CN105802985A - Method for achieving bacillus licheniformis gene knockout rapidly - Google Patents
Method for achieving bacillus licheniformis gene knockout rapidly Download PDFInfo
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Abstract
The invention relates to a method for achieving bacillus licheniformis gene knockout rapidly. The method comprises the following steps of 1, obtaining a gene fragment in a bacillus licheniformis to-be-knocked-out gene coding frame as a homologous arm sequence; 2, obtaining a resistance label gene fragment; 3, conducting an overlapped and extended PCR on the homologous arm sequence and the resistance label gene fragment, so that a fused gene sequence of the homologous arm sequence and the resistance label gene fragment is obtained, and treating the fused gene sequence as a gene knockout fragment for use; 4, conducting single digestion on the fused gene sequence, converting bacillus licheniformis competent cells, and conducting recovery culture and screening culture, so that bacillus licheniformis with a gene knocked out is obtained. According to the method, the bacillus licheniformis gene is knocked out based on the single exchange principle, in the knockout process, homologous recombination only needs to be generated once, and the integration process is high in efficiency; whole knockout and screening work can be completed within three or four days, the gene knockout cycle is short, and knockout cost is effectively lowered.
Description
Technical field
The present invention relates to a kind of method quickly realizing Bacillus licheniformis gene knockout, belong to technical field of biotechnology.
Background technology
Bacillus licheniformis (Bacilluslicheniformis), is a kind of Gram-positive thermophilic bacteria being widely present and resisting adverse circumstances at nature, is one of production bacterial strain important in biotechnological industries field.Currently mainly it is applied to medicine, the biological field such as detergent, nanotechnology, and shows up prominently in biological material degraded.By molecular modification, the metabolism of Bacillus licheniformis carries out regulation and control to realize purpose product accumulation and have been reported.
Gene knockout (geneknockout), also known as gene targeting, it it is a kind of novel Protocols in Molecular Biology, it utilizes DNA transformation technology, after the targeting vector built is imported target cell, by homologous DNA sequence on chromosome in recombinant vector DNA sequence and target cell, carrier DNA site-directed integration is entered a certain site determined on target cell genome, or a certain with on target cell genome determine that fragment is replaced, make the specific gene inactivation of body or disappearance, thus the method changing cell trait.At present, when Bacillus licheniformis is carried out gene knockout, mostly it is adopt the homologous double-crossover method being carrier with plasmid, namely genes of interest upstream and downstream two fragment gene sequence is obtained as homology arm, build shuttle vector or suicide vector and be transformed in purpose bacterial strain, need successively to occur twice single-swap after conversion, it is achieved knocking out of target gene.Although the method carrying out gene knockout with above-mentioned principle is widely used at present, but unitary construction flow process is complicated, and the operation cycle is long.
As Chinese patent literature CN103146739A (application number: CN201310070885) the serpin gene knockout carrier built is connected and composed by element head and the tail such as upstream homology arm (Up-serpin), resistant gene, downstream homology arm (Down-serpin) and pUC57, therefore only carrier construction is accomplished by enzyme action connection for several times and sequencing procedures, complete even more than one month several weeks consuming time of whole operation cycle, simultaneously because carrier structure is complicated, sequence is longer, and transformation efficiency is generally relatively low.Although said method is capable of the specific demands such as the traceless knockout of gene, but in the gene knockout being verified as purpose with gene function operates, such method often becomes the speed limit link of whole experiment flow.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a kind of method quickly realizing Bacillus licheniformis gene knockout.The method utilizes round pcr two step to build and knocks out fragment, converts after enzyme action to target Bacillus licheniformis, by a homologous single-crossover, inserts one section of sequence containing riddled basins, it is achieved knocking out of genes of interest inside genes of interest.
Technical solution of the present invention is as follows:
A kind of method quickly realizing Bacillus licheniformis gene knockout, it is characterised in that comprise the following steps:
(1) design primers F 1 and R1, with Bacillus licheniformis genome for template, by round pcr, intending knocking out a segment length in gene code frame in acquisition Bacillus licheniformis is that the genetic fragment more than 400bp is as homology arm sequence；
(2) design primers F 2 and R2, with the carrier containing resistance label gene or sequence for template, by round pcr, obtains resistance label genetic fragment；
(3) the resistance label genetic fragment prepared by the homology arm sequence that step (1) is prepared and step (2) carries out Overlap extension PCR, obtain and comprise homology arm sequence and the fusion gene sequence of resistance label genetic fragment, and use as gene knockout fragment, obtained fusion fragment two end all to contain by primers F 1/R2 or the F2/R1 identical restriction enzyme digestion sites introduced, and this restriction enzyme site is not knocked out in gene and resistance label genetic fragment in plan；
(4) the fusion gene sequence that step (3) is prepared carries out single endonuclease digestion, then being concentrated into concentration is 300～5000 μ g/ml, convert Bacillus licheniformis competent cell, cultivate and after screening and culturing through recovery, prepare the Bacillus licheniformis of gene knockout.
According to currently preferred, intending in described step (1) knocking out gene is glycosidehydrolase, and during nucleotide sequence such as SEQIDNO.1, the nucleotide sequence of primers F 1 and R1 is as follows:
F1:CGGGATCCAACCGTTCTTTTATCCGCAGT
R1:CCAGCAAAATTCAGCATCTCTCGGTTAAGCA
Wherein, underscore mark is BamHI restriction enzyme site.
According to the present invention it is further preferred that in described step (1) PCR amplification system as follows:
Primer R12.0 μ l, the template 2.0 μ l of the primers F 12.0 μ l, 10 μm of ol/L of 2 × HiFi-PCRmaster25 μ l, 10 μm of ol/L, uses ddH2O supplies 50 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
According to currently preferred, in described step (2), resistance label gene is chloromycetin, and when nucleotide sequence is such as shown in SEQIDNO.2, the nucleotide sequence of primers F 2 and R2 is as follows:
F2:CCGAGAGATGCTGAATTTTGCTGGCCTTTTGCTCA
R2:AACGTATCATGGGATCCTAGTGACTGGCGATGCTG
Wherein, underscore mark is BamHI restriction enzyme site.
According to the present invention it is further preferred that in described step (2) PCR amplification system as follows:
2 × HiFi-PCRmaster25 μ l, the primers F of 10 μm of ol/L2The primer R of 2.0 μ l, 10 μm of ol/L22.0 μ l, template 2.0 μ l, use ddH2O supplies 50 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 3min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
According to currently preferred, in described step (3), the first amplification system of over-lap PCR is as follows:
GH1 fragment 2 μ l；CmrFragment 2 μ l；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 5 circulations；72 DEG C extend 2min；
The supplementary amplification system of described over-lap PCR is as follows:
The forward primer F of 10 μm of ol/L12μl；The downstream primer R of 10 μm of ol/L22μl；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
According to currently preferred, in described step (4), the preparation process of Bacillus licheniformis competent cell is as follows:
The fresh Bacillus licheniformis list bacterium colony of picking, 35～38 DEG C, 200～240rpm when amplification culture to cell concentration OD600It is 0.7～0.9, is placed in cooled on ice, centrifugal after cooling, then turn buffer solution thalline 3～5 times with the electricity of pre-cooling, subpackage, to the aseptic EP pipe of pre-cooling, prepares Bacillus licheniformis Electrocompetent cells；
Described amplification culture base is that addition sorbitol to concentration is 0.4～0.6M on the basis of LB culture medium；
It is as follows that described electricity turns buffer composition:
0.4～0.6M sorbitol, 0.4～0.6M mannitol, 0.05M～0.16M glycerol, water is prepared.
According to currently preferred, being converted into electricity conversion in described step (4), electricity conversion condition is 1800～2200V electric shock 4～6ms, add liquid resuscitation culture medium culturing 3～4h immediately after, after centrifugal, taking thalline, suspension is cultivated containing antibiotic plate screening；
Described liquid resuscitation culture medium is add sorbitol to concentration on the basis of LB culture medium to be 0.4～0.6M and mannitol to concentration is 0.4～0.5M.
According to currently preferred, a kind of method quickly realizing Bacillus licheniformis glycosidehydrolase gene knockout, it is characterised in that comprise the following steps:
I () extracts the DNA of Bacillus licheniformis body, with DNA for template, use primers F1And R1Carrying out pcr amplification, obtain the homology arm GH1 for GH gene knockout, described PCR primer sequence is as follows:
F1:CGGGATCCAACCGTTCTTTTATCCGCAGT
R1:CCAGCAAAATTCAGCATCTCTCGGTTAAGCA
(ii) extract the DNA of the PHT01 of shuttle plasmid, with DNA for template, use primers F2And R2Carry out pcr amplification, obtain chloramphenicol resistance gene fragment Cmr, described PCR primer sequence is as follows:
F2:CCGAGAGATGCTGAATTTTGCTGGCCTTTTGCTCA
R2:AACGTATCATGGGATCCTAGTGACTGGCGATGCTG
(iii) by Cm prepared to GH1 fragment prepared for step (i) and step (ii)rFragment carries out over-lap PCR, prepares GH1-CmrFragment；
The first amplification system of described over-lap PCR is as follows:
GH1 fragment 2 μ l；CmrFragment 2 μ l；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 5 circulations；72 DEG C extend 2min；
The supplementary amplification system of described over-lap PCR is as follows:
The forward primer F of 10 μm of ol/L12μl；The downstream primer R of 10 μm of ol/L22μl；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
(iv) by GH1-Cm prepared for step (iii)rFragment restricted enzyme BamHI digests, and utilize electricity conversion instrument to convert to Bacillus licheniformis competent cell, electricity conversion condition is 1800～2200V electric shock 4～6ms, add liquid resuscitation culture medium culturing 3～4h immediately after, after centrifugal, taking thalline, the cell obtained is coated on the LuriaBroth solid medium containing chloromycetin, cultivating 1～2 day at 37 DEG C, screening has the transformant of chlorampenicol resistant.
Beneficial effect
The present invention carries out Bacillus licheniformis gene knockout based on single-swap principle, and the process that knocks out only needs to produce a homologous recombination (single-swap), and integration process is efficient；The fragment that knocks out of present invention structure only comprises two sections of sequences (homology arm and antibiotic resistance label gene), builds flow process simple；Bacillus licheniformis gene knockout method provided by the present invention, whole knocking out can complete with screening operation in 3～4 days, and the gene knockout cycle is short, and effectively reduction knocks out cost, improves work efficiency.
Accompanying drawing explanation
Fig. 1 is the structure of Bacillus licheniformis fragment and knocks out flow chart；
Fig. 2 is the electrophoresis detection result photo of homology arm GH1；
Swimming lane M is DNA molecular amount labelling (DNAmarker), and swimming lane 1～3 is positive recombinant the result.
Fig. 3 is CmrThe electrophoresis detection result photo of fragment；
Swimming lane M is DNA molecular amount labelling (DNAmarker), and swimming lane 1～4 is positive recombinant the result.
Fig. 4 be Bacillus licheniformis glycosidehydrolase gene knock out the result photo；
Swimming lane M is DNA molecular amount labelling (DNAmarker), and swimming lane 1～5 is positive recombinant the result.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to this.
Biological material source:
Shuttle plasmid PHT01 is purchased from Bao Sai bio tech ltd, Hangzhou；
Bacillus licheniformis (Bacilluslicheniformis) is purchased from Chinese industrial Culture Collection (CICC)；Bacterium numbering is CICC20085；
LB nutrient media components is as follows, is all weight percentage:
Peptone 1%, yeast leaching powder 0.5%, sodium chloride 1%, surplus is water, pH7.0～7.4.
Embodiment 1
Gene knockout fragment builds
I () extracts the DNA of Bacillus lichenformis thalline, with DNA for template, carry out pcr amplification, obtain homology arm GH1；
Described PCR primer sequence is as follows:
F1:CGGGATCCAACCGTTCTTTTATCCGCAGT
R1:CCAGCAAAATTCAGCATCTCTCGGTTAAGCA
Wherein, underscore is designated BamHI restriction enzyme site；
Described PCR amplification system is 50 μ l:
Primer R12.0 μ l, the template 2.0 μ l of the primers F 12.0 μ l, 10 μm of ol/L of 2 × HiFi-PCRmaster25 μ l, 10 μm of ol/L, uses ddH2O supplies 50 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, length is that (SEQIDNO.3, as shown in Figure 2), uses SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai) and carry out glue recovery, reclaim product-20 DEG C preservation 549bp, standby；
(ii) extract the DNA of shuttle plasmid pHT01, with DNA for template, carry out pcr amplification, obtain CmrFragment；
Described PCR primer sequence is as follows:
F2:CCGAGAGATGCTGAATTTTGCTGGCCTTTTGCTCA
R2:AACGTATCATGGGATCCTAGTGACTGGCGATGCTG
Wherein, underscore is designated BamHI restriction enzyme site
Described PCR amplification system is 50 μ l:
2 × HiFi-PCRmaster25 μ l, the primers F of 10 μm of ol/L2The primer R of 2.0 μ l, 10 μm of ol/L22.0 μ l, template 2.0 μ l, use ddH2O supplies 50 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 3min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, length is that (SEQIDNO.2, as shown in Figure 3), uses SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai) and carry out glue recovery, reclaim product-20 DEG C preservation 1245bp, standby；
(iii) by Cm prepared to GH1 fragment prepared for step (i) and step (ii)rFragment carries out over-lap PCR, prepares GH1-CmrFragment；
The first amplification system of described over-lap PCR is 25 μ l:
GH1 fragment 2 μ l；CmrFragment 2 μ l；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 5 circulations；72 DEG C extend 2min；
The supplementary amplification system of described over-lap PCR is 25 μ l:
The forward primer F of 10 μm of ol/L12μl；The downstream primer R of 10 μm of ol/L22μl；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, length is 1794bp, uses SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai) and carries out glue recovery, reclaims product-20 DEG C preservation, standby；
After testing, the gene order knocked out is such as shown in SEQIDNO.1.
Embodiment 2
Preparation Bacillus licheniformis competence
I the Bacillus licheniformis list bacterium colony of the fresh LB solid culture primary surface of () picking, is inoculated in 10mLGM (expansion) culture medium, 37 DEG C, 220r/min, incubated overnight；
(ii) take the above-mentioned bacterium solution of 1mL and be transferred to 100mLGM (expansion) fluid medium, 37 DEG C, 220r/min be cultured to OD600=0.9；
(iii) bacterium solution is transferred to 100mL centrifuge tube, ice bath 20min, makes thalline stop growing；
(iv) after ice bath 4 DEG C, 5000g, 5min centrifugal, collect thalline；
The electricity of the thalline pre-cooling v () is centrifugal after turns buffer (ETM) and washs 3 times；
(vi), after washing terminates, 1000uL electricity is used to turn the resuspended thalline of buffer；
(vii) the competent cell subpackage 100 μ L prepared often is managed ,-80 DEG C of preservations, standby.
Wherein, GM:LB+0.5M sorbitol
ETM:0.5M sorbitol+0.5M mannitol+0.11M glycerol.
Embodiment 3
By GH1-CmrFragment converts Bacillus licheniformis cell
I GH1-Cm that embodiment 1 is prepared by ()rFragment restricted enzyme BamHI digests；
Enzyme action system (40uL) is as follows:
(ii) digestion products is concentrated and purified
(1) add 1/10 volume 3M sodium acetate and 2.5 times of volume dehydrated alcohol, be placed in-20 DEG C of refrigerator 20min；
(2) 12000r/min, centrifugal 5min must precipitate；
(3) 300 μ L percents by volume are the resuspended precipitation of ethanol of 75%；
(4) 12000r/min, centrifugal 5min, remove ethanol, 37 DEG C of air-dry 30min；
(5) 15～18 μ LddH are added2The resuspended DNA of O, is placed in-20 DEG C of preservations.
(iii) electricity converts
Nucleic acid ultramicrospectrophotometer is utilized to measure GH1-CmrFragment concentrations, electricity conversion is carried out after reaching concentration 2000 μ g/ml, electricity conversion condition is 2100V, 5ms, then cultivate in RM (liquid resuscitation) culture medium, take 100 μ L after the cell recovery obtained and be coated on the LB solid medium containing 30 μ g/ml chloromycetin, cultivating 1～2 day at 37 DEG C, screening has the transformant of chlorampenicol resistant；
RM:LB+0.5M sorbitol+0.38M mannitol.
Embodiment 4
The cultivation of positive recombinant bacterium and qualification
The above-mentioned positive restructuring bacterium colony of picking, is inoculated in the LB liquid medium containing chlorampenicol resistant 37 DEG C of overnight incubation, after having cultivated, uses the test kit that Shanghai biological engineering company limited provides to extract recombinant bacterium DNA, and with the genome of acquisition for template, F1And R2Carrying out pcr amplification for primer, amplified production utilizes agarose gel electrophoresis to be verified；
Described PCR primer sequence is as follows:
F1:CGGGATCCAACCGTTCTTTTATCCGCAGT
R2:AACGTATCATGGGATCCTAGTGACTGGCGATGCTG
Wherein, underscore mark is restriction enzyme site
Described PCR amplification system is 20 μ l:
2 × HiFi-PCRmaster10 μ l, primers F1(10 μm of ol/L) 1.0 μ l, primer R2(10 μm of ol/L) 1.0 μ l, template 1.0 μ l, uses ddH2O supplies 20 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 4min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations, agarose gel electrophoresis inspection PCR primer.
Testing result is as shown in Figure 2.By this result it can be seen that the present invention knocks out process only needs homologous recombination (single-swap) of generation, it is possible to effectively reduce knocking out cost, improve work efficiency.
Claims (9)
1. the method quickly realizing Bacillus licheniformis gene knockout, it is characterised in that comprise the following steps:
(1) design primers F 1 and R1, with Bacillus licheniformis genome for template, by round pcr, intending knocking out a segment length in gene code frame in acquisition Bacillus licheniformis is that the genetic fragment more than 400bp is as homology arm sequence；
(2) design primers F 2 and R2, with the carrier containing resistance label gene or sequence for template, by round pcr, obtains resistance label genetic fragment；
(3) the resistance label genetic fragment prepared by the homology arm sequence that step (1) is prepared and step (2) carries out Overlap extension PCR, obtain and comprise homology arm sequence and the fusion gene sequence of resistance label genetic fragment, and use as gene knockout fragment, obtained fusion fragment two end all to contain by primers F 1/R2 or the F2/R1 identical restriction enzyme digestion sites introduced, and this restriction enzyme site is not knocked out in gene and resistance label genetic fragment in plan；
(4) the fusion gene sequence that step (3) is prepared carries out single endonuclease digestion, then being concentrated into concentration is 300～5000 μ g/ml, convert Bacillus licheniformis competent cell, cultivate and after screening and culturing through recovery, prepare the Bacillus licheniformis of gene knockout.
2. the method for claim 1, it is characterised in that intending in described step (1) knocking out gene is glycosidehydrolase, and during nucleotide sequence such as SEQIDNO.1, the nucleotide sequence of primers F 1 and R1 is as follows:
F1:CGGGATCCAACCGTTCTTTTATCCGCAGT；
R1:CCAGCAAAATTCAGCATCTCTCGGTTAAGCA。
3. method as claimed in claim 2, it is characterised in that in described step (1), PCR amplification system is as follows:
Primer R12.0 μ l, the template 2.0 μ l of the primers F 12.0 μ l, 10 μm of ol/L of 2 × HiFi-PCRmaster25 μ l, 10 μm of ol/L, uses ddH2O supplies 50 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
4. the method for claim 1, it is characterised in that in described step (2), resistance label gene is chloromycetin, when nucleotide sequence is such as shown in SEQIDNO.2, the nucleotide sequence of primers F 2 and R2 is as follows:
F2:CCGAGAGATGCTGAATTTTGCTGGCCTTTTGCTCA；
R2:AACGTATCATGGGATCCTAGTGACTGGCGATGCTG。
5. method as claimed in claim 4, it is characterised in that in described step (2), PCR amplification system is as follows:
2 × HiFi-PCRmaster25 μ l, the primers F of 10 μm of ol/L2The primer R of 2.0 μ l, 10 μm of ol/L22.0 μ l, template 2.0 μ l, use ddH2O supplies 50 μ l；
Described pcr amplification program is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 3min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
6. the method for claim 1, it is characterised in that in described step (3), the first amplification system of over-lap PCR is as follows:
GH1 fragment 2 μ l；CmrFragment 2 μ l；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 5 circulations；72 DEG C extend 2min；
The supplementary amplification system of described over-lap PCR is as follows:
The forward primer F of 10 μm of ol/L12μl；The downstream primer R of 10 μm of ol/L22μl；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
7. the method for claim 1, it is characterised in that in described step (4), the preparation process of Bacillus licheniformis competent cell is as follows:
The fresh Bacillus licheniformis list bacterium colony of picking, 35～38 DEG C, 200～240rpm when amplification culture to cell concentration OD600It is 0.7～0.9, is placed in cooled on ice, centrifugal after cooling, then turn buffer solution thalline 3～5 times with the electricity of pre-cooling, subpackage, to the aseptic EP pipe of pre-cooling, prepares Bacillus licheniformis Electrocompetent cells；
Described amplification culture base is that addition sorbitol to concentration is 0.4～0.6M on the basis of LB culture medium；
It is as follows that described electricity turns buffer composition:
0.4～0.6M sorbitol, 0.4～0.6M mannitol, 0.05M～0.16M glycerol, surplus is water.
8. the method for claim 1, it is characterized in that, described step (4) is converted into electricity conversion, electricity conversion condition is 1800～2200V electric shock 4～6ms, add liquid resuscitation culture medium culturing 3～4h immediately after, after centrifugal, taking thalline, suspension is cultivated containing antibiotic plate screening；
Described liquid resuscitation culture medium is add sorbitol to concentration on the basis of LB culture medium to be 0.4～0.6M and mannitol to concentration is 0.4～0.5M.
9. the method quickly realizing Bacillus licheniformis glycosidehydrolase gene knockout, it is characterised in that comprise the following steps:
I () extracts the DNA of Bacillus licheniformis body, with DNA for template, use primers F1And R1Carrying out pcr amplification, obtain the homology arm GH1 for GH gene knockout, described PCR primer sequence is as follows:
F1:CGGGATCCAACCGTTCTTTTATCCGCAGT
R1:CCAGCAAAATTCAGCATCTCTCGGTTAAGCA
(ii) extract the DNA of the PHT01 of shuttle plasmid, with DNA for template, use primers F2And R2Carry out pcr amplification, obtain chloramphenicol resistance gene fragment Cmr, described PCR primer sequence is as follows:
F2:CCGAGAGATGCTGAATTTTGCTGGCCTTTTGCTCA
R2:AACGTATCATGGGATCCTAGTGACTGGCGATGCTG
(iii) by Cm prepared to GH1 fragment prepared for step (i) and step (ii)rFragment carries out over-lap PCR, prepares GH1-CmrFragment；
The first amplification system of described over-lap PCR is as follows:
GH1 fragment 2 μ l；CmrFragment 2 μ l；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 1.5min, 5 circulations；72 DEG C extend 2min；
The supplementary amplification system of described over-lap PCR is as follows:
The forward primer F of 10 μm of ol/L12μl；The downstream primer R of 10 μm of ol/L22μl；2×HiFi-PCRmaster12.5μl；ddH2O8.5μl；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturation 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
(iv) by GH1-Cm prepared for step (iii)rFragment restricted enzyme BamHI digests, and utilize electricity conversion instrument to convert to Bacillus licheniformis competent cell, electricity conversion condition is 1800～2200V electric shock 4～6ms, add liquid resuscitation culture medium culturing 3～4h immediately after, after centrifugal, taking thalline, the cell obtained is coated on the LuriaBroth solid medium containing chloromycetin, cultivating 1～2 day at 37 DEG C, screening has the transformant of chlorampenicol resistant.
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| CN107641631A (en) * | 2017-09-07 | 2018-01-30 | 浙江工业大学 | A kind of method that bacillus coli gene is knocked out based on CRISPR/Cas9 systems by chemical conversion mediation |
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