CN107760704A - Based on recombinant vector pGPE57 recombinant bacterial strain E.coli GPE10 and its construction method and application - Google Patents
Based on recombinant vector pGPE57 recombinant bacterial strain E.coli GPE10 and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of recombinant vector pGPE57 and recombinant bacterial strain E.coli GPE10 structure and application, belong to biological technical field, plasmid pGPE57 and recombinant bacterial strain E.coli GPE10 contain Amp, AraC, Pbad, gam, pfu Ssod7 and exo Genetic elements.Recombinant vector pGPE57 and recombinant bacterial strain E.coli GPE10 can recombinate enzyme system by arabinose induced expression, and completing intracellular DNA by the restructuring between homologous sequence assembles.Multiple clips DNA assembling is carried out using recombinant bacterial strain E.coli GPE10, it is a kind of simple, quick and efficient DNA directed clonings technology, digestion enzyme company need not be carried out, DNA fragmentation directly can be transferred to E.coli GPE10 competent cells and realize molecular cloning, do not limited by restriction enzyme site, it can be a kind of quick clone technology independent of coupled reaction by any site of target DNA fragment directed cloning to any carrier, multiple clips DNA efficient sequential concatenation can be realized simultaneously.
Description
Technical field
The present invention relates to a kind of recombinant vector pGPE57 and recombinant bacterial strain E.coli GPE10 and its construction method and application,
Linear DNA fragment with homologous sequence is imported in E.coli GPE10 competent cells and completes DNA assemblings, belongs to biological skill
Art field.
Background technology
DNA clone and package technique are one of the most frequently used instruments of Molecular Biology Lab.In recent years, with rear gene
The arrival in group epoch, life science is fast-developing, and gene function, controlling gene element are analyzed in terms of synthetic biology
Also deepened continuously with biological approach is explored, just seem particularly critical using molecule clone technology come assembled dna element.Traditional enzyme
It is most widely used molecule clone technology to cut connection method, but this method is confined to the selection of restriction enzyme site, and is operated numerous
It is trivial, take time and effort.With the development of molecule clone technology, some simple efficient gene clone methods have also closely come out, such as
Gateway recombinant clones technology (Katzen F. recombinational cloning:a biological
operating system[J].Expert opinion on drug discovery,2007,2(4):571-589.) and
Gibson connection methods (Gibson D G, et al.Enzymatic assembly of DNA molecules up to
several hundred kilobases[J].Nature methods,2009,6(5):343-345.).Such as gateway
Clone technology is that the target gene of donor vehicle is transferred on purpose carrier using the recombinant technique of specific site, and process is numerous
It is trivial, and anti-ccdB special bacterial strain is needed to convert.For Gibson connection methods, then it is anti-to participate in connection to need a series of enzymes
Should, while, it is necessary to external 50 degree of connections 1h, both expends the time, sinks money again.
Red homologous recombination systems from bacteriophage lambda are mainly made up of a series of albumen such as Exo, Beta, Gam
(Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in
Escherichia coli K-12using PCR products[J].Proceedings of the National
Academy of Sciences,2000,97(12):6640-6645.), Red systems have for linear and annular homologous recombination
Higher efficiency, but then some deficiencies in the restructuring of linear fragment.
The content of the invention
For the practical problem and demand in molecular cloning, the invention provides a kind of recombinant vector pGPE57, to incite somebody to action
Bet elements in pKD46 plasmids are substituted for pfu-Ssod7DNA sequences and form the restructuring matter that full length sequence is SEQ ID NO.17
Grain pGPE57.
The present invention also provides a kind of plasmid pUC-YZ based on above-mentioned recombinant vector pGPE57, for by pGPE57 plasmids
It is SEQ ID that repA101 and OriR101 elements, which are substituted for LacY, pUC ori and LacZ DNA sequence dnas formation full length sequence,
NO.18 plasmid pUC-YZ.
Further, the present invention also provides a kind of recombinant bacterial strain E.coli based on above-mentioned recombinant vector pGPE57
GPE10, for above-mentioned plasmid pUC-YZ is incorporated into DH10B, formed containing Amp, AraC, Pbad, gam, pfu-Ssod7 and
The recombinant bacterial strain E.coli GPE10 of exo Genetic elements.
The present invention provides above-mentioned recombinant vector pGPE57 construction method, including design of primers, gene cloning and carrier structure
Build, pfu-Ssod7DNA sequences and pKD46 purpose fragments expanded by synthetic primer PCR, by pfu-Ssod7DNA sequences and
PKD46 purpose fragments connect, and obtain recombinant plasmid pGPE57.
The present invention provides above-mentioned plasmid pUC-YZ construction method, including design of primers, gene cloning and vector construction, leads to
Synthetic primer PCR amplifications LacY, pUC ori and LacZ and pGPE57 purpose fragments are crossed, by LacY, pUC ori, LacZ sequences
Connected with pGPE57 purpose fragments, obtain recombinant plasmid pUC-YZ.
The present invention provides above-mentioned recombinant bacterial strain E.coli GPE10 construction method, and the pUC-YZ plasmids after digestion are converted
Enter DH10B competent cells, Amp, AraC, Pbad, gam, pfu-Ssod7 and exo Genetic elements are incorporated into DH10B genomes
On, form recombinant bacterial strain E.coli GPE10.
Applications of the above-mentioned recombinant bacterial strain E.coli GPE10 in multiple clips DNA assemblings, including：Induced by arabinose
The competent cell of E.coli GPE10 afterwards, available for the multiple pcr amplification products for having homologous sequence in E.coli GPE10
It is intracellular to be directly oriented DNA assemblings, it can also be used to which that pcr amplification product and linearized enzyme digestion product are thin in E.coli GPE10
Intracellular is directly oriented DNA assemblings, realizes molecular cloning.
The present invention constructs a kind of recombinant vector pGPE57 and engineering strain for intracellular multiple clips DNA assemblings
E.coli GPE57, engineering bacteria is controlled in a series of recombinases of intracellular expression, such as gam, pfu- using inducible promoter
Ssod7, exo and DNA ligase.By way of homologous recombination, complete to divide by homologous recombination directly in competent cell
Son clone.On the one hand, this method does not need Ligation in vitro, saves experimental work amount, improves conventional efficient；On the other hand, keep away
Exempt from the use of enzyme, save the use of experiment fees.Meanwhile the technology is a kind of simple, quick and efficient DNA orientations
Clone technology, can be a kind of disconnected dependent form by any site of Insert Fragment PCR primer directed cloning to any carrier
Quick clone technology, it is possible to achieve the efficient sequential concatenation of multiple clips.PCR primer does not need body without any ferment treatment
Outer connection, linear DNA can be directly converted to competent cell, realizes molecular cloning.
Brief description of the drawings
Fig. 1 is the digestion verification figure of pGPE57 carriers, and M represents that marker, a represent HindIII digestions, and b represents SacI enzymes
Cut.
Fig. 2 is the pGPE57 plasmid maps that structure is completed.
Fig. 3 is double crossing over plasmid pUC-YZ digestion verification figures, and M represents that marker, a represent HindIII digestions, and b is represented
NdeI and KpnI digestions.
Fig. 4 is double crossing over plasmid pUC-YZ plasmid map.
Fig. 5 is recombinant bacterial strain E.coli GPE10 PCR proof diagrams.
Fig. 6 is recombinant plasmid pUC-gfp digestion verification figure, and M represents that marker, a represent KpnI digestions, and b is represented
HindIII digestions.
Fig. 7 is the green fluorescent protein figure under fluorescence microscope.
Fig. 8 is recombinant plasmid pCP-Ble digestion verification figure, and M represents that marker, a represent KpnI digestions, and b represents NdeI
Digestion.
Embodiment
With reference to embodiment and accompanying drawing, the invention will be further described.
Embodiment one：A kind of new homologous recombination bacterial strain E.coli GPE10 construction method
1st, recombinant bacterial strain pGPE57 structure, including design of primers, gene cloning and vector construction, operating procedure are as follows：
(1), design of primers：Engineer synthesizes specific primer sequence
pfuFor:5’-gattcagaggtataaaacgggaggcacggatcaatgattttag-3’；
pfuRev:5’-gtcatgctgccaccttctgcctactttttctgcttctccag-3’
KDFor:5’-ctggagaagcagaaaaagtaggcagaaggtggcagcatgac-3’；
KDRev:5’-ctaaaatcattgatccgtgcctcccgttttatacctctgaatc-3’
(2), PCR is expanded：PCR reaction systems configure as shown in Table 1.
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 extensions, 30 circulate；Finally
72 DEG C of extension 10min.The DNA profiling is pET-PfuX7 and pKD46, and primer is pfuFor/pfuRev and KDFor/KDRev,
Extension of time is 1.5min and 3min respectively.
(3), vector construction：The pcr amplification product that step 2 is expanded is separated by agarose gel electrophoresis, respectively
Reclaim pfu-Ssod7 and pKD46 purpose fragments.Pfu-Ssod7, pKD46 fragment are carried out with Gibson Assembly mode
Connection, and connection product is converted into escherichia coli DH5a, the checking of picking positive colony, obtain cloning vector pGPE57.Institute
It is the nucleic acid fragment that length is 2589bp to state purpose fragment pfu-Ssod7, and pKD46 is the nucleic acid fragment that length is 5606bp.Fine jade
Sepharose electrophoresis referring to《Molecular cloning》The method of middle agarose gel electrophoresis, glue reclaim use qiagen glue reclaim reagents
Box, chemical transformation referring to《Molecular cloning》Middle calcium chloride prepares and the method for competence conversion Escherichia coli.Linked system and
Method is referring to Gibson Assembly.PGPE57 plasmids are 8110bp with HindIII digestion post-fragment sizes, with SacI digestions
Post-fragment size is respectively 6071bp and 2039bp, and proof diagram is as shown in figure 1, pGPE57 plasmid maps such as Fig. 2 that structure is completed
It is shown.
2nd, homologous recombination bacterial strain E.coli GPE10 construction method, including design of primers, gene cloning, vector construction and
Strain construction；It is characterized in that operating procedure is as follows：
(1), design of primers：Engineer synthesizes specific primer sequence
pUCFor：5’-gaagatcgcactccagccagggtatcagctcactcaaagg-3’；
pUCRev：5’-gtcgtcaggtgaatgaagtcgcattggtaactgtcagacc-3’；
T1For:5’-ggtctgacagttaccaatgcgacttcattcacctgacgac-3’；
T1Rev:5’-ctgtcagaccaagtttactccgatttggctacatgacatc-3’；
T2For:5’-cgagtatcgagatggcacatcgaccagatgatcacactcg-3’；
T2Rev:5’-cctttgagtgagctgataccctggctggagtgcgatcttc-3’；
Amp-BPFor:5’-gatgtcatgtagccaaatcggagtaaacttggtctgacag-3’；
Amp-BPRev：5’-cgagtgtgatcatctggtcgatgtgccatctcgatactcg-3’；
GPE1For:5’-ggtaagccttcgcacatatc-3’；
GPE1Rev:5’-gcagtgctgccataaccatg-3’；
GPE2For:5’-cactttcgtctactccgttac-3’；
GPE2Rev:5’-gaatggcgaatggcgctttg-3’；
(2), 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 extend, and 30
Individual circulation；Last 72 DEG C of extensions 10min.The DNA profiling be plasmid pUC19, DH10B genome LacY, pGPE57 respectively with
And DH10B genomes LacZ.Primer is pUCFor/pUCRev, T1For/T1Rev, Amp-BPFor/Amp-BPRev and
T2For/T2Rev, extension of time are 1min, 1min, 3.5min and 1min respectively.
(3), vector construction：Pcr amplification product pUC ori that step 2 is expanded, LacY, only expand Amp, AraC,
Pbad, gam, pfu-Ssod7 and exo pGPE57, LacZ are separated by agarose gel electrophoresis, reclaim purpose fragment.
PUC ori, LacY, pGPE57 and LacZ purpose fragment are attached with Gibson Assembly mode, and by connection product
Conversion the checking of picking positive colony, obtains cloning vector pUC-YZ into escherichia coli DH5a.The purpose fragment pUC ori
Length be 915bp nucleic acid fragment, LacY length is 1197bp nucleic acid fragment, and pGPE57 length is 6163bp core
Acid fragment, LacZ length are 1196bp nucleic acid fragment.
(4), strain construction：With restriction enzyme PciI digestion pUC-YZ, linearized fragment is reclaimed, electricity is transformed into
DH10B competent cells, picking monoclonal checking, obtain recombinant bacterial strain E.coli GPE10.Recombinant bacterial strain E.coli GPE10
Verified by way of PCR, checking primer is GPE1For/GPE1FRev and GPE2For/GPE2Rev.Agarose gel electrophoresis
Refer to《Molecular cloning》The method of middle agarose gel electrophoresis, glue reclaim use qiagen glue reclaim kits, chemical conversion
Method referring to《Molecular cloning》Middle calcium chloride prepares and the method for competence conversion Escherichia coli.Electrotransformation referring to《Molecular cloning》
The electric method for transformation of middle Escherichia coli, linked system and method are referring to Gibson Assembly.PUC-YZ plasmids HindIII
Digestion post-fragment size is 9331bp, is respectively 3435bp and 5896bp with KpnI and NdeI digestion post-fragment sizes, digestion is tested
Card figure is as shown in figure 3, build the pGPE57 plasmid maps completed as shown in figure 4, recombinant bacterial strain E.coli GPE10PCR expand piece
Duan great little distinguishes 1817bp and 1378bp, and checking is as shown in Figure 5.
Embodiment two：It is a kind of to can be used for the method that intracellular linear DNA recombinates to carry out intracellular multiple clips assembling, including
Following steps：
Bacteria Culture：The DH5a containing plasmid pUC19 is inoculated with the μ g/ml of ampicillin 100 fluid nutrient medium, in
37 DEG C of culture 10h；The DH5a containing plasmid pKVC is inoculated with the μ g/ml of chloramphenicol 20 fluid nutrient medium, in 37 DEG C of cultures
10h, the DH5a containing plasmid pET29a is inoculated with the μ g/ml of kanamycins 50 fluid nutrient medium, in the μ g/ml of kanamycins 50
Fluid nutrient medium in be inoculated with the DH5a containing plasmid pTD103LuxI-sfgfp, respectively extract plasmid pUC19, pKVC,
PCM271rfp and pTD103LuxI-sfgfp.
Design of primers：Engineer synthesizes specific primer sequence
UCFor：5’-gatctcgaccgatgcccttgggtatcagctcactcaaagg-3’；
UCRev：5’-atgagggtgtcagtgaagtggcattggtaactgtcagacc-3’；
CmFor:5’-ggtctgacagttaccaatgccacttcactgacaccctcat-3’；
CmRev:5’-cagcgatacaatagtgtgactcgccgcacttatgactgtc-3’；
gfpFor：5’-gacagtcataagtgcggcgagtcacactattgtatcgctg-3’；
gfpRev:5’-gaattcgagctccgtcgacaactcgaggtgaagacgcctag-3’；
LacIFor:5’-ctaggcgtcttcacctcgagttgtcgacggagctcgaattc-3’；
LacIRev:5’-cctttgagtgagctgatacccaagggcatcggtcgagatc-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.The DNA profiling is pUC19 respectively, pKVC, pTD103LuxI-sfgfp and
PET29a, primer are UCFor/UCRev CmFor/CmRev, gfpFor/gfpRev and LacIFor/LacIRev respectively.
Pcr amplified fragment is separately recovered by agarose gel electrophoresis, above-mentioned purpose fragment pUC ori are that length is
935bp nucleic acid fragment, CmR are the nucleic acid fragments that length is 1616bp, and gfp is the nucleic acid fragment that length is 949bp, and LacI is
Length is 1783bp nucleic acid fragment.
It is prepared by E.coli GPE10 cells competence：In being inoculated with E.coli GPE10 monoclonals on fresh plate, in 30 DEG C
Overnight incubation, then 1% 20ml is transferred to containing in 50 μ g/ml penicillin fresh LBs, in 30 DEG C of 235rmp/min bar
Culture adds 20% arabinose 20ul, is further cultured for OD600 to 0.5 to OD600 to 0.2 under part；After ice bath 10min,
4000rmp/min, 5min is centrifuged at 4 DEG C and collects cell.Then 0.1mol chlorination Calcium treatment 20min is used；In 4000rmp/
Min, 5min is centrifuged at 4 DEG C and collects cell.Finally, it is suspended in 0.1mol calcium chloride of the 1ml containing 10% glycerine.
Cotransformation：By four nucleic acid fragment cotransformations such as pUC ori, CmR, gfp and LacI to Competent
In E.coli GPE10 cells, in 37 DEG C of recovery culture 1h, it is incubated overnight, the checking of picking monoclonal, obtains in 37 DEG C of incubators
Obtain positive colony pUC-gfp.
Result of the test shows：Utilize intracellular homologous recombination technique, it is not necessary to Ligation in vitro is carried out, can be directly by PCR
The direct Transformed E .coli GPE10 competent cells of recovery product, 4 DNA fragmentation assemblings, recombinant plasmid are completed in the cell
PUC-gfp is 5124bp with KpnI digestion post-fragment sizes, with HindIII digestion post-fragment sizes be respectively 1200bp and
3924bp, digestion verification figure is as shown in fig. 6, the form with recombinant plasmid pUC-gfp under fluorescence microscope is as shown in Figure 7.
Embodiment three：A kind of method available for intracellular DNA assembly restructuring clones large fragment DNA, including with
Lower step：
Bacteria Culture：The DH5a containing plasmid pCP20 is inoculated with the μ g/ml of chloramphenicol 20 fluid nutrient medium, in 37 DEG C
Cultivate 10h；The DH5a containing plasmid pPICZa-A is inoculated with the μ g/ml of bleomycin 50 fluid nutrient medium, in 37 DEG C of cultures
10h.Plasmid pCP20 and pPICZa-A are extracted respectively.
Design of primers：Engineer synthesizes specific primer sequence
pPIFor：5’-ggtgagaatggcaaaagcttgatacactagcagcagaccg-3’
pPIRev：5’-ctattctctagggggatcctcgttccactgagcgtcagac-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.The DNA profiling is pPICZa-A, and primer is pPIFor/pPIRev.Use agarose
Gel electrophoresis glue reclaim pPICZa-A fragments 3500bp.
Carrier digestion：PCP20 DNAs are subjected to digestion, glue reclaim linearized fragment with restriction enzyme XbaI
PCP20, nucleic acid fragment size are 9497bp.
It is prepared by E.coli GPE10 cells competence：In being inoculated with E.coli GPE10 monoclonals on fresh plate, in 30 DEG C
Overnight incubation, then 1% 20ml is transferred to containing in 50 μ g/ml penicillin fresh LBs, in 30 DEG C of 235rmp/min bar
Culture adds 20% arabinose 20ul, is further cultured for OD600 to 0.5 to OD600 to 0.2 under part；After ice bath 10min,
4000rmp/min, 5min is centrifuged at 4 DEG C and collects cell.Then 0.1mol chlorination Calcium treatment 20min is used；In 4000rmp/
Min, 5min is centrifuged at 4 DEG C and collects cell.Finally, it is suspended in 0.1mol calcium chloride of the 1ml containing 10% glycerine.
Cotransformation：By pPICZa-A and pCP20 linear fragments cotransformation to Competent E.coli GPE10 cells
In, in 37 DEG C of recovery culture 1h, it is incubated overnight in 37 DEG C of incubators, the checking of picking monoclonal, obtains positive colony pCP-
Ble。
Result of the test shows：Utilize the restructuring enzyme system of cell inner expression, it is not necessary to carry out Ligation in vitro, can directly by
The direct Transformed E .coli GPE10 competent cells of PCR recovery products, complete large fragment DNA assembling, and recombinant plasmid pCP-Ble is used
KpnI digestion post-fragments size is 12953bp, is respectively with NdeI digestion post-fragments size and 5928bp and 7025bp, digestion are tested
Card figure is as shown in Figure 8.
Claims (9)
1. a kind of recombinant vector pGPE57, it is characterised in that be that the bet elements in pKD46 plasmids are substituted for pfu-
Ssod7DNA sequences form the recombinant plasmid pGPE57 that full length sequence is SEQ ID NO.17.
2. the plasmid pUC-YZ based on recombinant vector pGPE57 as claimed in claim 1, it is characterised in that be by pGPE57 matter
It is SEQ that repA101 and OriR101 elements in grain, which are substituted for LacY, pUC ori and LacZ DNA sequence dnas formation full length sequence,
ID NO.18 plasmid pUC-YZ.
3. the recombinant bacterial strain E.coli GPE10 based on plasmid pUC-YZ as claimed in claim 2, it is characterised in that be by matter
Grain pUC-YZ is incorporated into DH10B, forms the restructuring containing Amp, AraC, Pbad, gam, pfu-Ssod7 and exo Genetic elements
Bacterial strain E.coli GPE10.
4. recombinant vector pGPE57 as claimed in claim 1 construction method, it is characterised in that expanded by synthetic primer PCR
Increase pfu-Ssod7DNA sequences and pKD46 purpose fragments, pfu-Ssod7DNA sequences are connected with pKD46 purpose fragments, obtained
Recombinant plasmid pGPE57.
5. plasmid pUC-YZ as claimed in claim 2 construction method, it is characterised in that expanded by synthetic primer PCR
LacY, pUC ori and LacZ and pGPE57 purpose fragments, LacY, pUC ori, LacZ sequences and pGPE57 purpose fragments are connected
Connect, obtain recombinant plasmid pUC-YZ.
6. recombinant bacterial strain E.coli GPE10 as claimed in claim 3 construction method, it is characterised in that after digestion
PUC-YZ plasmids are transformed into DH10B competent cells, and Amp, AraC, Pbad, gam, pfu-Ssod7 and exo Genetic elements are whole
Close on DH10B genomes, form recombinant bacterial strain E.coli GPE10.
7. applications of the recombinant bacterial strain E.coli GPE10 as claimed in claim 3 in multiple clips DNA assemblings.
8. applications of the recombinant bacterial strain E.coli GPE10 as claimed in claim 7 in multiple clips DNA assemblings, its feature exist
In E.coli GPE10 of the multiple pcr amplification products for having homologous sequence after being induced through arabinose is directly entered into the cell
Row orientation DNA assemblings.
9. applications of the recombinant bacterial strain E.coli GPE10 as claimed in claim 7 in multiple clips DNA assemblings, its feature exist
In the E.coli GPE10 of pcr amplification product and linearized enzyme digestion product after being induced through arabinose are directly entered into the cell
Row orientation DNA assemblings.
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