CN106755046B - A method of transformation bacillus gene group - Google Patents
A method of transformation bacillus gene group Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a method of transformation bacillus gene group.The present invention is adaptable, and extensively (it is low that plasmid converts difficulty, pBTS plasmid can be replicated in a variety of gram-positive bacterias, the genome of a variety of bacterium is transformed), same bacterial strain repeated multiple times can be transformed, and (there are two homologous recombination segments for recombinant plasmid tool, homologous recombination twice successively occurs, wild type may be restored to after second of homologous recombination, also available transformation bacterial strain, but the segment that any influence continues transformation is not remained, such as resistance marker, plasmid replication element etc.), and it can be transformed and (cre recombinase and second of resistance marker system are introduced to the gene that bacterial growth is affected, obtained transformation bacterial strain can be recombinated for the second time by resistance screening, kill the strong wild-type strain of survival ability, cre recombinase is expressed later, cut away second of resistance marker, remaining segment will not influence right again Bacterial strain is transformed).
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a method of transformation bacillus gene group.
Background technique
Protein expression techniques are one of core technologies of modern biology, and expression albumen cannot be only used for biological study,
Commercialized protein product, such as recombinant vaccine, Recombulin, cell factor product can also be provided.Currently used table
There are Escherichia coli, yeast, insect cell and mammalian cell etc. up to system, but they all respectively there are apparent advantage and disadvantage.Greatly
The research of enterobacteria expression system is most abundant, and there are many selections, and the most commonly used is the pET expression systems of Novagen, utilizes phagocytosis
The t7 rna polymerase of body can transcribe the target gene after T7 promoter in specific manner.At optimum conditions, destination protein can reach
To 50% or more of e. coli total protein.Although having expression efficiency high, the advantages such as toxigenic capacity is low, disadvantage is also very
It is obvious: albumen inclusion body easy to form, renaturation difficulty and higher cost；Escherichia coli cannot carry out albumen glycosylation modified；
Cell wall contains lipopolysaccharides (endotoxin), is not easy to completely remove.Another common expression system is yeast expression system,
It with expression quantity height, can induce, protein secretion is easy to purify to extracellular, and excellent with certain posttranslational modification ability etc.
Point, but a disadvantage is that part expression product is degradable, expression quantity is uncontrollable, and the albumen greater than 30KDa can hardly be secreted.Animal
The characteristics of cell and insect cell expression system is that have complete modification system, and expression product has or similar natural work
Property, without contaminated with endotoxins, but expression quantity is low, and the period is long, and technical requirements are high, high production cost.
Bacillus subtilis be it is a kind of be widely present in water body, air, the gram-positive bacteria in soil, can be in ring
Spore is generated when border is unfavorable, spore can resist the extreme environments such as high temperature, arid, sprout progress again when environment is suitable for
Nutrient growth.The secretion capacity of bacillus subtilis is strong, and when carrying out high density fermentation, protein secretion can achieve 20-
25g/L.The products such as protease, amylase, inosine, the riboside of its fermenting and producing enter our day already
Often life.Since it is with biological safety, by FDA judging panel's GRAS class additive, the probiotics of production and its production is utilized
Fermented product be widely used to medicine and aquaculture in.
Last century the eighties begin to carry out protein expression using bacillus subtilis, and expression product not only includes thin
The various enzymes in bacterium source, such as amylase, protease, endo-dextranase, lipase, further include hEGF, IFN-alpha 2,
The albumen of the separate sources such as Proinsulin, Streptavidin, cathelicidin-BF.Utilize point of bacillus subtilis
Secrete approach, can will expression protein secretion into fermentation liquid, significantly reduce the difficulty that the later period isolates and purifies.Bacillus subtilis
Belong to gram-positive bacteria, does not contain lipopolysaccharides, injection drug easy to produce.The nutritional requirement of bacillus subtilis is simple,
It is produced since it has been carried out technical scale metaplasia, large-scale culture technical difficulty is low, and toxigenic capacity is low.Currently, bacillus subtilis
Multiple bacterial strains in bacterium and multiple kinds in bacillus all have been completed that gene order-checking works, and genetic background understands,
It is highly-safe, it is also convenient for carrying out genome manipulation.But bacillus subtilis expression system is not a widely applied table
Up to system, there are many more disadvantage is to be overcome.
The wild-type strain of 168 bacterium of mode bacterium of bacillus subtilis has been lost, and the bacterial strain being currently available all is
Its mutant.Although its requirement for being satisfied with scientific research, protein secretion ability is poor, is auxotrophic mutations
Characteristic is not able to satisfy the requirement for establishing commercialization expression system.The conversion system established at present is all based on type strain 168
It establishes, although the bacillus subtilis strain of the bacterial strain in wild type source and production application is very more, is not easy conversion and all hinders
The further transformation to these bacterial strains is hindered.The plasmid in bacillus subtilis source is also nearly all stealthy plasmid, is not easy to construct
Carrier；Plasmid majority from other gram-positive bacterias is all rolling-circle replication property grain, is replicated in bacillus subtilis
Unstable, easy to be lost, copy number is less；These characteristics all limit the possibility for directly stablizing expression system using plasmid construction
Property.Bacillus subtilis is there are four types of secretory pathway, Sec approach, Tat approach, Com system and abc transport approach, research at present compared with
It is clear that Sec approach, some expression systems also utilize Sec approach secreting, expressing albumen.But Sec approach has regulation machine
System can inhibit the protein secretion of incorrect folding, and foreign protein generally folds relatively slowly, and folding process needs the ginseng of chaperone
With, be easy degraded by Sec secretory pathway.Bacillus subtilis can also secrete up to eight kinds of protease into fermentation liquid, same
Can degrade the destination protein of expression.
Overcoming the effective way of disadvantages mentioned above is: without using the carrier of plasmid gene and expression system element as a purpose,
It directly will be in target gene and expression system element insertion Bacillus subtilis genes group；Its genome is transformed, is knocked out
The gene of some inhibition or influence protein secretion.At present there are two ways to transformed bacillus bacillus gene group, the first
It is that direct conversion double chain DNA fragment enters cell while carrying out homologous recombination, it is high to transformation efficiency requirement, it is currently only used for
168 mode bacterium.Second is using temperature-sensitive plasmid, and converting, there is the temperature-sensitive plasmid of homologous recombination segment to enter carefully
Then born of the same parents cultivate under restrictive temperature, the bacterium of homologous recombination can not occur for the bacterium recombinated due to matter by screening
Grain is lost, and screening cannot be passed through.The conversion of this method and reconstitution steps separate, and reduce the requirement to transformation efficiency.It can select
The temperature-sensitive plasmid selected is pE194, but the plasmid is the plasmid that can only be replicated in gram-positive bacteria, Bu Neng
It is replicated in Escherichia coli, i.e., cannot quickly carry out plasmid construction work using Escherichia coli.Solution first is that structure
Fusion plasmid, the i.e. fusion plasmid of pE194 and pBR322 are built, which possesses two sets of dubbing systems, and difference can be in withered grass bud
It is replicated in spore bacillus and Escherichia coli.But the plasmid when being replicated in Escherichia coli stability it is poor, if polyclonal
When site insertion is less than the segment of 1.5k, plasmid can stablize duplication；If Insert Fragment be greater than 2k, the plasmid duplication when just
It can there is a phenomenon where fragment loss.Another normal temperature sensitive plasmid is pKS1, which is host range plasmid more than one, can
To replicate in most gram-positive bacterias and negative bacterium, but the plasmid stability is poor, and copy number is lower.Document
(Rational Design of a Plasmid Origin That Replicates Efficiently in Both
Gram-Positive and Gram-Negative Bacteria, 2010) the original plasmid pWV01 of pKS1 is transformed,
Its stability and copy number in Escherichia coli is increased, vector construction is convenient for.But not to transformation plasmid in text
The temperature-sensing property of pBAV1K is studied, and temperature of the pBAV1K-T5-GFP in bacillus subtilis is found in our work
Degree sensitive copy characteristic still remains, but restricted cultivation temperature is increased to 45 DEG C from 37 DEG C of pKS1.According to pBAV1K-
These characteristics of T5-GFP plasmid, the method that a set of transformed bacillus bacillus gene group can be developed.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of method that bacillus gene group is transformed, more places are utilized
Main high copy number plasmid pBAV1K stability in Escherichia coli is high, and copy number is more, is easy to the characteristic of transformation and in bacillus
Temperature sensitive copy characteristic, construct the method for a set of transformed bacillus bacillus gene group, adaptable wide, transformation
Plasmid is easy to convert, and can be replicated in a variety of bacillus, a variety of bacillus are transformed；It can repeat that same bacterium is transformed
Strain, not remaining resistance, reproduction element etc. after transformation every time influences the segment of transformation next time；Can also be transformed influences strain growth
Biggish gene introduces cre recombinase and the second resistance screening label.
Solve the method that bacillus gene group is transformed in one of present invention of the above technical problem, it is characterised in that:
The following steps are included:
(1) it is inserted into multiple cloning sites (MCS) segment in EcoR I and the Apa I site of pBAV1K-T5-GFP plasmid, and
By same sense mutation, NdeI restriction enzyme site therein is deleted, plasmid pBTS is obtained.
PBTS plasmid can be replicated in a variety of gram-positive bacterias, and the genome of a variety of bacterium is transformed.
(2) restriction enzyme site in the left end in the site MCS is inserted into the homologous recombination segment A of 500-800bp, right side restriction enzyme site
It is inserted into the homologous recombination segment B of 500-800bp, intermediate insertion mutation segment or target fragment or is not inserted into any segment, both
The segment for needing to be transformed between two homologous recombination regions, obtains plasmid pBTS-X.
Here the restriction enzyme site of left end and right end can be any restriction enzyme site, it is only necessary to guarantee A before B just；
A, B respectively represents two sections of sequences, specifically may refer to Technology Roadmap, and A, B are any two sections of sequences；Mutant fragments, purpose piece
Section can be any segment or you think the segment of transformation, only be indicated here with X, referring specifically to Technology Roadmap or implementation
Example.
(3) pBTS-X is transferred in bacillus by electrotransformation, obtains positive transformants bacterial strain；
(4) picking positive transformants bacterium colony, which enters in fluid nutrient medium, is cultivated, and cultivation temperature is 45 DEG C, and incubation time is big
In for 24 hours.
(5) bacterium solution 100ul -500ul in 4 is taken, is applied on plate containing kanamycin, cultivation temperature is 45 DEG C, into
Row is incubated overnight.
(6) bacterium colony in picking 5, which enters in fluid nutrient medium, is cultivated, and every 8-16h passage is primary, until screening
Nonreactive bacterial strain.The i.e. same bacterial strain, does not grow on containing resistant plate, grows on the plate of nonreactive.
(7) bacterium solution in 6 is taken to be diluted coated plate, dilution 105-106Times, it is incubated overnight.
(8) bacterium colony in picking 7 carries out Resistance Identification, until obtaining 5-10 plants of nonreactive bacterial strains.
(9) it takes the nonreactive bacterial strain in 8 to be inoculated into fluid nutrient medium, is incubated overnight, extract bacterial genomes, carry out PCR
Identification；Positive strain continues sequencing identification, and sequencing qualification result is that positive strain is the bacterial strain for completing genome manipulation.
Bacillus is bacillus subtilis 168, Z12 bacterial strain in the step (3), also includes other bacillus, such as
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus),
The bacterial strains such as clothing bacillus (Bacillus licheniformis) or other pBTS plasmids have temperature sensitive copy characteristic
Gram-positive bacteria.
The gram-positive bacteria is streptococcus pneumonia (Streptococcus pneumoniae), Lactococcus lactis
(Lactococcus lactis) or series bacillus (Paenibacillus sp) bacterial strain.
The pBTS nucleotide sequence is as shown in SEQ ID NO.1.
For in 6-9 step in the present invention, second of recombination can obtain wild-type strain and transformation bacterial strain, if wild
The case where growth ability of type bacterial strain is better than transformation bacterial strain, is not easy to screen transformation bacterial strain, the strain improvement scheme of design optimization,
Steps are as follows:
(1) in the homologous recombination segment A of the site the MCS left-hand end insertion 500-800bp of pBTS, right-hand end is inserted into 500-
The homologous recombination segment B of 800bp, intermediate insertion mutation segment or target fragment or is not inserted into segment, then in B and intermediate transformation
Insertion has blasticidin resistance segment between segment or A and the transformation segment of centre, and blasticidin resistance segment both ends contain
Lox71 the and lox72 segment for having cre recombinase to identify, the plasmid are named as pBTS-X-Z.Resistance fragments herein are not limited only to
Blasticidin resistance segment also includes the resistance fragments such as similar tetracycline, chloramphenicol, spectinomycin.
(2) conversion of pBTS-X-Z plasmid is entered in bacillus by electrotransformation, bacillus herein includes withered grass
The bacterial strains such as bacillus 168, Z12 also include other bacillus, such as bacillus amyloliquefaciens, bacillus pumilus, lichens bud
The bacterial strains such as spore bacillus also include the gram-positive bacteria that other pBTS plasmids have temperature sensitive copy characteristic, such as pneumonia streptococcus
Bacterium (Streptococcus pneumoniae), Lactococcus lactis (Lactococcus lactis), series bacillus
Bacterial strains such as (Paenibacillus sp.).
(3) the positive transformants bacterium colony in picking (1), which enters in fluid nutrient medium, is cultivated, and cultivation temperature is 45 DEG C, training
The time is supported greater than for 24 hours.
(4) bacterium solution 100ul -500ul in (3) is taken, is applied on containing kanamycin or bleomycin plate, is trained
Supporting temperature is 45 DEG C, is incubated overnight.
(5) bacterium colony in picking (4) enters in the LB liquid medium containing blasticidin resistance and is cultivated, and every 8-
16h passage is primary, until screening bacterium colony only with bleomycin.
(6) bacterium solution in (5) is taken to be diluted coated plate, plate contains blasticidin resistance, dilution 105-106Times, it trains overnight
It supports.
(7) bacterium colony in picking (6) carries out Resistance Identification, only has blasticidin resistance until obtaining 3-5, does not have card
The bacterial strain of that chloramphenicol resistance.
(8) it takes the monoclonal antibody strain inoculated in (7) into culture medium, is incubated overnight, extract bacterial genomes, carry out PCR mirror
It is fixed.Positive strain continues sequencing identification, and the positive strain that identification is sequenced enters in next step.
(9) conversion pBTS-IC plasmid, which enters, is accredited as positive monoclonal antibody bacterial strain in (8), IC is that lactose operator regulates and controls cre
Recombinate expression of enzymes segment, cre recombinase can characteristic identify the site lox71 and lox66, recombinated, left in genome
It cannot be cut lower segment by the site loxP that cre recombinase identifies and be contained the site lox72 more easily identified.
(10) the positive transformants bacterial strain in picking (9), connects bacterium and is cultivated, utilize IPTG inducing expression or leakage expression
Cre recombinase cut blasticidin resistance segment.
(11) bacterium solution in (10) is taken, is inoculated into fluid nutrient medium, cultivation temperature is 45 DEG C, and incubation time is greater than for 24 hours.
(12) bacterium solution cultivated in (11), dilution 10 are taken5-106On coated plate to the plate of nonreactive, cultivation temperature is 45 DEG C, mistake
Night culture；
(13) single colonie in (12) is taken, Resistance Identification is carried out, until screening 2-5 plants of nonreactive bacterial strains.
(14) nonreactive bacterial strain in (13) is taken, is inoculated into culture medium, is incubated overnight, genome is extracted, carries out PCR mirror
It is fixed.Positive strain carries out sequencing identification again, and being still accredited as positive bacterial strain is the bacterial strain for completing genome manipulation.
The pBTS-IC nucleotide sequence is as shown in SEQ ID NO.3.
The skeleton of pBTS plasmid is that stability is high, the pBAV1K plasmid more than copy number, multiple relative to other responsive to temperature type
Plasmid processed, it is short (containing only a kind of dubbing system element and kalamycin resistance label) with dubbing system element segment, it can
To be replicated in Escherichia coli and gram-positive bacteria, duplicate copy number is high, stability height (segment of insertion 6K or so,
The imagination of fragment loss will not occur).
The method of homologous recombination transformation bacterial genomes in the present invention based on pBTS, adaptable wide (plasmid conversion
Difficulty is low, and pBTS plasmid can be replicated in a variety of gram-positive bacterias), it can the repeated multiple times same bacterial strain of transformation (weight
There are two homologous recombination segments for group plasmid tool, and homologous recombination twice successively occurs, may be restored to open country after second of homologous recombination
Raw type, also available transformation bacterial strain, but the segment that any influence continues transformation is not remained, such as resistance marker, plasmid replication
Element etc.), and can be transformed and (cre recombinase and second of resistance marker system are introduced to the bacterial strain that bacterial growth is affected
System can recombinate for the second time obtained transformation bacterial strain by resistance screening, kill the strong wild-type strain of survival ability, express later
Cre recombinase cuts away second of resistance marker, and remaining segment, which will not influence, is again transformed bacterial strain).
Detailed description of the invention
Fig. 1 is Technology Roadmap in the present invention
Fig. 2 is the Technology Roadmap optimized in the present invention
Fig. 3 is the pBTS structure chart in the present invention
Fig. 4 is the pBTS-IC structure chart in the present invention
Specific embodiment
The present invention is described in further detail With reference to embodiment:
Embodiment 1
(1) pBAV1K-T5-GFP plasmid is cut by EcoR I and Apa I restriction endonuclease, is carried out with the MCS segment of synthesis same
Source recombination, obtains plasmid pBAV1K.
Restriction endonuclease in this experiment and follow-up test uses the quick restriction endonuclease of Thermo company, and segment recycling uses Chengdu
The plastic recovery kit (DE-02011) of Fu Ji biotech firm, MCS segment are the synthesis of purchase Jin Weizhi Biotechnology Co., Ltd
Segment, homologous recombination uses the EsayGeno Quick Casting Cloning Kit (VI201-02) of Tiangeng, and coli strain is
Top10, preparation competence use KCM method, and plasmid extraction uses the universal plasmid Mini Kit (DE- of good fortune border biology
01001)。
(2) design primer, the pBAV1K segment of Nde I restriction enzyme site is deleted in amplification same sense mutation, using homologous recombination gram
Grand mode junction fragment obtains plasmid and is named as pBTS.
Primer in this experiment and subsequent experimental is synthesized by Jin Weizhi Biotechnology Co., Ltd.
Embodiment 2:
Full genome synthesizes IC segment, is inserted between XbaI the and Pst I of pBTS by homologous recombination, obtains plasmid
pBTS-IC.Here what is obtained is the pBTS-IC plasmid in preferred embodiment；PBTS-FR-X may refer to following transformation xylAB
The plasmid of gene.
Embodiment 3:
1, the segment xyl-F of the xylose xylAB operon upstream 493bp of design primer amplification Z12 bacterial strain, is inserted into pBTS's
Between the site EcoR I and BamHI, plasmid pBTS-xyl-F is obtained；The segment xyl-R of downstream 620bp is expanded, plasmid pBTS- is inserted into
Between BamHI the and Hind III of xyl-F, plasmid pBTS-xyl plasmid (knocking out xylAB operon) is obtained.High fidelity enzyme uses
The KOD-Plus hi-fi polymerase (KOD-201) of toyobo.
2, design primer amplification Z12 bacterial strain pgsBCA operon upstream 693bp segment, be inserted into pBTS EcoR I and
Between the site BamHI, obtain plasmid pBTS-pgs-F amplification downstream 558bp segment, be inserted into plasmid pBTS-pgs-F BamHI and
Between Hind III, plasmid pBTS-pgs is obtained.Full genome synthesizes zeo segment, is inserted into the site BamHI of pBTS-pgs, obtains
Plasmid pBTS-pgs-Z (knocks out pgsBCA operon, but marks among homologous recombination segment containing blasticidin resistance).
Embodiment 4:
Bacterial strain method for transformation is referring to hypertonic conversion method (High osmolarity improves the electro-
transformation efficiency of the gram-positive bacteria Bacillus subtilis and
Bacillus licheniformis, 1999).The new scribing line single colonie for taking Z12 bacterial strain is inoculated into the (mountain LB+0.5M about 3ml GM
Pears sugar alcohol) in culture medium, 37 DEG C, 180rpm is incubated overnight.The next morning is inoculated into 50ml GM culture medium by 1:100, and 37
DEG C, 180rpm culture when bacterium solution grows into OD and reaches between 0.85-0.95, bacterium solution is put into, 10min is pre-chilled on ice；4 DEG C,
Supernatant is removed in 5000g, 5min, centrifugation, and with the EM being pre-chilled in equal volume, (0.5M D-sorbite+0.5M+10% glycerol of mannitol is water-soluble
Liquid) thallus is resuspended, 4 DEG C again, 5000g, 5min, supernatant is removed in centrifugation, washes repeatedly 4 times altogether；The EM of about 1/40 volume is added
Thallus is resuspended, guarantees bacterial concentration in 1-1.3 × 1010Between cfu/ml.The above-mentioned bacterium solution of 60ul is taken, 1ul plasmid to be transformed is added
(pBTS-xyl and pBTS-pgs-Z), plasmid concentration are greater than 100ng/ul, and piping and druming mixes, and the 1mm electric shock cup of pre-cooling is then added.
Electric converter (Eppendorf Eporator) parameter setting, 2.1kV is practical to shock by electricity the time in 4.0-5.0ms, has been likely to
Transformed bacteria is born length.After electric shock.Immediately plus 1ml RM (LB+0.5M D-sorbite+0.38M mannitol), piping and druming are mixed, are transferred to
In 5ml sterile centrifugation tube, 37 DEG C, 180rpm cultivates 3h.Bacterium solution is centrifuged, is diluted by a certain percentage after removing supernatant, 200ul is taken to apply
On cloth to the LB plate containing 30mg/L kanamycins, 37 DEG C are incubated overnight, while being coated with the bacterium solution without electrotransformation and being done feminine gender
Control.The next morning observes bacterium colony growing state, if reformer plate has bacterium colony growth, negative control does not have, then shows to be converted to
Function.The bacterial strain that name obtains is Z12-pBTS-xyl or Z12-pBTS-pgs-Z.
Embodiment 5:
(1) bacterium Z12-pBTS-xyl is taken to be inoculated into 3ml LB culture medium, 45 DEG C, 180rpm, culture is for 24 hours；Separately connect bacterium
Z12-pBTS is compareed.
(2) the bacterium solution 200ul in 1 is taken to be applied on the LB plate containing 30mg/L kanamycins, 45 DEG C are incubated overnight.If
Z12-pBTS does not have bacterium colony growth, and Z12-pBTS-xyl has bacterium colony growth, then the bacterial strain obtained is completed to recombinate for the first time, is named as
Z12-pBTS-xyl-45。
(3) it connects in bacterium Z12-pBTS-xyl-45 to 3ml LB culture medium, is incubated overnight, (Chengdu good fortune border is raw for extracting genome
Object Technology Co., Ltd., bacterial genomes DNA extraction kit, DE-05311), PCR amplification xyl segment is verified (Chengdu
Fu Ji Bioisystech Co., Ltd, 2 × fast PCR react premix system, DP-20041.Primer sequence, F:
CAAAATGTCTTTCGTTATTTCTGGAG, R:AGTGTTCGCTGACAAAATACGGTTC, 60 DEG C of annealing, 2min extend).
(4) it takes and is verified as positive Z12-pBTS-xyl-45 bacterial strain in 3, be inoculated into 3ml LB base 37 DEG C, 180rpm training
It supports, it is primary every 8-16h passage, after continuous passage 8 times, bacterium solution is taken to dilute 106Times, it is coated on the LB plate of nonreactive, obtains
Single colonie.
(5) single colonie in 4 is taken, while being crossed on LB and LB plate containing 30mg/L kanamycins, nonreactive is screened
Bacterium colony, need to obtain 5-10 plants of nonreactive bacterium colonies.
(6) the nonreactive bacterium colony in 5 is taken, is inoculated into 3ml LB culture medium, 37 DEG C, 180rpm is incubated overnight, and extracts bacterium
Genome, by the verifying of PCR amplification xyl segment, (Chengdu Fu Ji Bioisystech Co., Ltd, 2 × fast PCR react premixed material
System, DP-20041.Primer sequence, F:CAAAATGTCTTTCGTTATTTCTGGAG, R:
AGTGTTCGCTGACAAAATACGGTTC, 60 DEG C of annealing, 2min extend) (wild type knocks out xyl base to homologous recombination result
Cause).Positive segment is taken, Song Jinwei intelligence Biotechnology Co., Ltd further progress sequence verification obtains positive bacterial strain i.e.
For bacterial strain --- the Z12 (△ xylAB) for knocking out xyl gene.
(7) Z12 (△ xylAB) and Z12 bacterial strain are taken, according to the wood-sugar fermentation experimental verification in primary Jie Shi Bacteria Identification handbook
The ability of its fermenting xylose, the results showed that Z12 can be with fermenting xylose, but Z12 (△ xylAB) is unable to fermenting xylose.
Embodiment 6:
(1) bacterium Z12-pBTS-pgs-Z is taken to be inoculated into 3ml LB culture medium, 45 DEG C, 180rpm, culture is for 24 hours；Separately connect bacterium
Z12-pBTS is compareed.
(2) the bacterium solution 200ul in 1 is taken to be applied on the LB plate containing 30mg/L kanamycins, 45 DEG C are incubated overnight.If
Z12-pBTS does not have bacterium colony growth, and Z12-pBTS-pgs-Z has bacterium colony growth, then the bacterial strain obtained is completed to recombinate for the first time, name
For Z12-pBTS-pgs-Z-45.
(3) it connects in bacterium Z12-pBTS-xyl-45 to 3ml LB culture medium, is incubated overnight, extract genome, PCR amplification pgs
Segment verified (primer sequence, F:AGCTGTTCCGATTTTATACTGGTC, R:TAGCCAAAACAGCTCCTCCTTG, 60
DEG C annealing, 2min extend).
(4) it takes and is verified as positive Z12-pBTS-pgs-Z-45 bacterial strain in 3, be inoculated into containing 30mg/L bleomycin
37 DEG C in 3mlLB base, 180rpm culture is primary every 8-16h passage, after continuous passage 10 times, bacterium solution is taken to dilute 106Times, it applies
It is distributed on the LB plate containing 30mg/L bleomycin, obtains single colonie.
(5) single colonie in 4 is taken, while being crossed to the sum containing 30mg/L kanamycins containing 30mg/L bleomycin
On LB plate, screening contains only the bacterium colony of blasticidin resistance, needs to obtain 3-5 plants of monoclonal antibody bacterium colonies.
(6) the monoclonal antibody bacterium colony in 5 is taken, is inoculated into 3ml LB culture medium, 37 DEG C, 180rpm is incubated overnight, and extracts bacterium
Genome, by PCR amplification pgs segment verifying (primer sequence, F:AGCTGTTCCGATTTTATACTGGTC, R:
TAGCCAAAACAGCTCCTCCTTG, 60 DEG C of annealing, 2min extend) homologous recombination result (wild type or knocking out pgs gene).
Naming the positive strain is Z12-pgs-Z.
(7) Z12-pgs-Z bacterial strain is entered by electrotransformation pBTS-IC plasmid, is named as Z12-pgs-Z-pBTS-IC.
(8) bacterium Z12-pgs-Z-pBTS-IC is met into the 3ml LB culture medium containing 1mM IPTG, 37 DEG C, 180rpm, mistake
Night culture.
(9) bacterium solution in 8 is taken to be inoculated into 3ml LB culture medium, 45 DEG C, 180rpm, culture is for 24 hours.Bacterium solution dilution 106Times,
It is applied on the LB plate of nonreactive, 45 DEG C, is incubated overnight, obtains single colonie.
(10) it takes the single colonie in 9 while being crossed to containing 30mg/L kanamycins, 30mg/L bleomycin and nonreactive
On LB plate, the bacterial strain of nonreactive is screened.
(11) nonreactive bacterial strain in 10 is taken, is inoculated into 3ml LB culture medium, 37 DEG C, 180rpm is incubated overnight.Extract base
Because of group, verify whether correctly to cut blasticidin resistance segment by PCR amplification pgs segment.To positive segment, Jin Weizhi is sent
Biotechnology Co., Ltd carries out sequencing identification, knocks out pgsBCA operon and the bacterial strain without containing blasticidin resistance label is
For the bacterial strain for completing transformation, it is named as Z12 (△ pgsBCA).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
SEQUENCE LISTING
<110>Chengdu Mei Yide Bioisystech Co., Ltd
<120>a kind of method that bacillus gene group is transformed
<160>3
<170>PatentIn version 3.3
<210>1
<211>2847
<212>DNA
<213>artificial synthesized
<220>
<223>pBTS sequence
<400>1
gacgtcgaattcgaggtacctgcggccgcaggatccatctagacgctcgagagctgcagtgaagcttc
agggcccgatcgatgccgccgcttaattaattaatccagaggcatcaaataaaacgaaaggctcagtcgaaagact
gggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtg
tcattttatttcccccgtttcagcatcaagaacctttgcataacttgctctatatccacactgataattgccctca
aaccataatctaaaggcgctagagtttgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccg
ctcccctaaggcgaataaaagccattaaatcttttgtatttaccaaattatagtcatccactatatctaagagtaa
attcttcaattctcttttttggctttcatcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaata
tcttttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatgagtcaaatattcataagaacctt
tgatataatcaagtatctcaacatgagcaactgaactattccccaattttcgcttaatcttgttcctaacgctttc
tattgttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatagtgctttccatttcgtataaca
tcactactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggactgacagccatagatacgccca
aactctctaatttttccttccaatcattaggaattgagtcaggatataataaaaatccaaaatttctagctttagt
atttttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgtatccttctaaaaacgcgagct
ttcgcttattttttttgttctgattcctttcttgcatattcttctatagctaacgccgcaaccgcagattttgaaa
aacctttttgtttcgccatatctgttaattttttatcttgctcttttgtcagagaaatcataactctttttttcga
ttctgaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgtatcactttgtaatcataaaaac
aacaataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggcgattcgttggcgattgaaaaa
ccccttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggcactcggcacttaatggggggtc
gtagtacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaatttttttcaaaaattttccag
cgctaccgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaatttcattccctcatactccctt
gagcctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcatcggctttcataatctaacaga
caacatcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataacgcctgttttaacgattatgcc
gataactaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgttttaataaaaaatcgcctagtgct
tggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcatt
actggatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccagtaaaatataatattttatttt
ctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatc
gaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactt
tgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgt
ctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggcc
agatcgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcgtatagggacaatccgatatgt
cgatggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagggctttgttcatcttcataccc
ttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacc
tttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttat
accggctgtccgtcatttttaaatataggatttcattttctcccaccagcttatataccttagcaggagacattcc
ttccgtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgatattctcattttagccatttat
tatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcact
gttccttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgttttcaaagttggcgtataacatag
tatcgacggagccgattttgaaaccacaattatgatagaattt 2847
<210>2
<211>3344
<212>DNA
<213>artificial synthesized
<220>
<223>pBTS-Z sequence
<400>2
gacgtcgaattcgaggtacctgcggccgcaggatcctaccgttcgtatagcatacattatacgaagtt
atcttgatatggctttttatatgtgttactctacatacagaaaggaggaactaaatatggccaagttgaccagtgc
cgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggac
ttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtgg
tgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtc
cacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctg
cgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgaataacttcgtatagcatacattata
cgaacggtaGtctagacgctcgagagctgcagtgaagcttcagggcccgatcgatgccgccgcttaattaattaat
ccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaac
gctctcctgagtaggacaaatccgccgccctagacctagtgtcattttatttcccccgtttcagcatcaagaacct
ttgcataacttgctctatatccacactgataattgccctcaaaccataatctaaaggcgctagagtttgttgaaac
aatatcttttacatcattcgtatttaaaattccaaactccgctcccctaaggcgaataaaagccattaaatctttt
gtatttaccaaattatagtcatccactatatctaagagtaaattcttcaattctcttttttggctttcatcaagtg
ttatatagcggtcaatatcaaaatcattaatgttcaaaatatcttttttgtcgtatatatgtttattcttagcaat
agcgtcctttgattcatgagtcaaatattcataagaacctttgatataatcaagtatctcaacatgagcaactgaa
ctattccccaattttcgcttaatcttgttcctaacgctttctattgttacaggatttcgtgcaatatatataacgt
gatagtgtggttttttatagtgctttccatttcgtataacatcactactattccatgtatctttatcttttttttc
gtccatatcgtgtaaaggactgacagccatagatacgcccaaactctctaatttttccttccaatcattaggaatt
gagtcaggatataataaaaatccaaaatttctagctttagtatttttaatagccatgatataattaccttatcaaa
aacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcgcttattttttttgttctgattcctttcttgc
atattcttctatagctaacgccgcaaccgcagattttgaaaaacctttttgtttcgccatatctgttaatttttta
tcttgctcttttgtcagagaaatcataactctttttttcgattctgaaatcaccatttaaaaaactccaatcaaat
aattttataaagttagtgtatcactttgtaatcataaaaacaacaataaagctacttaaatatagatttataaaaa
acgttggcgaaaacgttggcgattcgttggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgt
ttttggcacaaaaattggcactcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcctttcccccc
atttttttccaaattccaaatttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaa
aatcaaacccatgagggaatttcattccctcatactcccttgagcctcctccaaccgaaatagaagggcgctgcgc
ttattatttcattcagtcatcggctttcataatctaacagacaacatcttcgctgcaaagccacgctacgctcaag
ggcttttacgctacgataacgcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgtctcag
aaacgattttgagacgttttaataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcggcaac
cgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcgg
tactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaa
atagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtc
cgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccagg
tcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaa
atggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaa
gcggccgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatac
agctcgatagtcttttcagggctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatga
gcagattgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcat
catgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggatttca
ttttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatca
gttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagata
ccccaagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaac
agccttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatga
tagaattt3344
<210>3
<211>5528
<212>DNA
<213>artificial synthesized
<220>
<223>pBTS-IC sequence
<400>3
gacgtcgaattcgaggtacctgcggccgcaggatccatctagataactcacattaattgcgttgcgct
cactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcgg
tttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctg
gccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttgac
ggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccgg
actcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctc
attcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatt
tgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaaca
gcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataat
actgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatg
gcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctt
tacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaat
cgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgccc
gccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcg
cagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgta
taacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctt
tccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactca
aacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaagg
aaatgatgacctcgtttccaccggaattagcttgcatgcctaatcgccatcttccagcaggcgcaccattgcccct
gtttcactatccaggttacggatatagttcatgacaatatttacattggtccagccaccagcttgcatgatctccg
gtattgaaactccagcgcgggccatatctcgcgcggctccgacacgggcactgtgtccagaccaggccaggtatct
ctgaccagagtcatccttagcgccgtaaatcaatcgatgagttgcttcaaaaatcccttccagggcgcgagttgat
agctggctggtggcagatggcgcggcaacaccattttttctgacccggcaaaacaggtagttattcggatcatcag
ctacaccagagacggaaatccatcgctcgaccagtttagttacccccaggctaagtgccttctctacacctgcggt
gctaaccagcgttttcgttctgccaatatggattaacattctcccaccgtcagtacgtgagatatctttaaccctg
atcctggcaatttcggctatacgtaacagggtgttataagcaatccccagaaatgccagattacgtatatcctggc
agcgatcgctattttccatgagtgaacgaacctggtcgaaatcagtgcgttcgaacgctagagcctgttttgcacg
ttcaccggcatcaacgttttcttttcggatccgccgcataaccagtgaaacagcattgctgtcacttggtcgtggc
agcccggaccgacgatgaagcatgtttagctggcccaaatgttgctggatagtttttactgccagaccgcgcgcct
gaagatatagaagataatcgcgaacatcttcaggttctgcgggaaaccatttccggttattcaacttgcaccatgc
cgcccacgaccggcaaacggacagaagcattttccaggtatgctcagaaaacgcctggcgatccctgaacatgtcc
atcaggttcttgcgaacctcatcactcgttgcatcgaccggtaatgcaggcaaattttggtgtacggtcagtaaat
tggacatacctgcttcctccttaagcttaattgttatccgctcacaattccacacattatgccacaccttgtagat
aaagtcaacaactttttgcaaaatttttcaggaattttagcagaggttgttctggatgtagaacaaaacatctttc
cgctcttgtgctgttaggatatctttcttggaagctaggtaggcaagggctacctcaaataaatcttcttcagggt
gcgctatttttaaggtgcctagtgaggtcttgaccacttcacccataatttcagtgccgaatagtctggactgggc
tgtgtactgcagtgaagcttcagggcccgatcgatgccgccgcttaattaattaatccagaggcatcaaataaaac
gaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaa
tccgccgccctagacctagtgtcattttatttcccccgtttcagcatcaagaacctttgcataacttgctctatat
ccacactgataattgccctcaaaccataatctaaaggcgctagagtttgttgaaacaatatcttttacatcattcg
tatttaaaattccaaactccgctcccctaaggcgaataaaagccattaaatcttttgtatttaccaaattatagtc
atccactatatctaagagtaaattcttcaattctcttttttggctttcatcaagtgttatatagcggtcaatatca
aaatcattaatgttcaaaatatcttttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatgag
tcaaatattcataagaacctttgatataatcaagtatctcaacatgagcaactgaactattccccaattttcgctt
aatcttgttcctaacgctttctattgttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatag
tgctttccatttcgtataacatcactactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggac
tgacagccatagatacgcccaaactctctaatttttccttccaatcattaggaattgagtcaggatataataaaaa
tccaaaatttctagctttagtatttttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgt
atccttctaaaaacgcgagctttcgcttattttttttgttctgattcctttcttgcatattcttctatagctaacg
ccgcaaccgcagattttgaaaaacctttttgtttcgccatatctgttaattttttatcttgctcttttgtcagaga
aatcataactctttttttcgattctgaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgta
tcactttgtaatcataaaaacaacaataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggc
gattcgttggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggca
ctcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaa
tttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaat
ttcattccctcatactcccttgagcctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcat
cggctttcataatctaacagacaacatcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataac
gcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgtttt
aataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatcc
agatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccag
taaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttct
tccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaa
gatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaag
ttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccag
ttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcg
tatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagg
gctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatca
tgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttcca
catcataggtggtccctttataccggctgtccgtcatttttaaatataggatttcattttctcccaccagcttata
taccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgat
attctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataa
caagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgtttt
caaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgatagaattt5528
Sequence table
SEQUENCE LISTING
<110>Chengdu Mei Yide Bioisystech Co., Ltd
<120>a kind of method that bacillus gene group is transformed
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 2847
<212> DNA
<213>artificial synthesized
<220>
<223>pBTS sequence
<400> 1
gacgtcgaattcgaggtacctgcggccgcaggatccatctagacgctcgagagctgcagtgaagcttc
agggcccgatcgatgccgccgcttaattaattaatccagaggcatcaaataaaacgaaaggctcagtcgaaagact
gggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtg
tcattttatttcccccgtttcagcatcaagaacctttgcataacttgctctatatccacactgataattgccctca
aaccataatctaaaggcgctagagtttgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccg
ctcccctaaggcgaataaaagccattaaatcttttgtatttaccaaattatagtcatccactatatctaagagtaa
attcttcaattctcttttttggctttcatcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaata
tcttttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatgagtcaaatattcataagaacctt
tgatataatcaagtatctcaacatgagcaactgaactattccccaattttcgcttaatcttgttcctaacgctttc
tattgttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatagtgctttccatttcgtataaca
tcactactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggactgacagccatagatacgccca
aactctctaatttttccttccaatcattaggaattgagtcaggatataataaaaatccaaaatttctagctttagt
atttttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgtatccttctaaaaacgcgagct
ttcgcttattttttttgttctgattcctttcttgcatattcttctatagctaacgccgcaaccgcagattttgaaa
aacctttttgtttcgccatatctgttaattttttatcttgctcttttgtcagagaaatcataactctttttttcga
ttctgaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgtatcactttgtaatcataaaaac
aacaataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggcgattcgttggcgattgaaaaa
ccccttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggcactcggcacttaatggggggtc
gtagtacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaatttttttcaaaaattttccag
cgctaccgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaatttcattccctcatactccctt
gagcctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcatcggctttcataatctaacaga
caacatcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataacgcctgttttaacgattatgcc
gataactaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgttttaataaaaaatcgcctagtgct
tggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcatt
actggatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccagtaaaatataatattttatttt
ctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatc
gaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactt
tgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgt
ctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggcc
agatcgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcgtatagggacaatccgatatgt
cgatggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagggctttgttcatcttcataccc
ttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacc
tttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttat
accggctgtccgtcatttttaaatataggatttcattttctcccaccagcttatataccttagcaggagacattcc
ttccgtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgatattctcattttagccatttat
tatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcact
gttccttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgttttcaaagttggcgtataacatag
tatcgacggagccgattttgaaaccacaattatgatagaattt 2847
<210> 2
<211> 3344
<212> DNA
<213>artificial synthesized
<220>
<223>pBTS-Z sequence
<400> 2
gacgtcgaattcgaggtacctgcggccgcaggatcctaccgttcgtatagcatacattatacgaagtt
atcttgatatggctttttatatgtgttactctacatacagaaaggaggaactaaatatggccaagttgaccagtgc
cgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggac
ttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtgg
tgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtc
cacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctg
cgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgaataacttcgtatagcatacattata
cgaacggtaGtctagacgctcgagagctgcagtgaagcttcagggcccgatcgatgccgccgcttaattaattaat
ccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaac
gctctcctgagtaggacaaatccgccgccctagacctagtgtcattttatttcccccgtttcagcatcaagaacct
ttgcataacttgctctatatccacactgataattgccctcaaaccataatctaaaggcgctagagtttgttgaaac
aatatcttttacatcattcgtatttaaaattccaaactccgctcccctaaggcgaataaaagccattaaatctttt
gtatttaccaaattatagtcatccactatatctaagagtaaattcttcaattctcttttttggctttcatcaagtg
ttatatagcggtcaatatcaaaatcattaatgttcaaaatatcttttttgtcgtatatatgtttattcttagcaat
agcgtcctttgattcatgagtcaaatattcataagaacctttgatataatcaagtatctcaacatgagcaactgaa
ctattccccaattttcgcttaatcttgttcctaacgctttctattgttacaggatttcgtgcaatatatataacgt
gatagtgtggttttttatagtgctttccatttcgtataacatcactactattccatgtatctttatcttttttttc
gtccatatcgtgtaaaggactgacagccatagatacgcccaaactctctaatttttccttccaatcattaggaatt
gagtcaggatataataaaaatccaaaatttctagctttagtatttttaatagccatgatataattaccttatcaaa
aacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcgcttattttttttgttctgattcctttcttgc
atattcttctatagctaacgccgcaaccgcagattttgaaaaacctttttgtttcgccatatctgttaatttttta
tcttgctcttttgtcagagaaatcataactctttttttcgattctgaaatcaccatttaaaaaactccaatcaaat
aattttataaagttagtgtatcactttgtaatcataaaaacaacaataaagctacttaaatatagatttataaaaa
acgttggcgaaaacgttggcgattcgttggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgt
ttttggcacaaaaattggcactcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcctttcccccc
atttttttccaaattccaaatttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaa
aatcaaacccatgagggaatttcattccctcatactcccttgagcctcctccaaccgaaatagaagggcgctgcgc
ttattatttcattcagtcatcggctttcataatctaacagacaacatcttcgctgcaaagccacgctacgctcaag
ggcttttacgctacgataacgcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgtctcag
aaacgattttgagacgttttaataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcggcaac
cgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcgg
tactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaa
atagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtc
cgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccagg
tcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaa
atggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaa
gcggccgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatac
agctcgatagtcttttcagggctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatga
gcagattgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcat
catgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggatttca
ttttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatca
gttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagata
ccccaagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaac
agccttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatga
tagaattt 3344
<210> 3
<211> 5528
<212> DNA
<213>artificial synthesized
<220>
<223>pBTS-IC sequence
<400> 3
gacgtcgaattcgaggtacctgcggccgcaggatccatctagataactcacattaattgcgttgcgct
cactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcgg
tttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctg
gccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttgac
ggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccgg
actcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctc
attcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatt
tgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaaca
gcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataat
actgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatg
gcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctt
tacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaat
cgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgccc
gccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcg
cagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgta
taacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctt
tccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactca
aacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaagg
aaatgatgacctcgtttccaccggaattagcttgcatgcctaatcgccatcttccagcaggcgcaccattgcccct
gtttcactatccaggttacggatatagttcatgacaatatttacattggtccagccaccagcttgcatgatctccg
gtattgaaactccagcgcgggccatatctcgcgcggctccgacacgggcactgtgtccagaccaggccaggtatct
ctgaccagagtcatccttagcgccgtaaatcaatcgatgagttgcttcaaaaatcccttccagggcgcgagttgat
agctggctggtggcagatggcgcggcaacaccattttttctgacccggcaaaacaggtagttattcggatcatcag
ctacaccagagacggaaatccatcgctcgaccagtttagttacccccaggctaagtgccttctctacacctgcggt
gctaaccagcgttttcgttctgccaatatggattaacattctcccaccgtcagtacgtgagatatctttaaccctg
atcctggcaatttcggctatacgtaacagggtgttataagcaatccccagaaatgccagattacgtatatcctggc
agcgatcgctattttccatgagtgaacgaacctggtcgaaatcagtgcgttcgaacgctagagcctgttttgcacg
ttcaccggcatcaacgttttcttttcggatccgccgcataaccagtgaaacagcattgctgtcacttggtcgtggc
agcccggaccgacgatgaagcatgtttagctggcccaaatgttgctggatagtttttactgccagaccgcgcgcct
gaagatatagaagataatcgcgaacatcttcaggttctgcgggaaaccatttccggttattcaacttgcaccatgc
cgcccacgaccggcaaacggacagaagcattttccaggtatgctcagaaaacgcctggcgatccctgaacatgtcc
atcaggttcttgcgaacctcatcactcgttgcatcgaccggtaatgcaggcaaattttggtgtacggtcagtaaat
tggacatacctgcttcctccttaagcttaattgttatccgctcacaattccacacattatgccacaccttgtagat
aaagtcaacaactttttgcaaaatttttcaggaattttagcagaggttgttctggatgtagaacaaaacatctttc
cgctcttgtgctgttaggatatctttcttggaagctaggtaggcaagggctacctcaaataaatcttcttcagggt
gcgctatttttaaggtgcctagtgaggtcttgaccacttcacccataatttcagtgccgaatagtctggactgggc
tgtgtactgcagtgaagcttcagggcccgatcgatgccgccgcttaattaattaatccagaggcatcaaataaaac
gaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaa
tccgccgccctagacctagtgtcattttatttcccccgtttcagcatcaagaacctttgcataacttgctctatat
ccacactgataattgccctcaaaccataatctaaaggcgctagagtttgttgaaacaatatcttttacatcattcg
tatttaaaattccaaactccgctcccctaaggcgaataaaagccattaaatcttttgtatttaccaaattatagtc
atccactatatctaagagtaaattcttcaattctcttttttggctttcatcaagtgttatatagcggtcaatatca
aaatcattaatgttcaaaatatcttttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatgag
tcaaatattcataagaacctttgatataatcaagtatctcaacatgagcaactgaactattccccaattttcgctt
aatcttgttcctaacgctttctattgttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatag
tgctttccatttcgtataacatcactactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggac
tgacagccatagatacgcccaaactctctaatttttccttccaatcattaggaattgagtcaggatataataaaaa
tccaaaatttctagctttagtatttttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgt
atccttctaaaaacgcgagctttcgcttattttttttgttctgattcctttcttgcatattcttctatagctaacg
ccgcaaccgcagattttgaaaaacctttttgtttcgccatatctgttaattttttatcttgctcttttgtcagaga
aatcataactctttttttcgattctgaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgta
tcactttgtaatcataaaaacaacaataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggc
gattcgttggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggca
ctcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaa
tttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaat
ttcattccctcatactcccttgagcctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcat
cggctttcataatctaacagacaacatcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataac
gcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgtttt
aataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatcc
agatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccag
taaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttct
tccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaa
gatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaag
ttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccag
ttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcg
tatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagg
gctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatca
tgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttcca
catcataggtggtccctttataccggctgtccgtcatttttaaatataggatttcattttctcccaccagcttata
taccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgat
attctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataa
caagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgtttt
caaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgatagaattt 5528
Claims (3)
1. a kind of method that bacillus gene group is transformed, it is characterised in that: the following steps are included:
(1) it is inserted into multiple cloning sites segment in EcoR I and the Apa I site of pBAV1K-T5-GFP plasmid, and by synonymous
Mutation, deletes NdeI restriction enzyme site therein, obtains plasmid pBTS；The pBTS nucleotide sequence is as shown in SEQ ID NO.1；
(2) restriction enzyme site in the left end of multiple cloning sites is inserted into the homologous recombination segment A of 500-800bp, right side restriction enzyme site
It is inserted into the homologous recombination segment B of 500-800bp, intermediate insertion mutation segment, target fragment or is not inserted into any segment, both two
The segment for needing to be transformed between homologous recombination region obtains plasmid pBTS-X；
(3) pBTS-X is transferred in bacillus by electrotransformation, obtains positive transformants bacterial strain；
(4) the positive transformants bacterium colony in picking (3), which enters in fluid nutrient medium, is cultivated, and cultivation temperature is 45 DEG C, when culture
Between be greater than for 24 hours；
(5) bacterium solution 100ul -500ul in step (4) is taken, is applied on the LB plate of the kanamycins containing 30mg/L, cultivation temperature
It is 45 DEG C, is incubated overnight；
(6) bacterium colony in picking step (5), which enters in fluid nutrient medium, is cultivated, and every 8-16h passage is primary, until screening
To nonreactive bacterial strain；
(7) bacterium solution in step (6) is taken to be diluted coated plate, dilution 105-106Times, it is incubated overnight；
(8) bacterium colony in picking step (7) carries out Resistance Identification, until obtaining 5-10 plants of nonreactive bacterial strains；
(9) nonreactive bacterial strain is inoculated into fluid nutrient medium, is incubated overnight, extract bacterial genomes, carry out PCR identification；It is positive
Bacterial strain continues sequencing identification, and sequencing qualification result is positive strain up to Bacillus strain.
2. a kind of method that bacillus gene group is transformed according to claim 1, it is characterised in that: the step (3)
Middle bacillus is bacillus subtilis 168, Z12 bacterial strain.
3. a kind of method that bacillus gene group is transformed according to claim 1, it is characterised in that: including following step
It is rapid:
(1) in the homologous recombination segment A of the multiple cloning sites left-hand end insertion 400-800bp of pBTS, right-hand end is inserted into 400-
The homologous recombination segment B of 800bp, intermediate insertion mutation segment or target fragment or is not inserted into segment, then in B and intermediate transformation
Insertion has H chloramphenicol resistance segment between segment or A and the transformation segment of centre, and cre weight is contained at H chloramphenicol resistance segment both ends
Lox71 the and lox72 segment of group enzyme identification, the plasmid are named as pBTS-X-Z；The H mycin is bleomycin, tetracycline, chlorine
It is any in mycin and spectinomycin；
(2) conversion of pBTS-X-Z plasmid is entered in bacillus by electrotransformation, obtains positive transformants bacterial strain；
(3) the positive transformants bacterium colony in picking step (2), which enters in fluid nutrient medium, is cultivated, and cultivation temperature is 45 DEG C, training
The time is supported greater than for 24 hours；
(4) bacterium solution 100ul -500ul in (3) is taken in step, is applied on containing kanamycin or H mycin plate, 45 DEG C of trainings
It is incubated overnight in supporting；
(5) bacterium colony in picking (4) enters in the LB liquid medium containing H chloramphenicol resistance and is cultivated, every 8-16h passage
Once, until screening bacterium colony only with H chloramphenicol resistance；
(6) bacterium solution in (5) is taken to be diluted coated plate, plate contains H chloramphenicol resistance, dilution 105-106Times, it is incubated overnight；
(7) bacterium colony in picking (6) carries out Resistance Identification, only has H chloramphenicol resistance until obtaining 5-10 plants, that is mould without blocking
The bacterial strain of plain resistance；
(8) it takes monoclonal antibody strain inoculated into fluid nutrient medium, is incubated overnight, extract bacterial genomes, carry out PCR identification, it is positive
Bacterial strain continues sequencing identification, and sequencing is accredited as positive bacterial strain and enters in next step；
(9) conversion pBTS-IC plasmid, which enters, is accredited as positive monoclonal antibody bacterial strain in (8), IC is that lactose operator regulates and controls cre recombination
The segment of expression of enzymes, cre recombinase can characteristic identify the site lox71 and lox66, recombinated, leaving in genome cannot
The site loxP identified by cre recombinase cuts lower segment and contains the site lox72 more easily identified；The pBTS-IC nucleosides
Acid sequence is as shown in SEQ ID NO.3；
(10) the positive transformants bacterial strain in picking (9), connects bacterium and is cultivated, and utilizes IPTG inducing expression or leakage expression
Cre recombinase cuts H chloramphenicol resistance segment；
(11) bacterium solution in (10) is taken, is inoculated into fluid nutrient medium, cultivation temperature is 45 DEG C, and incubation time is greater than for 24 hours；
(12) bacterium solution cultivated in (11), dilution 10 are taken5-106On coated plate to the plate of nonreactive；
(13) single colonie in (12) is taken, Resistance Identification is carried out, until screening 2-5 plants of nonreactive bacterial strains；
(14) nonreactive bacterial strain in (13) is taken, is inoculated into culture medium, is incubated overnight, genome is extracted, carries out PCR identification, sun
Property bacterial strain carry out sequencing identification again, being still accredited as positive bacterial strain is the bacterial strain for completing genome manipulation.
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| PCT/CN2017/091056 WO2018099063A1 (en) | 2016-11-30 | 2017-06-30 | Method for efficiently secreting and expressing foreign protein using bacillus |
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| CN109337855A (en) * | 2018-11-20 | 2019-02-15 | 成都美溢德生物技术有限公司 | A kind of efficient method for reducing fermentation of bacillus liquid viscosity |
| CN112239742B (en) * | 2020-08-24 | 2022-10-18 | 天津科技大学 | Bacillus licheniformis for producing alkaline protease and application thereof |
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| CN102021164A (en) * | 2010-11-09 | 2011-04-20 | 南京农业大学 | Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene |
| CN102311970A (en) * | 2011-08-05 | 2012-01-11 | 浙江大学 | Expression cassette mazF-cassette for expressing escherichia coli toxin protein mazF gene and construction method and application thereof |
| CN104388460A (en) * | 2014-11-26 | 2015-03-04 | 成都美溢德生物技术有限公司 | Method for transforming bacillus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021164A (en) * | 2010-11-09 | 2011-04-20 | 南京农业大学 | Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene |
| CN102311970A (en) * | 2011-08-05 | 2012-01-11 | 浙江大学 | Expression cassette mazF-cassette for expressing escherichia coli toxin protein mazF gene and construction method and application thereof |
| CN104388460A (en) * | 2014-11-26 | 2015-03-04 | 成都美溢德生物技术有限公司 | Method for transforming bacillus |
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