CN104388460B

CN104388460B - A kind of method of transforming bacillus

Info

Publication number: CN104388460B
Application number: CN201410686389.XA
Authority: CN
Inventors: 任钧
Original assignee: Chengdu Mei Yide Bioisystech Co Ltd
Current assignee: Chengdu Mei Yide Bioisystech Co Ltd
Priority date: 2014-11-26
Filing date: 2014-11-26
Publication date: 2017-12-19
Anticipated expiration: 2034-11-26
Also published as: CN104388460A

Abstract

The present invention relates to biological technical field, specially a kind of method of transforming bacillus.This method induces the state that bacillus entrance can be converted first, then is co-cultured with the Escherichia coli containing recombinant plasmid, during which adds polymyxins, kills Escherichia coli, discharges plasmid.Bacterial strain after co-cultivation is coated being screened on corresponding antibiotic flat board, the bacterial strain being converted.The method for transformation is simple to operate, it is not necessary to special equipment；It cannot be only used for the conversion of the type strain of bacillus subtilis, it may also be used for the bacillus subtilis conversion of wild-type strain and the Bacterial Transformation of other bacillus, wide adaptability.

Description

A kind of method of transforming bacillus

Technical field

The present invention relates to biological technical field, specially a kind of method of transforming bacillus.

Background technology

Before thousands of years, the mankind have begun to make the food such as wine, pickles, Yoghourt using fermentation.But with modern micro- The development of biology, we can by the more products of fermenting and producing, including：1st, thalline, probiotics, dusty yeast etc., 2, just Level metabolite, such as protease, amylase, cellulase, lipase, ethanol, citric acid, acetone, glutamic acid, lysine, core Thuja acid, vitamin etc., 3, secondary metabolite, such as penicillin, kanamycins, tetracycline, antibacterial peptide material, 4, product turns Change, such as convert milk into Yoghourt, Starch Conversion is ethanol etc..These products all significantly improve our life.

For fermentation technique in addition to needing specific fermentation work production skill, the key point of fermentation technique is the bacterium for possessing uniqueness Strain.Under the promotion of modern microbiology, the bacterial strain for possessing peculiar property can be obtained by bacterial strain screening and mutagenesis.But The Period Process is longer, and with uncertainty.With the rise of molecular biology, bacterial strain is transformed by genetic engineering, can It is exactly that bacterium turns quickly and efficiently to obtain one of the bacterial strain for possessing peculiar property, the premise technology of the renovation technique of genetic engineering Change technology, only the purpose fragment for needing to transform is imported into after aimed strain and aimed strain could be transformed.

Bacillus is a kind of gram-positive bacteria, does not contain endotoxin.The bacillus subtilis strain of bacillus subordinate The bacterial strain with biological safety is also thought by FDA.Bacillus can be used for the products such as production protease, amylase at present, It can be added to as probiotics in food.Therefore establishing the method for transformation of bacillus and genetic modification method has important section Grind and economic value.

The method for transformation of bacillus can be divided into chemical transformation, protoplast transformation and electrotransformation.Chemistry turns Change method is to be established by Spizizen at last century the fifties end, and this method is transferred to using bacillus from rich medium After being grown on low nutrition culture medium, the principle that can be converted state, compound calcium ion and magnesium ion thorn are presented within a period of time Sharp method, DNA fragmentation or plasmid can be transferred in bacterium.But the method for transformation transformation efficiency is relatively low, is only used for withered The specific bacterial strains such as careless bacillus type strain 168.Protoplasm body is primary by the generation of lysozyme bacterium for degrading cell membrane Plastid, extracellular DNA is transferred into the cell using material incentive cans such as PEG.But this method is cumbersome, condition It is not easily controlled, transformation efficiency is not high, seldom uses.Electrotransformation is the side that last century late nineteen eighties are begun setting up Method, the bacillus for growing into certain phase is put into specific electric conversion fluid, applies highfield, punctures cell membrane, allows and turns The DNA changed in liquid enters cell.Up to the present, the electric method for transformation for bacillus of document report is as many as tens of Kind.Still someone established in 1999 in the method for transformation, xue et al. for improving bacillus and a kind of utilized sweet dew recent years Alcohol and sorbierite prepare hypertonic conversion fluid, are remarkably improved transformation efficiency（High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis, Journal of Microbiological Methods, 1999, 34:183–191）.2006, Romero et al. delivered a kind of method for transformation of electricity conversion protoplast, Ke Yizhuan Change some wild-type strains for being not easy to convert（Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation, Journal of Microbiological Methods, 2006, 66:556–559）.Cao et al. with the addition of trehalose on the basis of hypertonic conversion method, can improve and turn Change efficiency（A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides, Biotechnol Lett (2011) 33:1047–1051）.Fatma et al. adds glycine betaine, is remarkably improved electric shock on the basis of hypertonic conversion method The survival rate of bacterium afterwards, improve transformation efficiency.But the strain specificity of electrotransformation is higher, a kind of method for transformation can only convert Some bacterial strains.

In actual gene is engineered, a small amount of transformation can only be carried out to bacterial strain, it is therefore desirable to select characteristic relatively Good wild-type strain is transformed.But above-mentioned method for transformation limitation is very big, can only convert a small number of wild-type strains.Cause This establishes a kind of wider bacillus method for transformation of adaptability, can solve the problems, such as that wild bacillus can not be converted.

The content of the invention

For above technical problem, the present invention establishes a kind of method for transformation of brand-new bacillus.This method is first The state that induction bacillus entrance can be converted, then co-cultured with the Escherichia coli containing recombinant plasmid, during which add Polymyxins, Escherichia coli are killed, discharge plasmid.Bacterial strain after co-cultivation is coated carrying out on corresponding antibiotic flat board Screening, the bacterial strain being converted.The method for transformation is simple to operate, it is not necessary to special equipment；It cannot be only used for withered grass gemma The conversion of the type strain of bacillus, it may also be used for the bacillus subtilis conversion of wild-type strain is thin with other bacillus Bacterium converts, wide adaptability.

The object of the invention is realized by following technical proposals：

A kind of method of transforming bacillus, it is characterised in that comprise the following steps：

（1）, by bacterial strain streak inoculation to be transformed to LB flat boards, cultivate 16-24h under conditions of 37 DEG C；It will contain and need The Escherichia coli streak inoculation of plasmid is converted to containing on the LB flat boards for corresponding to antibiotic, grows 16-24h at 37 DEG C；

（2）, picking step（1）In the single bacterium colony of bacterial strain to be transformed be inoculated into teat glass, every teat glass contains 3ml MA culture mediums, 15-18h is cultivated under 37 DEG C, 200rpm, obtains bacterial strain to be transformed；By step（1）In Escherichia coli connect Kind is into teat glass, and often LB culture medium 4-6ml of the pipe containing corresponding antibiotic, 16-22h is cultivated under 37 DEG C, 200rpm；Institute The MA culture mediums stated are made up of following component：2 × MA base, 1 × MA salt, 50% glucose solution of 1% volume, wherein MA base and MA salt are with 10 × mother liquor, 4 DEG C of preservations.10 × MA base are by 10g/L yeast extracts and 2g/L pancreas eggs White peptone is formed.10 × MA salt are made up of following component：150mM ammonium sulfate, 800mM dipotassium hydrogen phosphates, 440mM biphosphates Potassium, 35mM trisodium citrates and 10mM magnesium sulfate.

（3）Take step（2）In bacterial strain 300ul to be transformed, be inoculated into 2.7ml MA culture mediums, be subsequently placed in glass examination Guan Zhong, 37 DEG C, 200rpm cultures 3h；

（4）Take step（3）In bacterial strain to be transformed, then dilute 2-20 times be inoculated into MB culture mediums, final volume is 3ml, it is placed in teat glass, 30 DEG C -37 DEG C, 90min is cultivated under 200rpm；MB culture mediums are made up of following component：1× MA base, 1 × MA salt, 50% glucose solution of 1% volume, 0.5mM calcium chloride, 2.5mM magnesium chlorides.Wherein MA Base and MA salt are with 10 × mother liquor, 4 DEG C of preservations；Calcium chloride, magnesium chloride are with 100 × mother liquor, 4 DEG C of preservations.

（5）Take step（2）In the Escherichia coli bacteria liquid containing plasmid, 13400 × g centrifugation 1min, remove supernatant, cultivated with MB Base washes twice, and removes remnants antibiotic；

（6）In step（5）In Escherichia coli in add 0.2-1 times of initial volume 4 in bacterium solution to be transformed, final volume 1ml, final concentration of 5-30mg/L polymyxin Bs are added, the 1/50-1/10 of normal screening concentration corresponding antibiotic, are placed in glass In glass test tube, 37 DEG C, 200rpm cultures 30-90min；Treated preferably, this step is chosen in the 4 of 0.5 times of initial volume Transformed bacteria solution, final volume 1ml add final concentration of 10mg/L polymyxin Bs, the corresponding antibiotic of normal screening concentration 1/20, Cultivate 1h；

（7）Take step（6）In bacterium solution 200-500ul, be coated on containing 5-30 mg/L polymyxin Bs and containing corresponding On the flat board of antibiotic, 20-40h is cultivated.Picking growth single bacterium colony identified, the bacillus converted, as excellent Choosing, is coated on containing 10 mg/L polymyxin Bs.

The positive effect of the present invention is embodied in：

This method induces the state that bacillus entrance can be converted first, then enters with the Escherichia coli containing recombinant plasmid Row co-cultures, and during which adds polymyxins, kills Escherichia coli, discharges plasmid.It is right that bacterial strain after co-cultivation is coated Answer and screened on antibiotic flat board, the bacterial strain being converted.The method for transformation is simple to operate, it is not necessary to special equipment； Cannot be only used for the conversion of the type strain of bacillus subtilis, it may also be used for the conversion of the bacillus subtilis of wild-type strain and The Bacterial Transformation of other bacillus, wide adaptability.

Embodiment

This specification（Including any accessory claim, summary）Disclosed in any feature, unless specifically stated otherwise, Replaced by other equivalent or with similar purpose alternative features.I.e., unless specifically stated otherwise, each feature is a series of An example in equivalent or similar characteristics.

Use below the compound method of article for：

Solution is prepared

10×MA base：1g yeast extracts (OXOID) are weighed, 0.2g tryptones (OXOID), add water constant volume to arrive 100ml, 121 DEG C, after 20min sterilizings, 4 DEG C of preservations.

10×MA salt：Weigh 1.98g ammonium sulfate, the hypophosphite monohydrate hydrogen dipotassiums of 18.26g tri-, 5.99g potassium dihydrogen phosphates, 0.90g two citric acid monohydrate trisodiums, 0.25g bitter salts, add water constant volume to 100ml, 121 DEG C, after 20min sterilizings, 4 DEG C Preserve.

50% glucose solution：50g glucose is weighed, adding water constant volume, 121 DEG C, after 20min sterilizings, 4 DEG C are protected to 100ml Deposit.

250mM magnesium chlorides：The water of 5.08g six and magnesium chloride are weighed, adds water constant volume to 100ml, 121 DEG C, after 20min sterilizings, 4 DEG C preserve.

50mM calcium chloride：0.55g anhydrous calcium chlorides are weighed, add water constant volume to 100ml, 121 DEG C, after 20min sterilizings, 4 DEG C Preserve.

MA culture mediums：The glucose solution of 10 × MA of 2ml, 10 × MA of base, 1ml salt, 100ul 50% is taken, adds nothing Bacterium water 6.9ml, it is now with the current.

MB culture mediums：Take the glucose solution of 10 × MA of 1ml, 10 × MA of base, 1ml salt, 100ul 50%, 100ul 250mM magnesium chlorides, 100ul 50mM calcium chloride, sterilized water 7.7ml is added, it is now with the current.

LB culture mediums：10g tryptones (OXOID), yeast extract 5g (OXOID), sodium chloride 10g are weighed, constant volume arrives 1L, pH are adjusted to 7.0.After packing, 121 DEG C, 20min sterilizings, room temperature preservation.1.5% fine jade is added before solid LB media sterilizing Cosmetics, the culture medium for adding antibiotic are now with the current.

10mg/ml polymyxin Bs：Weigh 0.25g aerosporins（Polymyxin B Sulfate, 6000U/mg, Amresco）, add water constant volume to 25ml, filtration sterilization, packing, 4 DEG C of preservations.

20mg/ml kanamycins：0.5g kanamycin sulfates are weighed, add water constant volume filtration sterilization, to be dispensed, -20 to 25ml DEG C preserve.

100mg/ml ammonia benzyl mycins：2.5g ammonia benzyl mycins are weighed, add water constant volume filtration sterilization, to be dispensed, -20 DEG C to 25ml Preserve.

100mg/ml spectinomycins：2.5g spectinomycins are weighed, add water constant volume filtration sterilization, to be dispensed, -20 DEG C to 25ml Preserve.

25mg/ml bleomycins：0.625g bleomycins are weighed, add water constant volume filtration sterilization, to be dispensed, -20 DEG C to 25ml Preserve.

The reagent used above, unless otherwise indicated, it is purchased from Shanghai Sheng Gong bioengineering limited company.

Comparative example 1：

A, on streak inoculation type strain Bs168 to LB flat boards, pBAV1K-T5-GFP is contained（NCBI：HQ191434）'s Escherichia coli Top10 bacterial strains are on the LB flat boards containing 40mg/L kanamycins, 37 DEG C of culture 24h.

B, in three to five 168 bacterial strain single bacterium colonies of picking in afternoon, it is inoculated into 3ml MA culture mediums, 37 DEG C, 200rpm mistakes Night cultivates；One Top10 single bacterium colony of picking, is inoculated into 5ml 40mg/L containing kanamycins LB culture mediums, 37 DEG C, 200rpm It is incubated overnight.

C, 9 points of next day, 168 bacterium solutions in 300ul B are taken, be inoculated into the fresh MA culture mediums of 2.7ml, 37 DEG C, 200rpm trainings Support 3h.

D, bacterium solution in 1.5ml C is taken, adds 1.5ml MB culture mediums, 37 DEG C, 200rpm cultivates 90min.

E, the Top10 bacterium solutions being incubated overnight in 1ml B are taken, 13400 × g centrifugation 1min, remove supernatant.Add 1ml MB cultures Base, piping and druming mix, and 13400 × g centrifugation 1min, remove supernatant, repeat the step once.

F, in the thalline in E, the bacterium solution in addition 1ml D, addition final concentration 20mg/L polymyxin B, 2mg/L's Kanamycins, 37 DEG C, 200rpm cultures 30min.

G, the bacterium solution 200ul being mixed in F is taken, is coated on the polymyxin B containing 20mg/L, that is mould for 20mg/L card On the LB flat boards of element.After cultivating 24h, picking colony carries out identification statistics.Because pBAV1K-T5-GFP plasmids contain green fluorescence GFP, all viridescent fluorescence of the bacterium colony grown, picking part bacterium colony enter performing PCR identification, are as a result all positive bacterium colonies.

Note：The bacterium solution culture mentioned in above-mentioned steps, is carried out in 15mm*150mm teat glass.

Comparative example 2：

Except step D is changed in the present embodiment：Bacterium solution in 100ul, 300ul, 500ul, 800ul, 1.5ml C is taken, respectively It is inoculated into 2.9ml, 2.7ml, 2.5ml, 2.2ml, 1.5ml MB bacterium solutions, 37 DEG C, 200rpm cultures 90min.

Remaining step is same as Example 1.Bacterium colony count when, 300ul-2.7ml groups it is long go out carry green fluorescent protein Bacterium colony it is most.

Comparative example 3：

Except step D is changed in the present embodiment：Take bacterium solution in 300ul C, be inoculated into 2.7ml MB bacterium solutions, 37 DEG C or 30 DEG C, 200rpm cultures 90min.

Remaining step is identical with comparative example 1.When bacterium colony counts, the bacterium colony with green fluorescent protein that 30 DEG C of group leaders go out is more In 37 DEG C of groups.

Comparative example 4：

Except step D is changed to：Bacterium solution in 300ul C is taken, is inoculated into 2.7ml MB bacterium solutions, 30 DEG C, 200rpm cultures 90min。

Separately the addition bacterium solution ratio in step F, polymyxins concentration, kanamycins concentration and incubation time are carried out orthogonal Optimization.

Remaining step is identical with comparative example 1.In bacterium colony statistics, bacterium solution thinner ratio is 2, and polymyxins concentration is 10mg/ L, kanamycins concentration are 1mg/L, incubation time 1h, and the bacterium colony with green fluorescent protein grown on flat board is most.

Embodiment 1：

A, on streak inoculation type strain Bs168 to LB flat boards, pBAV1K-T5-GFP is contained（NCBI is numbered： HQ191434）Escherichia coli Top10 bacterial strains on the LB flat boards containing 40mg/L kanamycins, 37 DEG C of culture 24h.

D, bacterium solution in 300ul C is taken, adds 2.7ml MB culture mediums, 30 DEG C, 200rpm cultivates 90min.

E, the Top10 bacterium solutions being incubated overnight in 2ml B are taken, 13400 × g centrifugation 1min, remove supernatant.Add 1ml MB cultures Base, piping and druming mix, and 13400 × g centrifugation 1min, remove supernatant, repeat the step once.

F, in the thalline in E, the bacterium solution in addition 1ml D, addition final concentration 10mg/L polymyxin B, 1mg/L's Kanamycins, 37 DEG C, 200rpm cultures 1h.

G, the bacterium solution 200ul being mixed in F is taken, is coated on the polymyxin B containing 10mg/L, that is mould for 20mg/L card On the LB flat boards of element.After cultivating 24h, relatively more bacterium colonies with green fluorescence is obtained, picking part is carried out by bacterium colony PCR identifies that result is the positive.

Embodiment 2：

Except bacterial strain to be transformed is changed to CICC23708 in step A（Bacillus subtilis, it is purchased from Chinese industrial microorganism fungus kind Collection）Outside strain, remaining step is same as Example 2.It is homologous to obtain largely with green in last bacterium colony statistics identification The bacterium colony of color fluorescence, unnecessary 168 bacterial strain of total plate count.Picking part bacterium colony enters performing PCR identification, is all as a result the positive.

Embodiment 3：

Except bacterial strain to be transformed is changed to CICC10266 in step A（Bacillus licheniformis, it is purchased from Chinese industrial microorganism fungus kind Collection）Outside strain, remaining step is same as Example 2.It is homologous to obtain largely with green in last bacterium colony statistics identification The bacterium colony of color fluorescence, total plate count are less than CICC23708 bacterial strains.Picking part bacterium colony enters performing PCR identification, is all as a result the positive.

Embodiment 4：

Except bacterial strain to be transformed is changed to CICC 21077 in step A（Bacillus pumilus, it is purchased from Chinese industrial microorganism fungus kind Collection）Outside strain, remaining step is same as Example 2.It is homologous to obtain largely with green in last bacterium colony statistics identification The bacterium colony of color fluorescence, total plate count are less than CICC23708 bacterial strains.Picking part bacterium colony enters performing PCR identification, is all as a result the positive.

Embodiment 5：

Except bacterial strain to be transformed is changed to BsA189 in step A（Bacillus subtilis, this laboratory are screened from field）Outside strain, Remaining step is same as Example 2.It is homologous to obtain a large amount of bacterium colonies for carrying green fluorescence, bacterium in last bacterium colony statistics identification Fall sum and be less than CICC23708 bacterial strains.Picking part bacterium colony enters performing PCR identification, is all as a result the positive.

Embodiment 6：

Except bacterial strain to be transformed is changed to CICC23708 in step A（Bacillus subtilis, it is purchased from Chinese industrial microorganism fungus kind Collection）, plasmid to be transformed is changed to pBAV1K-FpZR（The recombinant plasmid that laboratory is built on the basis of pBAV1K, greatly Small is 6kb, the 3.6kb more than pBAV1K-T5-GFP）.Remaining step is same as Example 1.In last bacterium colony statistics identification, It is homologous to obtain a large amount of white colonies, total plate count minority embodiment 1.Picking part bacterium colony enters performing PCR identification, is all as a result sun Property.

Embodiment 7：

A, streak inoculation type strain CICC 23708 contains pDGICZ on LB flat boards（NCBI is numbered： EU864235） Escherichia coli Top10 bacterial strains on the LB flat boards containing 100mg/L ammonia benzyl mycins, 37 DEG C of culture 24h.

B, in three to five 168 bacterial strain single bacterium colonies of picking in afternoon, it is inoculated into 3ml MA culture mediums, 37 DEG C, 200rpm mistakes Night cultivates；One Top10 single bacterium colony of picking, is inoculated into the LB culture mediums of 5ml ammonia containing 100mg/L benzyl mycin, 37 DEG C, 200rpm is incubated overnight.

F, in the thalline in E, the bacterium solution in addition 1ml D, addition final concentration 10mg/L polymyxin B, 5mg/L's Spectinomycin, 37 DEG C, 200rpm cultures 1h.

G, the bacterium solution 200ul being mixed in F is taken, is coated on the polymyxin B containing 10mg/L, 100mg/L's is grand On the LB flat boards of mycin.After cultivating 24h, picking colony carries out identification statistics.Relatively more white colonies, picking part are obtained Bacterium colony enters performing PCR identification, is the positive；Picking part colony inoculation is to being given birth on the LB flat boards containing 25mg/L bleomycins Long identification, is the positive.

Claims

A kind of 1. method of transforming bacillus, it is characterised in that comprise the following steps：

（1）, by bacterial strain streak inoculation to be transformed to LB flat boards, cultivate 16-24h under conditions of 37 DEG C；It will contain to be transformed The Escherichia coli streak inoculation of plasmid grows 16-24h to containing on the LB flat boards for corresponding to antibiotic at 37 DEG C；

（2）, picking step（1）In the single bacterium colony of bacterial strain to be transformed be inoculated into teat glass, every teat glass contains 3ml MA culture mediums, 15-18h is cultivated under 37 DEG C, 200rpm, obtain bacterial strain to be transformed；By step（1）In Escherichia coli be inoculated into In teat glass, often LB culture medium 4-6ml of the pipe containing corresponding antibiotic, 16-22h is cultivated under 37 DEG C, 200rpm；

（3）Take step（2）In bacterial strain 300ul to be transformed, be inoculated into 2.7ml MA culture mediums, be subsequently placed in teat glass In, 37 DEG C, 200rpm cultures 3h；

（4）Take step（3）In bacterial strain to be transformed, then dilute 2-20 times be inoculated into MB culture mediums, final volume 3ml, put In teat glass, 30 DEG C -37 DEG C, 90min is cultivated under 200rpm；MB culture mediums are made up of following component：1 × MA base, 1 × MA salt, 50% glucose solution of 1% volume, 0.5mM calcium chloride, 2.5mM magnesium chlorides；Wherein MA base and MA Salt is with 10 × mother liquor, 4 DEG C of preservations；Calcium chloride, magnesium chloride are with 100 × mother liquor, 4 DEG C of preservations；

（5）Take step（2）In the Escherichia coli bacteria liquid containing plasmid, 13400 × g centrifugation 1min, remove supernatant, washed with MB culture mediums Wash the antibiotic for twice, removing remnants；

（6）In step（5）In Escherichia coli in add 0.2-1 times of initial volume the step of（4）In bacterium solution to be transformed, whole body Product 1ml, adds final concentration of 5-30mg/L polymyxin Bs, the 1/50-1/10 of normal screening concentration corresponding antibiotic, is placed in In teat glass, 37 DEG C, 200rpm cultures 30-90min；

（7）Take step（6）In bacterium solution 200-500ul, be coated on containing 5-30 mg/L polymyxin Bs and containing corresponding antibiosis On the flat board of element, 20-40h is cultivated,

Picking growth single bacterium colony identified, the bacillus converted；

Step（2）Described in MA culture mediums be made up of following component：2 × MA base, 1 × MA salt, the 50% of 1% volume Glucose solution, wherein MA base and MA salt are with 10 × mother liquor, 4 DEG C of preservations.
2. the method for transforming bacillus according to claim 1, it is characterised in that：Step（4）In to take step（3）In Bacterial strain to be transformed, then dilute 10 times be inoculated into MB culture mediums, final volume 3ml, be placed in teat glass, in 30 DEG C, 90min is cultivated under conditions of 200rpm.
3. the method for transforming bacillus according to claim 1, it is characterised in that：Described 10 × MA base by with Lower composition is formed：10g/L yeast extracts, 2g/L tryptones, 10 × MA salt are made up of following component：150mM sulfuric acid Ammonium, 800mM dipotassium hydrogen phosphates, 440mM potassium dihydrogen phosphates, 35mM trisodium citrates, 10mM magnesium sulfate.
4. the method for transforming bacillus according to claim 1, it is characterised in that：Step（6）In for 0.5 times of volume Bacterium solution to be transformed, 10mg/L polymyxin B, the corresponding antibiotic of normal screening concentration 1/20, cultivate 1h.
5. the method for transforming bacillus according to claim 1, it is characterised in that：Polymyxin B is 10 in step (7) mg/L。