CN109609425A - A method of integrative recombinant is screened using the activation recovering of the enzyme of bacillus subtilis integration site - Google Patents
A method of integrative recombinant is screened using the activation recovering of the enzyme of bacillus subtilis integration site Download PDFInfo
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Abstract
Present invention relates particularly to a kind of methods of the activation recovering of enzyme using bacillus subtilis integration site screening integrative recombinant.Step is: 1) constructing resistant gene integrated plasmid, convert bacillus subtilis, the gene 3 ' that screening obtains the enzyme of integration site holds the bacillus subtilis for lacking and inactivating；2) exogenous origin gene integrator plasmid is constructed；3) exogenous origin gene integrator plasmid step of converting 1) obtain bacillus subtilis, using the enzyme of integration site can digest using substance Solid agar culture as screening and culturing medium, by the enzymatic activity of antibiotic resistance inactivation, integration site, the recombination engineering that exogenous origin gene integrator plasmid is integrated into bacillus subtilis by homologous double-crossover is filtered out.The invention has the advantages that: do not need selection markers of the antibiotics resistance gene as recombined bacillus subtilis；The recombined bacillus subtilis of acquisition keeps the enzymatic activity of integration site.
Description
[technical field]
The invention belongs to field of biotechnology, are related to a kind of activation recovering of enzyme using bacillus subtilis integration site
The method for screening integrative recombinant.
[background technique]
Bacillus subtilis (Bacillus subtilis) is nonpathogenic, not toxin producing and pyrogenicity allergic protein matter, is
A kind of strain of food safety (GRAS:Generally Recognized as Safe), is classified as food-grade microorganisms scope.
And bacillus subtilis has the advantage that 1. have very strong protein secreting function, does not need brokenly as expression system
Chopping fine born of the same parents extract protein, it is only necessary to which relatively simply handling fermented supernatant fluid can be obtained purer target protein, at present
Secreting, expressing is realized in bacillus subtilis there are many foreign protein.2. without apparent codon-bias, simultaneously
Expression product is also not easily formed inclusion body.3. fermentation condition is simple.Bacillus subtilis is developed and used to have as expression system
There is profound significance.
Different according to the type of used carrier, bacillus subtilis expression pattern can be divided into reproducible plasmid expression and dye
Colour solid integrant expression.Both usual expression patterns require antibiotics resistance gene to screen.In addition, plasmid replication usually exists
Be not in bacillus subtilis it is very stable, generally need in process of production using antibiotic, and antibacterial activity is food enzyme
The important examination requirements of preparation.In addition, it was discovered by researchers that antibiotics resistance gene present in genetically engineered food can shift
Into the bacterium in human body intestinal canal microorganism.Have some national governments now in order to avoid this gene contamination, clearly advises
It is fixed, forbid the genetically engineered food for making and selling any antibiotic resistant gene.
It is mostly using resistant gene as selection markers, moreover, if by homologous double in integrated plasmid used at present
Exchange is integrated into bacillus subtilis DNA sequence, and the enzyme of integration site can be made to inactivate.Such as integration site is alphalise starch
Plasmid pDG364, pMLK83 of enzyme gene, pDG1661, pDG1662, pDG1728, pDG1730, pDL, pDK, pSG1154,
PSG1192, pSG1193, pSG1729, pSG1190, pSG1191 homologous double-crossover are integrated into bacillus subtilis chromosomal gene
Group needs to be gone out with antibiotic-screenings such as chloramphenicol, neomycin or Togoplus recombinant bacterium, and the bacillus subtilis weight obtained
The alpha-amylase inactivation of group bacterium；Integration site is that plasmid pDG1663, pDG1664, pDG1729, pDG1731 of thrC gene are same
Source double crossing over is integrated into bacillus subtilis DNA sequence, needs to filter out recombinant bacterium with erythromycin or Togoplus, and
And the thrC enzyme inactivation of the bacillus subtilis recombinant bacterium obtained；And integration site is the plasmid of beta-galactosidase gene
PAX01, pA-spac homologous double-crossover are integrated into bacillus subtilis DNA sequence, need to be screened with antibiotic erythromycin
Recombinant bacterium out, and the beta galactosidase inactivation of the bacillus subtilis recombinant bacterium obtained.
[summary of the invention]
The object of the present invention is to provide a kind of screenings of the activation recovering of enzyme using bacillus subtilis integration site to integrate
The method of recon.It is related to a kind of side of the activation recovering screening integrative recombinant of enzyme using bacillus subtilis integration site
Method.Step is: 1) constructing resistant gene integrated plasmid, convert bacillus subtilis, screening obtains the gene of the enzyme of integration site
The bacillus subtilis of 3 ' end missings and inactivation；2) exogenous origin gene integrator plasmid is constructed；3) exogenous origin gene integrator plasmid conversion step
It is rapid 1) obtain bacillus subtilis, using the enzyme of integration site can digest using substance Solid agar culture as screen
Culture medium filters out exogenous origin gene integrator plasmid and passes through homologous double cross by the enzymatic activity of antibiotic resistance inactivation, integration site
Change the recombination engineering for being integrated into bacillus subtilis.
To achieve the goals above, The technical solution adopted by the invention is as follows:
A method of integrative recombinant being screened using the activation recovering of the enzyme of bacillus subtilis integration site, it is specific to walk
Suddenly it is:
(1) resistant gene integrated plasmid is constructed, bacillus subtilis is converted, screening obtains the gene 3 ' of the enzyme of integration site
End lacks and the bacillus subtilis of inactivation is as host strain, and concrete operations are as follows:
S1, building make the end of the gene 3 ' missing of the enzyme of integration site and the resistant gene integrated plasmid of inactivation, the integration matter
The DNA sequence dna of the homologous double-crossover of grain is A-ARG-C；
Wherein, A indicates upstream homology arm, is the section of DNA sequence that the enzyme gene 5 ' of bacillus subtilis integration site is held；
C indicate downstream homology arm, be integration site enzyme gene terminator codon after section of DNA sequence；
It is antibiotics resistance gene ARG between homology arm；
S2, screen integration site enzyme gene 3 ' end missing and inactivate bacillus subtilis: the resistance base of linearisation
Because integrated plasmid converts bacillus subtilis, cultivate, screen on antibiotic solid medium, by antibiotic resistance and
The enzyme of integration site inactivates, and obtains the end of the gene 3 ' missing of the enzyme of integration site and the bacillus subtilis of inactivation；
(2) exogenous origin gene integrator plasmid is constructed, wherein the DNA sequence dna of homologous double-crossover is AB-EGEU-C；
Wherein, AB is the complete sequence of the enzyme gene of bacillus subtilis integration site；
C indicate downstream homology arm, be integration site enzyme gene terminator codon after section of DNA sequence；
It is exogenous gene expression unit EGEU between homology arm；
(3) bacillus subtilis of the exogenous gene integration to bacillus subtilis integration site: the external source of linearisation
Gene integration plasmid converts the end of the gene 3 ' missing of the enzyme of integration site and the bacillus subtilis of inactivation, with the enzyme of integration site
Can digest using substance Solid agar culture as screening and culturing medium, pass through antibiotic resistance inactivation, integration site
Enzymatic activity filters out the recombination engineering that exogenous origin gene integrator plasmid is integrated into bacillus subtilis by homologous double-crossover.
It is further preferred that the bacillus subtilis is 168 derivative strain of bacillus subtilis, including 1A751,
WB600 and WB800.
In the present invention, it further illustrates:
One, the enzyme of the integration site is bacillus subtilis alpha-amylase；
The DNA sequence dna AB is the complete sequence of bacillus subtilis alpha-amylase gene, is expanded by AmyS/AmyA primer
Increase and obtains；
The DNA sequence dna A is the section of DNA sequence that bacillus subtilis alpha-amylase gene 5 ' is held, A containing DNA sequence dna
Plasmid fragments by the plasmid pUC1ABC of the complete sequence containing the alpha-amylase gene containing bacillus subtilis through I digestion of Pst, pure
Change and obtains；
The DNA sequence dna C is the section of DNA sequence after bacillus subtilis alpha-amylase gene terminator codon, by
AEndS/AEndA primer amplification and obtain；
The antibiotics resistance gene ARG is neomycin resistance gene, Togoplus resistant gene, chlorampenicol resistant base
One of cause, erythromycin resistance gene；
The antibiotic solid medium is the LB solid medium of the 20ug/mL containing neomycin in step s 2, or
The LB solid medium of the 100ug/mL containing Togoplus, or the LB solid medium of the 5ug/mL containing chloramphenicol, or contain erythromycin
The LB solid medium of 0.5ug/mL；
The Solid agar culture that the substance utilized can be digested described in step (3) is only containing the fine jade of soluble starch
Rouge solid medium.
It further illustrates, AmyS, AmyA, AEndS, AEndA primer sequence is as follows:
AmyS:5′-TTCGACGTCTCAAATAAGGAGTGTCA-3′；
AmyA:5′-TAGGAGCTCCCTCAATGGGGAAGAGA-3′；
AEndS:5′-AGGAAGCTTGGGACTTACCGAAAGAA-3′；
AEndA:5′-AATCAGCTGCTGCTTCCAACAAAACC-3′。
The enzyme of integration site described in two, is bacillus subtilis invertase；
The DNA sequence dna AB is the complete sequence of bacillus subtilis saccharase gene, by InvS/InvA primer amplification
And it obtains；
The DNA sequence dna A is the section of DNA sequence that bacillus subtilis saccharase gene 5 ' is held, and is drawn by InvS/InvP
Object is expanded and is obtained；
The DNA sequence dna C is the section of DNA sequence after bacillus subtilis saccharase gene terminator codon, by
IEndS/IEndA primer amplification and obtain；
The antibiotics resistance gene ARG is neomycin resistance gene, Togoplus resistant gene, chlorampenicol resistant base
One of cause, erythromycin resistance gene；
The antibiotic solid medium is the LB solid medium of the 20ug/mL containing neomycin in step s 2, or
The LB solid medium of the 100ug/mL containing Togoplus, or the LB solid medium of the 5ug/mL containing chloramphenicol, or contain erythromycin
The LB solid medium of 0.5ug/mL；
The Solid agar culture of the substance utilized can be digested described in step (3) as the only agar solid containing sucrose
Culture medium.
It further illustrates, InvS, InvA, InvP, IEndS, IEndA primer sequence is as follows:
InvS:5′-GAGGACGTCATGACAGCACATGACCAGGA-3′；
InvA:5′-TAGGAGCTCTTTCTACATAAGTGTCCAAATTCC-3′；
InvP:5′-CAACTGCAGCCCGCTTCCAATTCACA-3′；
IEndS:5′-CCTCTGCAGTTCTTATGTGAAATCTGAGC-3′；
IEndA:5′-AATAAGCTTTTGCAGATTTCCTCAAA-3′。
In conclusion by adopting the above-described technical solution, the beneficial effects of the present invention are:
(1) selection markers of the antibiotics resistance gene as recombined bacillus subtilis are not needed；
(2) recombined bacillus subtilis obtained keeps the enzymatic activity of integration site and as selection markers.
[Detailed description of the invention]
Fig. 1 is the Technology Roadmap that the present embodiment specifically constructs；
Fig. 2 is II/Sac of plasmid Aat, I restriction enzyme digestion and electrophoresis figure,
Wherein M is Marker D2000 molecular weight standard, band be followed successively by from top to bottom 2000bp, 1000bp, 750bp,
500bp,250bp,100bp；Number 1 is II/Sac of plasmid Aat, I restriction enzyme digestion and electrophoresis figure；
Fig. 3 is III/Pvu of plasmid Hind, II restriction enzyme digestion and electrophoresis figure,
Wherein M is Marker III molecular weight standard, band be followed successively by from top to bottom 4500bp, 3000bp, 2000bp,
1200bp,800bp,500bp,200bp；Number 1 is III/Pvu of plasmid Hind, II restriction enzyme digestion and electrophoresis figure；
Fig. 4 is I restriction enzyme digestion and electrophoresis figure of plasmid Pst,
Wherein M is Marker III molecular weight standard, band be followed successively by from top to bottom 4500bp, 3000bp, 2000bp,
1200bp,800bp,500bp,200bp；Number 1 is I restriction enzyme digestion and electrophoresis figure of plasmid Pst；
Fig. 5 is III/Kpn of plasmid Hind, I restriction enzyme digestion and electrophoresis figure,
Wherein M is Marker III molecular weight standard, band be followed successively by from top to bottom 4500bp, 3000bp, 2000bp,
1200bp,800bp,500bp,200bp；Number 1 is III/Kpn of plasmid Hind, I restriction enzyme digestion and electrophoresis figure；
Fig. 6 is I/Hind of plasmid Pst, III double digestion electrophoretogram,
Wherein M is Marker III molecular weight standard, band be followed successively by from top to bottom 4500bp, 3000bp, 2000bp,
1200bp,800bp,500bp,200bp；Number 1 is I/Hind of plasmid Pst, III double digestion electrophoretogram；
Fig. 7 is II/Pst of plasmid Aat, I double digestion electrophoretogram,
Wherein M is Marker D2000 molecular weight standard, band be followed successively by from top to bottom 2000bp, 1000bp, 750bp,
500bp,250bp,100bp；Number 1 is II/Pst of plasmid Aat, I double digestion electrophoretogram；
Fig. 8 is I restriction enzyme digestion and electrophoresis figure of plasmid Pst,
Wherein M is Marker D2000 molecular weight standard, band be followed successively by from top to bottom 2000bp, 1000bp, 750bp,
500bp,250bp,100bp；Number 1 is I restriction enzyme digestion and electrophoresis figure of plasmid Pst；
Fig. 9 is II/Sac of plasmid Aat, I double digestion electrophoretogram,
Wherein M is Marker III molecular weight standard, band be followed successively by from top to bottom 4500bp, 3000bp, 2000bp,
1200bp,800bp,500bp,200bp；Number 1 is II/Sac of plasmid Aat, I double digestion electrophoretogram；
Figure 10 is I/Kpn of plasmid Sal, I double digestion electrophoretogram,
Wherein M is Marker III molecular weight standard, band be followed successively by from top to bottom 4500bp, 3000bp, 2000bp,
1200bp,800bp,500bp,200bp；Number 1 is I/Kpn of plasmid Sal, I double digestion electrophoretogram.
[specific embodiment]
Below with reference to specific embodiment, description is of the invention in further detail.It should be understood that these embodiments are intended merely to
It illustrates the present invention, rather than limits the scope of the invention in any way.
Embodiment 1: the enzyme of bacillus subtilis integration site is utilized --- the activation recovering screening mango second of alpha-amylase
The method that alkene acceptor gene ETRlb is integrated into the recon of bacillus subtilis
1. the building of integrated plasmid
Synthetic primer:
AmyS:TTCGACGTCTCAAATAAGGAGTGTCA (underscore is II restriction enzyme site of Aat)
AmyA:TAGGAGCTCCCTCAATGGGGAAGAGA (underscore is I restriction enzyme site of Sac)
AEndS:AGGAAGCTTGGGACTTACCGAAAGAA (underscore is III restriction enzyme site of Hind)
AEndA:AATCAGCTGCTGCTTCCAACAAAACC (underscore is II restriction enzyme site of Pvu)
Wherein AmyS/AmyA amplification is bacillus subtilis alpha-amylase gene sequence, and AEndS/AEndA amplification is
Section of DNA sequence after bacillus subtilis alpha-amylase gene terminator codon.With bacterial genomes DNA extraction kit
(TIANGEN) extract bacillus subtilis WB600 genomic DNA and as template, with primer AmyS/AmyA, AEndS/
AEndA and archaeal dna polymerase PrimeSTAR HS DNA Polymerase (TAKARA) are expanded respectively, and reaction system and condition are such as
Under:
Reaction system is designed as 100uL total system, specifically 5 × PCR Buffer (buffer) 20uL, concentration 2.5mM
DNTPmix (deoxyribonucleoside triphosphate mixture) 5uL, concentration is each 2uL of upstream and downstream primer of 10mM, and concentration is
2.5U/uL PrimeSTAR HS DNA Polymerase (TAKARA) 2uL, DNA profiling 2uL (about 20ng), is mended with aqua sterilisa
Sufficient 100uL system.
Reaction condition are as follows: 94 DEG C of 3min initial denaturations, the interior 94 DEG C of 30s denaturation of circulation, 60 DEG C of annealing 30s, 72 DEG C of extensions
1.5min, 30 circulations；Continue to extend 10min for 72 DEG C after PCR reaction cycle, then 16 DEG C of preservations.
Amplified production carries out Ago-Gel recovery purifying, AmyS/ with DNA purification and recovery kit (TIANGEN) respectively
The target fragment about 2.0kb of AmyA recycling, is bacillus subtilis alpha-amylase gene sequence, is named as AB (in addition, A conduct
The upstream homology arm of integrated plasmid, the plasmid fragments of the A containing DNA sequence dna are obtained after digestion by the plasmid containing AB in the present embodiment)；
The target fragment about 0.2kb of AEndS/AEndA recycling, is one section after bacillus subtilis alpha-amylase gene terminator codon
DNA sequence dna is named as C (downstream homology arm of the C as integrated plasmid).
Plasmid pUC19, plasmid pUC19 and DNA fragmentation AB, which are extracted, with the small extraction reagent kit of plasmid (TIANGEN) uses Aat respectively
II/Sac, I double digestion carries out Ago-Gel recovery purifying with DNA purification and recovery kit (TIANGEN) after digestion, wherein
PUC19 recycles about 2.2kb segment, and DNA fragmentation AB recycles about 2.0kb segment, the connection of the two segments, conversion bacillus coli DH 5
α, is coated on Amp containing 100ug/mL, surface added in the LB screening flat board of 40mL X-gal liquid storage and 4 μ l IPTG liquid storages,
37 DEG C are incubated overnight.When single colonie in screening flat board grows to suitable size, plate is placed in 4 DEG C of a few hours, keeps colour developing complete.
It chooses white single colonie to be cultivated, upgrading grain, with II/Sac of Aat, I digestion, obtains the band of about 2.0kb Yu about 2.2kb size,
(Fig. 2) in the same size with theory, institute's upgrading grain is pUC1AB.
Plasmid pUC1AB and DNA fragmentation C uses III/Pvu of Hind, II double digestion respectively, and DNA purification and recovery reagent is used after digestion
Box (TIANGEN) carries out Ago-Gel recovery purifying, and wherein pUC1AB recycles about 4.0kb segment, and DNA fragmentation C is recycled about
0.2kb segment, the connection of the two segments, conversion bacillus coli DH 5 alpha, is coated in the LB screening flat board of the Amp containing 100ug/mL,
37 DEG C are incubated overnight.It when single colonie in screening flat board grows to suitable size, chooses single colonie and is cultivated, upgrading grain uses Hind
III/Pvu, II digestion obtains the band of about 4.0kb Yu about 0.2kb size, (Fig. 3) in the same size with theory, and institute's upgrading grain is through surveying
After sequence verifying, it is named as pUC1ABC.
Plasmid pUC1ABC and plasmid pVK73 uses I digestion of Pst respectively, and DNA purification and recovery kit is used after digestion
(TIANGEN) Ago-Gel recovery purifying is carried out.Wherein pUC1ABC recycles about 3.0kb segment, this segment remains withered grass
The Duan Xulie that Bacillus alpha-amylase gene 5 ' is held；PVK73 recycles about 1.4kb segment, this segment is antibiotic resistance
Gene A RG (Antibiotic Resistance Genes), specifically neomycin resistance gene.The connection of the two segments, conversion
Bacillus coli DH 5 alpha screens, picking single colonie, upgrading grain in the LB solid panel of the 50ug/mL containing kanamycins, with I enzyme of Pst
It cuts, obtains the band of about 2.9kb Yu about 1.4kb size, (Fig. 4) in the same size with theory, institute's upgrading grain is pUC1AC-NEO.
In the present embodiment, foreign gene is the mango Ethylene receptor gene ETRlb of this laboratory preservation.Plasmid pUC1ABC
And the cloned plasmids pUC19-ETRlb of the mango Ethylene receptor gene ETRlb of this laboratory preservation uses III/Kpn of Hind, I enzyme respectively
It cuts, carries out Ago-Gel recovery purifying with DNA purification and recovery kit (TIANGEN) after digestion, wherein pUC1ABC is recycled about
4.2kb segment, pUC19-ETRlb recycle about 2.3kb segment, and the connection of the two segments, conversion bacillus coli DH 5 alpha are coated on and contain
In the LB screening flat board of 100ug/mL Amp, 37 DEG C are incubated overnight.When single colonie in screening flat board grows to suitable size, picking
Single colonie, upgrading grain obtains the band of about 4.2kb Yu about 2.3kb size with III/Kpn of Hind, I digestion, in the same size with theory
(Fig. 5), institute's upgrading grain are pUC1ABC-ETRlb.
2. the building of the bacillus subtilis of alpha-amylase inactivation
Plasmid pUC1AC-NEO is after limitation restriction endonuclease Aat II is linearized, with DNA product purification kit (TIANGEN)
Purifying converts bacillus subtilis WB600, is coated on the LB plate of the neomycin containing 20ug/mL and screens, the single colonie grown
In parallel contact plate to two LB plates containing 1% (w/v) soluble starch, after 37 DEG C of overnight incubations, wherein plate leather is taken
The colour developing of Lan Shi iodine solution obtains transformant without hydrolysis circle screening after developing the color by Gram's iodine solution.
Synthesize following primer: aam1:GGTCTGATCGATGGGATGTC；Aam2:TCATCATCGCTCATCCATGT.It will turn
After beggar is incubated overnight in LB culture medium, total DNA is extracted.Then respectively with primer pair aam1/aam2 and aam1/AEndA into
Row PCR.PCR reaction condition difference is as follows:
Aam1/aam2:PCR reaction system 20ul:DNA template (transformant total DNA) 1ul (about 20ng), 10 × Taq
The forward and reverse primer of Buffer 2ul, 10pmol/ul dNTP 0.4ul, 10pmol/ul is respectively 0.5ul, 2.5U/ul Taq DNA
Polymerase 1ul adds ddH2O to 20ul.PCR response procedures: 94 DEG C of 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 are followed
Ring；72℃10min；4 DEG C of preservations；
Aam1/AEndA:PCR reaction system 20ul:DNA template (transformant total DNA) 1ul (about 20ng), 10 × Taq
Buffer 2ul, 10pmol/ul dNTP 0.4ul, 10pmol/ul forward and reverse primer is respectively 0.5ul, 2.5U/ul Taq DNA
Polymerase 1ul adds ddH2O to 20ul.PCR response procedures: 94 DEG C of 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 70s, 30 are followed
Ring；72℃10min；4 DEG C of preservations；
If a certain transformant with aam1/aam2 primer pair be PCR cannot about 470bp product, and use aam1/
AEndA primer pair is PCR and obtains the product of about 1.8kb, then this transformant is that foreign gene is whole in a manner of homologous double-crossover
Close the bacillus subtilis WB600Amy in DNA sequence-Neo+。
3. exogenous origin gene integrator is to bacillus subtilis DNA sequence
Plasmid pUC1ABC-ETRlb is after limitation restriction endonuclease Aat II is linearized, with DNA product purification kit
(TIANGEN) it purifies, converts bacillus subtilis WB600Amy-Neo+, it is coated on containing 1% (w/v) soluble starch, 1.5%
(w/v) in the screening flat board of agar, 37 DEG C are incubated overnight, and picking grows biggish single colonie, respectively parallel contact plate to LB plate,
LB plate containing 1% (w/v) soluble starch and on the LB plate of the neomycin containing 20ug/mL, after 37 DEG C of overnight incubations, contains 1%
(w/v) the LB plate of soluble starch is developed the color with Gram's iodine solution, and picking is removed from office in the LB plate containing 1% (w/v) soluble starch
There is hydrolysis to enclose after the colour developing of Lan Shi iodine solution, the single colonie that do not grow on the LB plate of the neomycin containing 20ug/mL obtains transformant, will
After single colonie is incubated overnight in LB culture medium, total DNA is extracted.Then PCR is carried out with primer pair AmyS/AEndA.PCR reaction
Condition difference is as follows:
PCR reaction system 20ul:DNA template (transformant total DNA) 1ul (about 20ng), 10 × Taq Buffer 2ul,
10pmol/ul dNTP 0.4ul, 10pmol/ul forward and reverse primer is respectively 0.5ul, 2.5U/ul Taq archaeal dna polymerase 1ul,
Add ddH2O to 20ul.PCR response procedures: 94 DEG C of 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃
10min；4 DEG C of preservations；
If a certain transformant is PCR with AmyS/AEndA primer pair and obtains the product of about 4.5kb (theoretically not successfully
The transformant PCR of integration obtains the product of about 2.3kb), then this transformant is that foreign gene is whole in a manner of homologous double-crossover
Close the bacillus subtilis WB600Amy in DNA sequence+[ETRlb].(1 constructing technology route of the present embodiment is shown in figure
1)。
Embodiment 2: the enzyme of bacillus subtilis integration site is utilized --- the activation recovering screening mango ethylene of invertase
The method that acceptor gene ETRlb is integrated into the recon of bacillus subtilis
1. the building of integrated plasmid
Synthetic primer:
InvS:GAGGACGTCATGACAGCACATGACCAGGA (underscore is II restriction enzyme site of Aat)
InvA:TAGGAGCTCTTTCTACATAAGTGTCCAAATTCC (underscore is I restriction enzyme site of Sac)
InvP:CAACTGCAGCCCGCTTCCAATTCACA (underscore is I restriction enzyme site of Pst)
IEndS:CCTCTGCAGTTCTTATGTGAAATCTGAGC (underscore is I restriction enzyme site of Pst)
IEndA:AATAAGCTTTTGCAGATTTCCTCAAA (underscore is III restriction enzyme site of Hind)
Wherein InvS/InvA amplification is bacillus subtilis saccharase gene sequence, and InvS/InvP amplification is withered grass
The section of DNA sequence that bacillus saccharase gene 5 ' is held, IEndS/IEndA amplification is bacillus subtilis saccharase gene
Section of DNA sequence after terminator codon.Bacillus subtilis is extracted with bacterial genomes DNA extraction kit (TIANGEN)
The genomic DNA of WB600 and as template, with primer I nvS/InvA, InvS/InvP, IEndS/IEndA and archaeal dna polymerase
PrimeSTAR HS DNA Polymerase (TAKARA) is expanded respectively, and reaction system and condition are as follows:
Reaction system is designed as 100uL total system, specifically 5 × PCR Buffer (buffer) 20uL, concentration 2.5mM
DNTPmix (deoxyribonucleoside triphosphate mixture) 5uL, concentration is each 2uL of upstream and downstream primer of 10mM, and concentration is
2.5U/uL PrimeSTAR HS DNA Polymerase (TAKARA) 2uL, DNA profiling 2uL (about 20ng), is mended with aqua sterilisa
Sufficient 100uL system.
Reaction condition are as follows: 94 DEG C of 3min initial denaturations, the interior 94 DEG C of 30s denaturation of circulation, 60 DEG C of annealing 30s, 72 DEG C of extensions
1.5min, 30 circulations；Continue to extend 10min for 72 DEG C after PCR reaction cycle, then 16 DEG C of preservations.
Amplified production carries out Ago-Gel recovery purifying, InvS/ with DNA purification and recovery kit (TIANGEN) respectively
The target fragment about 1.4kb of InvA recycling, is bacillus subtilis saccharase gene sequence, is named as AB；InvS/InvP recycling
Target fragment about 0.3kb, be bacillus subtilis saccharase gene 5 ' hold section of DNA sequence, be named as A (A as integration
The upstream homology arm of plasmid)；The target fragment about 0.3kb of IEndS/IEndA recycling is that bacillus subtilis saccharase gene is whole
The only section of DNA sequence after codon is named as C (downstream homology arm of the C as integrated plasmid).
With the small extraction reagent kit of plasmid (TIANGEN) extract plasmid pUC19, plasmid pUC19 and DNA fragmentation C use respectively Pst I/
III double digestion of Hind carries out Ago-Gel recovery purifying with DNA purification and recovery kit (TIANGEN) after digestion, wherein
PUC19 recycles about 2.7kb segment, and DNA fragmentation C recycles about 0.3kb segment, and the connection of the two segments converts bacillus coli DH 5 alpha,
It is coated on Amp containing 100ug/mL, surface is added in the LB screening flat board of 40mL X-gal liquid storage and 4 μ l IPTG liquid storages, and 37
It DEG C is incubated overnight.When single colonie in screening flat board grows to suitable size, plate is placed in 4 DEG C of a few hours, keeps colour developing complete.It chooses
White single colonie is cultivated, upgrading grain, with I/Hind of Pst, III double digestion, obtains the band of about 2.7kb Yu about 0.3kb size,
(Fig. 6) in the same size with theory, institute's upgrading grain is pUC2C.
Plasmid pUC2C and DNA fragmentation A uses II/Pst of Aat, I double digestion respectively, and DNA purification and recovery kit is used after digestion
(TIANGEN) Ago-Gel recovery purifying is carried out, wherein pUC2C recycles about 2.5kb segment, and DNA fragmentation A recycles about 0.3kb
Segment, the connection of the two segments, conversion bacillus coli DH 5 alpha, is coated in the LB screening flat board of the Amp containing 100ug/mL, 37 DEG C
It is incubated overnight.When single colonie in screening flat board grows to suitable size, picking single colonie, upgrading grain, with II/Pst of Aat, I pair of enzyme
It cuts, obtains the band of about 2.5kb Yu about 0.3kb size, (Fig. 7) in the same size with theory, institute's upgrading grain is pUC2AC.
Plasmid pUC2AC and plasmid pVK73 uses I digestion of Pst respectively, and DNA purification and recovery kit is used after digestion
(TIANGEN) Ago-Gel recovery purifying is carried out.Wherein pUC2AC recycles about 2.7kb segment；PVK73 recycles about 1.4kb piece
Section, this segment is antibiotics resistance gene ARG (Antibiotic Resistance Genes), specifically neomycin resistance
Gene.The connection of the two segments, conversion bacillus coli DH 5 alpha, are coated in the LB screening flat board of the 50ug/mL containing kanamycins, 37
It DEG C is incubated overnight.When single colonie in screening flat board grows to suitable size, picking single colonie, upgrading grain is obtained with I digestion of Pst
The band of about 2.7kb and about 1.4kb size, (Fig. 8) in the same size with theory, institute's upgrading grain is pUC2AC-NEO.
Plasmid pUC2C and DNA fragmentation AB uses II/Sac of Aat, I double digestion respectively, and DNA purification and recovery kit is used after digestion
(TIANGEN) Ago-Gel recovery purifying is carried out, wherein pUC2C recycles about 2.5kb segment, and DNA fragmentation AB recycles about 1.4kb
Segment, the connection of the two segments, conversion bacillus coli DH 5 alpha, is coated in the LB screening flat board of the Amp containing 100ug/mL, 37 DEG C
It is incubated overnight.When single colonie in screening flat board grows to suitable size, picking single colonie, upgrading grain, with II/Sac of Aat, I pair of enzyme
It cuts, obtains the band of about 2.5kb Yu about 1.4kb size, (Fig. 9) in the same size with theory, institute's upgrading grain is after sequence verification, life
Entitled pUC2ABC.
In the present embodiment, foreign gene is the mango Ethylene receptor gene ETRlb of this laboratory preservation.Plasmid pUC2ABC
And the cloned plasmids pUC19-ETRlb of the mango Ethylene receptor gene ETRlb of this laboratory preservation uses I/Kpn of Sal, I enzyme respectively
It cuts, carries out Ago-Gel recovery purifying with DNA purification and recovery kit (TIANGEN) after digestion, wherein pUC2ABC is recycled about
3.9kb segment, pUC19-ETRlb recycle about 2.2kb segment, and the connection of the two segments, conversion bacillus coli DH 5 alpha are coated on and contain
In the LB screening flat board of 100ug/mL Amp, 37 DEG C are incubated overnight.When single colonie in screening flat board grows to suitable size, picking
Single colonie, upgrading grain obtains the band of about 3.9kb Yu about 2.2kb size with I/Kpn of Sal, I double digestion, in the same size with theory
(Figure 10), institute's upgrading grain are pUC2ABC-ETRlb.
2. the building of the bacillus subtilis of invertase inactivation
Plasmid pUC2AC-NEO is after limitation restriction endonuclease Aat II is linearized, with DNA product purification kit (TIANGEN)
Purifying converts bacillus subtilis WB600, is coated on the LB plate of the neomycin containing 20ug/mL and screens, obtain transformant.
Synthesize following primer: InvN:CGCTGGCTCCGAGTGAT.After transformant is incubated overnight in LB culture medium, mention
Take total DNA.Then PCR is carried out with primer pair InvN/IEndA and InvS/IEndA respectively.PCR reaction condition difference is as follows:
InvN/IEndA:PCR reaction system 20ul:DNA template (transformant total DNA) 1ul (about 20ng), 10 × Taq
The forward and reverse primer of Buffer 2ul, 10pmol/ul dNTP 0.4ul, 10pmol/ul is respectively 0.5ul, 2.5U/ul Taq DNA
Polymerase 1ul adds ddH2O to 20ul.PCR response procedures: 94 DEG C of 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 are followed
Ring；72℃10min；4 DEG C of preservations；
InvS/IEndA:PCR reaction system 20ul:DNA template (transformant total DNA) 1ul (about 20ng), 10 × Taq
Buffer2ul, 10pmol/ul dNTP 0.4ul, 10pmol/ul forward and reverse primer are respectively 0.5ul, 2.5U/ul Taq DNA
Polymerase 1ul adds ddH2O to 20ul.PCR response procedures: 94 DEG C of 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 70s, 30 are followed
Ring；72℃10min；4 DEG C of preservations；
If a certain transformant with InvN/IEndA primer pair be PCR cannot about 1.5kb product, and use InvS/
IEndA primer pair is PCR and obtains the product of about 1.9kb, then this transformant is that foreign gene is whole in a manner of homologous double-crossover
Close the Strains B. subtilis WB600Ine in DNA sequence-Neo+。
3. exogenous origin gene integrator is to bacillus subtilis DNA sequence
Plasmid pUC2ABC-ETRlb is after limitation restriction endonuclease Aat II is linearized, with DNA product purification kit
(TIANGEN) it purifies, converts bacillus subtilis WB600Ine-neo+, it is coated on containing 1% (w/v) sucrose, 1.5% (w/v) fine jade
On the plate of rouge, 37 DEG C are incubated overnight, and are selected and are growed biggish single colonie, and the parallel contact plate of difference is to LB plate and containing 20ug/mL
On the LB plate of neomycin, after 37 DEG C of overnight incubations, picking is not grown on the LB plate of the neomycin containing 20ug/mL, in LB plate
The single colonie of upper growth, obtains transformant, after single colonie is incubated overnight in LB culture medium, extracts total DNA.Then primer is used
PCR is carried out to InvS/IEndA.PCR reaction condition difference is as follows:
PCR reaction system 20ul:DNA template (transformant total DNA) 1ul (about 20ng), 10 × Taq Buffer 2ul,
10pmol/ul dNTP 0.4ul, 10pmol/ul forward and reverse primer is respectively 0.5ul, 2.5U/ul Taq archaeal dna polymerase 1ul,
Add ddH2O to 20ul.PCR response procedures: 94 DEG C of 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃
10min；4 DEG C of preservations；
If a certain transformant is PCR with InvS/IEndA primer pair and obtains the product of about 4.0kb (theoretically not successfully
The transformant PCR of integration obtains the product of about 1.9kb), then this transformant is that foreign gene is whole in a manner of homologous double-crossover
Close the Strains B. subtilis WB600Ine in DNA sequence+[ETRlb] (2 constructing technology route of the present embodiment is shown in
Fig. 1).
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitation of the scope of the invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, protection scope of the present invention should be determined by the appended claims.
Sequence table
<110>Institute of Agro-Products Processing Science and Technology, Guangxi Zhuang Autonomous Region Academy af Agricultural Sciences
<120>method of the activation recovering screening integrative recombinant of a kind of enzyme using bacillus subtilis integration site
<141> 2018-12-29
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ttcgacgtct caaataagga gtgtca 26
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<213>artificial sequence (Artificial sequence Latin)
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taggagctcc ctcaatgggg aagaga 26
<210> 3
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<213>artificial sequence (Artificial sequence Latin)
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aggaagcttg ggacttaccg aaagaa 26
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aatcagctgc tgcttccaac aaaacc 26
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taggagctct ttctacataa gtgtccaaat tcc 33
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Claims (6)
1. a kind of method of the activation recovering screening integrative recombinant of enzyme using bacillus subtilis integration site, feature exist
In comprising the concrete steps that:
(1) resistant gene integrated plasmid is constructed, bacillus subtilis is converted, the end of gene 3 ' that screening obtains the enzyme of integration site lacks
Lose and inactivate bacillus subtilis as host strain, concrete operations are as follows:
S1, building make the end of the gene 3 ' missing of the enzyme of integration site and the resistant gene integrated plasmid of inactivation, the integrated plasmid
The DNA sequence dna of homologous double-crossover is A-ARG-C；
Wherein, A indicates upstream homology arm, is the section of DNA sequence that the enzyme gene 5 ' of bacillus subtilis integration site is held；
C indicate downstream homology arm, be integration site enzyme gene terminator codon after section of DNA sequence；
It is antibiotics resistance gene ARG between homology arm；
S2, screen integration site enzyme gene 3 ' end missing and inactivate bacillus subtilis: the resistant gene of linearisation is whole
Conjugative plasmid converts bacillus subtilis, cultivates, screens on antibiotic solid medium, passes through antibiotic resistance and integration
The enzyme in site inactivates, and obtains the end of the gene 3 ' missing of the enzyme of integration site and the bacillus subtilis of inactivation；
(2) exogenous origin gene integrator plasmid is constructed, wherein the DNA sequence dna of homologous double-crossover is AB-EGEU-C；
Wherein, AB is the complete sequence of the enzyme gene of bacillus subtilis integration site；
C indicate downstream homology arm, be integration site enzyme gene terminator codon after section of DNA sequence；
It is exogenous gene expression unit EGEU between homology arm；
(3) bacillus subtilis of the exogenous gene integration to bacillus subtilis integration site: the foreign gene of linearisation
Integrated plasmid converts the end of the gene 3 ' missing of the enzyme of integration site and the bacillus subtilis of inactivation, can enzyme with the enzyme of integration site
Solution using substance Solid agar culture as screening and culturing medium, pass through the enzyme activity of antibiotic resistance inactivation, integration site
Property, filter out the recombination engineering that exogenous origin gene integrator plasmid is integrated into bacillus subtilis by homologous double-crossover.
2. the activation recovering screening integration recombination of a kind of enzyme using bacillus subtilis integration site according to claim 1
The method of son, which is characterized in that the bacillus subtilis is 168 derivative strain of bacillus subtilis, including 1A751,
WB600 and WB800.
3. the activation recovering screening integration recombination of a kind of enzyme using bacillus subtilis integration site according to claim 1
The method of son, which is characterized in that
The enzyme of the integration site is bacillus subtilis alpha-amylase；
The DNA sequence dna AB is the complete sequence of bacillus subtilis alpha-amylase gene, by AmyS/AmyA primer amplification
?；
The DNA sequence dna A is the section of DNA sequence that bacillus subtilis alpha-amylase gene 5 ' is held, the matter of the A containing DNA sequence dna
Grain segment is obtained by the plasmid pUC1ABC of the complete sequence of the alpha-amylase gene containing bacillus subtilis through I digestion of Pst, purifying；
The DNA sequence dna C is the section of DNA sequence after bacillus subtilis alpha-amylase gene terminator codon, by
AEndS/AEndA primer amplification and obtain；
The antibiotics resistance gene ARG be neomycin resistance gene, Togoplus resistant gene, chloramphenicol resistance gene,
One of erythromycin resistance gene；
The antibiotic solid medium is the LB solid medium of the 20ug/mL containing neomycin in step s 2, or contains shape
The LB solid medium of miromycin 100ug/mL, or the LB solid medium of the 5ug/mL containing chloramphenicol, or 0.5ug/ containing erythromycin
The LB solid medium of mL；
It is solid for the only agar containing soluble starch that the Solid agar culture of the substance utilized can be digested described in step (3)
Body culture medium.
4. the activation recovering screening integration recombination of a kind of enzyme using bacillus subtilis integration site according to claim 3
The method of son, which is characterized in that AmyS, AmyA, AEndS, AEndA primer sequence is as follows:
AmyS:5′-TTCGACGTCTCAAATAAGGAGTGTCA-3′；
AmyA:5′-TAGGAGCTCCCTCAATGGGGAAGAGA-3′；
AEndS:5′-AGGAAGCTTGGGACTTACCGAAAGAA-3′；
AEndA:5′-AATCAGCTGCTGCTTCCAACAAAACC-3′。
5. the activation recovering screening integration recombination of a kind of enzyme using bacillus subtilis integration site according to claim 1
The method of son, which is characterized in that
The enzyme of the integration site is bacillus subtilis invertase；
The DNA sequence dna AB is the complete sequence of bacillus subtilis saccharase gene, by InvS/InvA primer amplification
?；
The DNA sequence dna A is the section of DNA sequence that bacillus subtilis saccharase gene 5 ' is held, and is expanded by InvS/InvP primer
Increase and obtains；
The DNA sequence dna C is the section of DNA sequence after bacillus subtilis saccharase gene terminator codon, by IEndS/
IEndA primer amplification and obtain；
The antibiotics resistance gene ARG be neomycin resistance gene, Togoplus resistant gene, chloramphenicol resistance gene,
One of erythromycin resistance gene；
The antibiotic solid medium is the LB solid medium of the 20ug/mL containing neomycin in step s 2, or contains shape
The LB solid medium of miromycin 100ug/mL, or the LB solid medium of the 5ug/mL containing chloramphenicol, or 0.5ug/ containing erythromycin
The LB solid medium of mL；
The Solid agar culture of the substance utilized can be digested described in step (3) as the only agar solid culture containing sucrose
Base.
6. the activation recovering screening integration recombination of a kind of enzyme using bacillus subtilis integration site according to claim 5
The method of son, which is characterized in that
InvS, InvA, InvP, IEndS, IEndA primer sequence is as follows:
InvS:5′-GAGGACGTCATGACAGCACATGACCAGGA-3′；
InvA:5′-TAGGAGCTCTTTCTACATAAGTGTCCAAATTCC-3′；
InvP:5′-CAACTGCAGCCCGCTTCCAATTCACA-3′；
IEndS:5′-CCTCTGCAGTTCTTATGTGAAATCTGAGC-3′；
IEndA:5′-AATAAGCTTTTGCAGATTTCCTCAAA-3′。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172433A (en) * | 2019-05-12 | 2019-08-27 | 华中农业大学 | It is a kind of produce pig's epidermal growth factor recombined bacillus subtilis engineering bacteria and application |
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