning Bacillus cercus 16srRNA genes.
Fig. 7 is the restriction enzyme mapping being used for using pUB857 carriers after Bacillus cercus construction of gene library.
Embodiment
For the scope of application that the present invention is furture elucidated, the present invention is illustrated with accompanying drawing with reference to embodiments,
But the application of the carrier does not limit to the present invention and determines scope.
Plasmid pCP20 of the present invention, pUC19, E.coli Top10F ' and bacillus coli DH 5 alpha are purchased by business
Buy.
Embodiment one：A kind of flat end cloning vector pUB857 of zero background construction method
A kind of flat end cloning vector pUB857 of zero background construction method, comprises the following steps：
(1) design of primers：Engineer synthesizes specific primer sequence
CIFor：5’-tggccgattcattaatgcagaccagaacaccttgccgatc-3’；
CIRev：5’-gtgtaaaccttaaactgcatgctatacaacctccttagtacatgc-3’
ccdBFor:5’-gcatgtactaaggaggttgtatagcatgcagtttaaggtttacac-3’；
ccdBRev:5’-tcttcgctattacgccagctatattccccagaacatcagg-3’
(2) PCR is expanded：PCR reaction systems are prepared as shown in table 1.
Table 1PCR reaction systems are with tabulation
| Reagent name | Storing solution solubility | Add the volume (μ L) of PCR reaction systems |
| 5×Q5PCR buffer | 5× | 10 |
| 5×enhancer | 5× | 10 |
| dNTP | 10mmol/L | 1 |
| Forward primer | 10μmol/L | 2.5 |
| Reverse primer | 10μmol/L | 2.5 |
| Q5 enzymes | 5U/μL | 0.5 |
| DNA profiling | 20ng/μL | 2 |
| ddH2O | Add ddH2O to the μ L of final volume 50 |
PCR programs are 98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 circulate；
Last 72 DEG C of extensions 10min.It is template and CIFor/CIRev primers expanding cI857-P to use pCP20 DNAsRPurpose
Fragment, and it is template and ccdBFor/ccdBRev primers expanding to use the genomic DNAs of E.coli Top10F ' bacterial strains
CcdB purpose fragments.The ccdB purpose fragments length is 346bp, cI857-PRPurpose fragment length is 872bp.
Vector construction：By the ccdB and cI857-P of above-mentioned amplificationRPurpose fragment is divided by agarose gel electrophoresis
From, and gel extraction.PUC19 DNAs are subjected to digestion, glue reclaim linearized fragment with restriction enzyme PvuII
pUC19.By ccdB, cI857-PR, pUC19 fragments be attached with Gibson Assembly mode, and by connection product turn
Change into bacillus coli DH 5 alpha, be coated on the ampicillin/LB plates containing 100 μ g/mL, cultivate 18h in 30 DEG C, select
It is cloned in ampicillin liquid culture medium, cultivates 8h at 30 DEG C, extract plasmid, and tested with Restriction enzyme Sma I digestion
Card, obtain cloning vector pUB857.
Agarose gel electrophoresis referring to《Molecular cloning》The method of middle agarose gel electrophoresis, glue reclaim use Qiagen glue
QIAquick Gel Extraction Kit, chemical transformation referring to《Molecular cloning》Middle calcium chloride prepares and the method for competence conversion Escherichia coli.Even
Junctor system and method are referring to Gibson Assembly.Size of the plasmid after NdeI single endonuclease digestions is 3502bp nucleic acid fragment, is passed through
Size after NdeI and PciI double digestions is respectively 2062bp and 1440bp nucleic acid fragment, and proof diagram is as shown in Figure 1.Build
Into pUB857 plasmid maps it is as shown in Figure 2.Bacillus coli DH 5 alpha containing pUB857 plasmids is when 30 DEG C (a) and 37 DEG C are cultivated
Growth curve it is as shown in Figure 3.Bacillus coli DH 5 alpha containing pUB857 plasmids is on 30 DEG C (a) and 37 DEG C of (b) culture plates
Upgrowth situation is as shown in Figure 4.
Embodiment two：A kind of flat end cloning vector pUB857 of zero background is used to clone answering for bleomycin resistance gene
With
A kind of applications of the flat end cloning vector pUB857 of zero background for cloning bleomycin resistance gene, including with
Lower step：
(1) Bacteria Culture：The DH5 α containing plasmid pUB857 are inoculated with the μ g/ml of ammonia benzyl 100 fluid nutrient medium, in 30
DEG C culture 8h；The DH5 α containing plasmid pDGICZ are inoculated with the μ g/ml of bleomycin 50 fluid nutrient medium, in 37 DEG C of cultures
8h.Two kinds of plasmids of pUB857 and pDGICZ are extracted respectively.
(2) design of primers：Engineer synthesizes specific primer sequence
BleFor：5’-tggtctgatcggatcctcag-3’
BleRev：5’-gttaggatccttgatatggc-3’
(3) PCR is expanded：PCR reaction systems are 5 × Q5PCR buffer 10 μ L, 5 × enhancer 10 μ l, dNTP
(10mmol/L) 1 μ L, primer (10 μm of ol/L) each 2.5 μ L, DNA (about 20ng/ μ L) 1 μ L, Q5DNA polymerase (5U/ μ L)
0.5 μ L, add H2O to 50 μ L.PCR programs are 98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s,
30 circulations；Last 72 DEG C of extensions 10min.PDGICZ DNAs are used as template and BleFor/BleRev primers, to expand
Increase bleomycin resistance gene (BleR), the target DNA fragments are 420bp.
(4) carrier digestion：PUB857 DNAs are subjected to digestion, glue reclaim linearisation piece with Restriction enzyme Sma I
Section pUB857.
(5) blunt end cloning：PUB857 will be linearized with BleR fragments with mol ratio 1:3 ratio is attached, in T4
In the presence of ligase, 1h is connected in 16 DEG C, is then transformed into DH5 α Competent cells, 12h is cultivated in 37 DEG C.
Result of the test shows：During using ccdB genes as selection markers, bleomycin resistance fragment is only successively inserted into
Clone can be survived under 37 DEG C of condition of culture, and from even carrier can because ccdB genes expression and apoptosis；Turning
60 monoclonals are selected on the Amp flat boards of change at random, are rule on bleomycin solid medium, as a result display is made using ccdB
For selection markers so that positive colony rate has reached 100%, and the result is as shown in Figure 5.
Embodiment three：A kind of flat end cloning vector pUB857 of zero background is used to clone Bacillus cercus 16srRNA
Gene
A kind of flat end cloning vector pUB857 of zero background is used to clone Bacillus cercus 16srRNA genes, including
Following steps：
(1) Bacteria Culture：The DH5 α containing pUB857 plasmids are inoculated with the μ g/ml of ammonia benzyl 100 fluid nutrient medium, in 30
DEG C culture 8h；Bacillus cercus is inoculated with LB fluid nutrient mediums simultaneously, 8h is cultivated in 37 DEG C.PUB857 plasmids are extracted respectively
DNA and Bacillus cercus genomic DNA.
(2) design of primers：16S universal primers
27F：5’-agagtttgatcctggctcag-3’
1492R：5’-ggttaccttgttacgactt-3’
(3) PCR is expanded：PCR reaction systems are 5 × Q5PCR buffer 10 μ L, 5 × enhancer 10 μ l, dNTP
(10mmol/L) 1 μ L, primer (10 μm of ol/L) each 2.5 μ L, DNA (about 20ng/ μ L) 1 μ L, Q5DNA polymerase (5U/ μ L)
0.5 μ L, add H2O to 50 μ L.PCR programs are 98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s,
30 circulations；Last 72 DEG C of extensions 10min.Use Bacillus cercus genomic DNA for template and 27F/1492R primers to
Expand the 16srRNA genetic fragments that size is 1.6kb.
(4) carrier digestion：PUB857 DNAs are subjected to digestion, glue reclaim linearisation piece with Restriction enzyme Sma I
Section pUB857.
(5) blunt end cloning：PUB857 will be linearized with 16srDNA fragments with mol ratio 1:3 ratio is attached,
In the presence of T4 ligases, 1h is connected in 16 DEG C, is then transformed into DH5 α Competent cells, cultivating 12h in 37 DEG C is
Can.
Result of the test shows：Utilize responsive to temperature type promoter cI857-PRTo control ccdB genes to be marked as reversely screening
Note, the clone that as a result display is successively inserted into bacillus 16srDNA fragments can survive under 37 DEG C of condition of culture, connect certainly
Carrier can because toxin gene ccdB expression and apoptosis；6 monoclonals are selected at random in Amp converts flat board carries out bacterium colony
PCR verifies that the result is the positive, it was demonstrated that ccdB positive colonies rate is 100%, and electrophoresis result is as shown in Figure 6.
Example IV：A kind of flat end cloning vector pUB857 of zero background is used for the application of genomic library construction
A kind of flat end cloning vector pUB857 of zero background is used for the application of genomic library construction, including following step
Suddenly：
(1) Bacteria Culture：The DH5 α containing plasmid pUB857 are inoculated with the μ g/ml of ammonia benzyl 100 fluid nutrient medium, in 30
DEG C culture 8h；Bacillus cercus is inoculated with LB fluid nutrient mediums simultaneously, 8h is cultivated in 37 DEG C.PUB857 plasmids are extracted respectively
With Bacillus cercus genomic DNA.
(2) carrier digestion：PUB857 DNAs are subjected to digestion, glue reclaim linearisation piece with Restriction enzyme Sma I
Section pUB857.
(3) genomic DNA digestion：Genomic DNA is subjected to partially digested, glue reclaim DNA with restriction enzyme A luI
Fragment.
(4) blunt end cloning：Will linearisation pUB857 with through Bacillus cercus genomic DNA partially digested AluI with
Mol ratio 1:3 ratio is attached, and in the presence of T4DNA ligases, 1h is connected in 16 DEG C, is then transformed into DH5 α chemistry
In competent cell, 12h is cultivated in 37 DEG C.
Result of the test shows：Utilize responsive to temperature type promoter cI857-PRTo control toxin gene ccdB as reversely sieve
Choosing mark, being successively inserted into the clone of DNA fragmentation can survive under 37 DEG C of condition of culture, and the carrier connected certainly can be because toxin base
The apoptosis because of ccdB expression；Random to select 10 clone's progress digestion verifications, restriction enzyme mapping shows that all clones contain
40kb or so Insert Fragments (as shown in Figure 7).It is used for joint efficiency during construction of gene library using the flat ends vectors of pUB857
Up to 107cfu/μg。
Claims (4)
1. the flat end cloning vector pUB857 of a kind of zero background, it is characterised in that to utilize PvuII digestion pUC19 plasmid shapes
Linear DNA, while connect ccdB and cI857-P in restriction enzyme siteRThe full length sequence of formation is SEQ IDNO.9 clone
Carrier, SmaI restriction enzyme sites are designed with described ccdB ORFs.
2. the flat end cloning vector pUB857 of zero background as claimed in claim 1 construction method, it is characterised in that pass through
Synthetic primer PCR expands cI857-PRPurpose fragment and ccdB purpose fragments, and by pUC19 DNA restriction enzymes
PvuII carries out digestion, linearized fragment pUC19 is reclaimed, finally by ccdB, cI857-PR, linearisation pUC19 fragments connected
Connect, obtain cloning vector pUB857.
3. applications of the flat end cloning vector pUB857 of zero background as claimed in claim 1 in gene cloning.
4. applications of the flat end cloning vector pUB857 of zero background as claimed in claim 1 in construction of gene library.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112011587A (en) * | 2020-08-07 | 2020-12-01 | 华东理工大学 | Erasable and rewritable living cell sensing recording system and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000052141A1 (en) * | 1999-03-02 | 2000-09-08 | Invitrogen Corporation | Cells resistant to toxic genes and uses thereof |
| WO2001025451A2 (en) * | 1999-10-01 | 2001-04-12 | Transgenomic, Inc. | Cloning vectors |
| CN1880460A (en) * | 2006-04-28 | 2006-12-20 | 清华大学 | Escherichia coli self-cracking method and its dedicated carrier and application |
| CN101182533A (en) * | 2007-11-15 | 2008-05-21 | 马跃 | Design and construction method of high efficiency temperature controlled positive screening cloning carrier |
| CN103497965A (en) * | 2013-10-17 | 2014-01-08 | 扬州大学 | Temperature-controlled zero background T-vector precursor and construction method and application thereof |
| CN104313044A (en) * | 2014-09-30 | 2015-01-28 | 天根生化科技（北京）有限公司 | Zero-background cloning vector as well as preparation method and application thereof |
| CN105400809A (en) * | 2015-12-21 | 2016-03-16 | 生工生物工程（上海）股份有限公司 | Cloning vector and preparation and application thereof |
-
2016
- 2016-08-23 CN CN201610709625.4A patent/CN107760703A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000052141A1 (en) * | 1999-03-02 | 2000-09-08 | Invitrogen Corporation | Cells resistant to toxic genes and uses thereof |
| WO2001025451A2 (en) * | 1999-10-01 | 2001-04-12 | Transgenomic, Inc. | Cloning vectors |
| CN1880460A (en) * | 2006-04-28 | 2006-12-20 | 清华大学 | Escherichia coli self-cracking method and its dedicated carrier and application |
| CN101182533A (en) * | 2007-11-15 | 2008-05-21 | 马跃 | Design and construction method of high efficiency temperature controlled positive screening cloning carrier |
| CN103497965A (en) * | 2013-10-17 | 2014-01-08 | 扬州大学 | Temperature-controlled zero background T-vector precursor and construction method and application thereof |
| CN104313044A (en) * | 2014-09-30 | 2015-01-28 | 天根生化科技（北京）有限公司 | Zero-background cloning vector as well as preparation method and application thereof |
| CN105400809A (en) * | 2015-12-21 | 2016-03-16 | 生工生物工程（上海）股份有限公司 | Cloning vector and preparation and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LI-LING HU等: "The use of the ccdB lethal gene for constructing a zero background vector in order to clone blunt-end PCR products", 《MOLECULAR BIOLOGY》 * |
| ORRANDER,J.等: "Cloning vector pUC19, complete sequence", 《GENBANK DATABASE》 * |
| 范凯荣等: "毒素蛋白CcdB在载体构建中的研究及应用", 《生物技术通讯》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011587A (en) * | 2020-08-07 | 2020-12-01 | 华东理工大学 | Erasable and rewritable living cell sensing recording system and application thereof |
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