CN101481709A - Method for transforming bacillus - Google Patents
Method for transforming bacillus Download PDFInfo
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- CN101481709A CN101481709A CNA2009100776435A CN200910077643A CN101481709A CN 101481709 A CN101481709 A CN 101481709A CN A2009100776435 A CNA2009100776435 A CN A2009100776435A CN 200910077643 A CN200910077643 A CN 200910077643A CN 101481709 A CN101481709 A CN 101481709A
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000001131 transforming effect Effects 0.000 title claims abstract description 10
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 24
- 230000009466 transformation Effects 0.000 claims abstract description 11
- 238000004520 electroporation Methods 0.000 claims abstract description 8
- 235000015097 nutrients Nutrition 0.000 claims description 68
- 239000007788 liquid Substances 0.000 claims description 35
- 230000005611 electricity Effects 0.000 claims description 22
- 229930006000 Sucrose Natural products 0.000 claims description 21
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 21
- 239000005720 sucrose Substances 0.000 claims description 21
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 244000063299 Bacillus subtilis Species 0.000 abstract description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 6
- 239000013612 plasmid Substances 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 210000001938 protoplast Anatomy 0.000 abstract 3
- 239000000463 material Substances 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 108010014251 Muramidase Proteins 0.000 description 5
- 102000016943 Muramidase Human genes 0.000 description 5
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 229960000274 lysozyme Drugs 0.000 description 5
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- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 229940049547 paraxin Drugs 0.000 description 3
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method of transforming bacillus. In the method, bacillus protoplast is used as a transformation receptor. An electroporation method is used for introducing DNA molecules into the bacillus protoplast. The bacillus transforming method uses bacillus subtilis or bacillus licheniformis protoplast as the receptor and introduces recombinant plasmids into the bacillus subtilis or the bacillus licheniformis. The transformation rate reaches 60 percent. The method is of great significance to a bacillus subtilis or bacillus licheniformis genetic engineering expression system. In addition, the transformation of the bacillus licheniformis is hard to be implemented under general conditions. The bacillus transforming method of the invention is also effective in transforming the bacillus licheniformis.
Description
Technical field
The present invention relates to a kind of method of transforming bacillus.
Background technology
Genus bacillus is proposed in 1872 first as a genus, existing more than 100 year so far.People have almost related to the every field that Gram-positive can be given birth to the spore bacterium to the research of genus bacillus at present.Especially carried out a large amount of work in competence, gemma formation and regulation and control thereof, genetic manipulation, strain improvement, biotechnology field.Use maximum subtilises that surely belongs in the scientific research.
It is that the genetics work early stage with it is closely-related that subtilis can be developed into first gene engineering expression system in the genus bacillus.Spizizen has found subtilis 168 bacterial strains for since can transforming bacterial strain in 1958, the genetics work of subtilis deepens continuously and develops.The Bacillus of Ohio State Univ-Columbus USA heredity preservation center (BGSC, http://www.bgsc.org) to the genetic mutation strain of 168 bacterial strains of preservation in 1999 just has 890.It has involved the mutant of many nutritional requirements, various enzyme, gemma formation and germination, competence, the Sigma factor, DNA reorganization and each side genes such as reparation and positive negative regulation.Invented the seventies after the DNA recombinant technology, the plasmid that has the resistance sign of particularly finding streptococcus aureus can be used as after the carrier of subtilis, overcome the difficulty that subtilis has only the crypticity plasmid, the engineered work of subtilis accelerated development especially.So far, clone and expressed a large amount of protokaryons and eukaryotic gene in subtilis and sibling species thereof, what wherein have is applied to suitability for industrialized production, has obtained many achievements.With carrying in the foreign gene carrier importing host bacteria is the prerequisite of bacterial gene engineering expression system.Colibacillary calcium chloride transformation is invalid to subtilis, so 168 bacterial strains and the mutant thereof that just can carry out competence conversion (Spizizen 1958) the most exactly before the invention of DNA recombinant technology that the host strain in the subtilis gene engineering expression system is used.The ultimate principle that the Spizizen competence transforms: be cell forms one section hunger, is easy to absorb exogenous dna fragment in the minimum salt culture medium of Spizizen period.General step is earlier it to be cultivated in the substratum than horn of plenty, is transferred to then in the barren substratum, makes it form competence (generally very of short duration).
With the bacillus subtilis bacterium competence cell is that the transformation efficiency that transforms of host is very low.
Cell electroporation has ubiquity, can be used for various types of cells such as animal, plant and microorganism, and efficient height, no residual toxicity, parameter are controlled easily.But it is that plasmid is directly imported host cell that general electricity transforms.
Summary of the invention
The purpose of this invention is to provide a kind of transforming bacillus method.
Genus bacillus method for transformation provided by the present invention is to be transformation receptor with the genus bacillus protoplastis, with electroporation dna molecular is imported in the genus bacillus protoplastis.
Wherein, the genus bacillus protoplastis can obtain with existing several different methods.Described genus bacillus protoplastis specifically can be prepared as follows: genus bacillus is cultivated at the PAB nutrient solution, collects described genus bacillus, and is resuspended with thalline protection liquid, adds N,O-Diacetylmuramidase then and obtains the genus bacillus protoplastis; Described PAB nutrient solution is 25% (volumn concentration), 4 * PAB nutrient solution, and described 4 * PAB nutrient solution is made up of following material: 5-7g/L extractum carnis, 5-7g/L yeast extract paste, 18-22g/L peptone, 4.3-6.5g/L KH
2PO
4, 13-15g/L NaCl, 17.5-20.3g/LK
2HPO
43H
2O, 3-5g glucose; Described thalline protection liquid is made up of following material: 19-22g/L BSA, 1.8-2.2M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution; 50% (volumn concentration), 2 * SMM nutrient solution, described 2 * SMM nutrient solution contains 3.8-5.0g/L toxilic acid, 7.5-9.0g/L MgCl
26H
2O, 1.8-2.2M sucrose, pH6.2-6.8.
Described genus bacillus is subtilis or Bacillus licheniformis.Described electroporation is at 25 μ F electric capacity, 400 Ω resistance, and 0.3-0.5kV electric shock electricity changes the subtilis protoplastis in the liquid.Perhaps, described electroporation is at 25 μ F electric capacity, 400 Ω resistance, and 0.6-0.8kV electric shock electricity changes the Bacillus licheniformis protoplastis in the liquid.
Described electricity changes liquid and is made up of following material: 19-22g/L BSA, 1.8-2.2M sucrose, 47%-55% (volumn concentration) 2 * SMM nutrient solution, 20-28% (volumn concentration) 4 * PAB nutrient solution.
Genus bacillus method for transformation of the present invention is an acceptor with subtilis or Bacillus licheniformis protoplastis, recombinant plasmid is imported in subtilis or the Bacillus licheniformis, transformation efficiency reaches 60%, and subtilis and Bacillus licheniformis gene engineering expression system are had great importance.In addition, the conversion of Bacillus licheniformis is difficult to carry out in the ordinary course of things, and genus bacillus method for transformation of the present invention also is effectively for the conversion of Bacillus licheniformis.
Embodiment
Embodiment 1, subtilis transform
One, the preparation of subtilis protoplastis
Subtilis (Bacillus subtilis) CICC 10157 (purchasing in Chinese industrial microbial strains preservation administrative center) is inoculated in the PAB nutrient solution 37 ℃ of shaking table overnight incubation.Cultivate 4 hour in transferring the PAB nutrient solution fresh according to 2.5ml/100ml next day, to logarithmic growth early stage.Centrifugal 10 minutes of 5000rpm collects thalline, and is resuspended with thalline protection liquid, add then N,O-Diacetylmuramidase (sigma company, Lysozyme, code0663) making its final concentration is 10mg/mL, 37 ℃, shaking table was cultivated 60 minutes, promptly formed protoplastis.
PAB nutrient solution: 100ml4 * PAB nutrient solution adds the 300ml aqua sterilisa.
4 * PAB nutrient solution is made up of following material: 6g extractum carnis, 6g yeast extract paste, 20g peptone, 5.5g KH
2PO
4, 14g NaCl, 19.5g K
2HPO
43H
2O and 4g glucose.
Described thalline protection liquid is made up of following material: 20g/L BSA, 2M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution, 50% (volumn concentration), 2 * SMM nutrient solution.
2 * SMM nutrient solution: 4.6g/L toxilic acid, 8.1g/L MgCl
26H
2O, 2M sucrose, pH6.5.
Two, subtilis is that acceptor transforms with the protoplastis
When the cell when 95% becomes protoplastis, 4 ℃, behind centrifugal 5 minutes of the 6000rpm, electricity commentaries on classics liquid (20g/L BSA, 1M sucrose, 50% (volumn concentration) 2 * SMM nutrient solution, 25% (volumn concentration), 4 * PAB nutrient solution) with precooling is washed twice, change the resuspended protoplastis of liquid with the 500ul electricity afterwards, making protoplastis concentration is 108cfu/mL; With protoplastis with every pipe 120 μ L packing, adding 0.3 μ g pBCJ164.3 (preserving center, Bacillus Genetic Stock Center (BGSC) The Ohio StateUniversity available from Ohio State Univ-Columbus USA's bacterial classification) back ice put 5 minutes; Be transferred to then in the electric shock cup of precooling, 25 μ F electric capacity are set, 400 Ω resistance, with different voltages (0.1,0.2,0.3,0.4,0.5,0.6,0.7 or 0.8kv) electric shock once; Adding the 1mL electricity after the electric shock immediately changes liquid, is transferred in the EP pipe, and 37 ℃, the 100rpm shaking table was cultivated 12 hours; Be applied to then to contain on the paraxin 4.0 μ g/ml DM3 substratum and screen.Experiment repeats 3 times.The result is as shown in table 1.
The DM3 substratum is made up of following material: 8g/L agar, 5g/L caseinhydrolysate, 5g/L yeast powder, 1.5g/LKH
2PO
4, 3.5g/L K
2HPO
4, 45.5g/L sorbyl alcohol, 10g/L starch, 5g/L glucose; 0.09g/L BSA and 0.02M MgCl
2
The positive colony that screens on the table 1.DM3 substratum
| Voltage kv | Dull and stereotyped A colony number (individual) | Dull and stereotyped B colony number (individual) | Dull and stereotyped C colony number (individual) |
| 0.1 | Do not have | Do not have | Do not have |
| 0.2 | 2 | 5 | 3 |
| 0.3 | 30 | 35 | 38 |
| 0.4 | 33 | 40 | 30 |
| 0.5 | 37 | 40 | 36 |
| 0.6 | 11 | 9 | 13 |
| 0.7 | 5 | Do not have | Do not have |
| 0.8 | Do not have | 2 | 5 |
Embodiment 2, subtilis transform
One, the preparation of subtilis protoplastis
Subtilis (Bacillus subtilis) 210 CICC 11210 (purchasing in Chinese industrial microbial strains preservation administrative center) are inoculated in the PAB nutrient solution 37 ℃ of shaking table overnight incubation.Cultivate 4 hour in transferring the PAB nutrient solution fresh according to 2.5ml/100ml next day, to logarithmic growth early stage.Centrifugal 10 minutes of 5000rpm collects thalline, and is resuspended with thalline protection liquid, add then N,O-Diacetylmuramidase (sigma company, Lysozyme, code0663) making its final concentration is 10mg/mL, 37 ℃, shaking table was cultivated 60 minutes, promptly formed protoplastis.
The PAB nutrient solution: 100ml 4 * PAB nutrient solution adds the 300ml aqua sterilisa.
4 * PAB nutrient solution is made up of following material: 5g extractum carnis, 5g yeast extract paste, 18g peptone, 4.3g KH
2PO
4, 13g NaCl, 17.5g K
2HPO
43H
2O and 3g glucose.
Thalline protection liquid: 19g/L BSA, 1.8M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution, 50% (volumn concentration), 2 * SMM nutrient solution.
2 * SMM nutrient solution: 3.8g/L toxilic acid, 7.5g/L MgCl
26H
2O, 1.8M sucrose, pH6.2.
Two, subtilis is that acceptor transforms with the protoplastis
When the cell when 95% becomes protoplastis, 4 ℃, behind centrifugal 5 minutes of the 6000rpm, electricity commentaries on classics liquid [19g/LBSA, 1.8M sucrose, 47% (volumn concentration) 2 * SMM nutrient solution, 20% (volumn concentration), 4 * PAB nutrient solution] with precooling is washed twice, change the resuspended protoplastis of liquid with the 500ul electricity afterwards, making protoplastis concentration is 10
8Cfu/mL; Protoplastis with every pipe 120 μ L packing, is added 0.5 μ g pBCJ164.3.Back ice was put 5 minutes; Be transferred to then in the electric shock cup of precooling, 25 μ F electric capacity are set, 400 Ω resistance, with the 0.3kv electric shock once; Adding the 1mL electricity after the electric shock immediately changes liquid, is transferred in the EP pipe, and 37 ℃, the 100rpm shaking table was cultivated 12 hours; Be applied to then on the paraxin 4.0 μ g/ml DM3 substratum and screen.Experiment repeats 3 times.
After the screening, the colony number on the flat board is 40 on the DM3 substratum.
Embodiment 3, subtilis transform
One, the preparation of subtilis protoplastis
Subtilis Bacillus subtilis 074 (BF-7658), CICC 10074 (purchasing in Chinese industrial microbial strains preservation administrative center) is inoculated in the PAB nutrient solution 37 ℃ of shaking table overnight incubation.Next day is according to cultivating 4 hours in 2.5ml/100ml switching as the fresh PAB nutrient solution, to logarithmic growth early stage.Centrifugal 10 minutes of 5000rpm collects thalline, and is resuspended with thalline protection liquid, add then N,O-Diacetylmuramidase (sigma company, Lysozyme, code0663) making its final concentration is 10mg/mL, 37 ℃, shaking table was cultivated 60 minutes, promptly formed protoplastis.
The PAB nutrient solution: 100ml 4 * PAB nutrient solution adds the 300ml aqua sterilisa.
4 * PAB nutrient solution is made up of following material: 7g/L extractum carnis, 7g/L yeast extract paste, 22g/L peptone, 6.5g/LKH
2PO
4, 15g/L NaCl, 20.3g/L K
2HPO
43H
2O, 5g glucose.
Thalline protection liquid is made up of following material: 22g/L BSA, 2.2M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution, 50% (volumn concentration), 2 * SMM nutrient solution.
2 * SMM nutrient solution: 5.0g/L toxilic acid, 9.0g/L MgCl
26H
2O, 2.2M sucrose, pH6.8.
Two, subtilis is that acceptor transforms with the protoplastis
When the cell when 95% becomes protoplastis, 4 ℃, behind centrifugal 5 minutes of the 6000rpm, electricity commentaries on classics liquid (22g/LBSA, 2.2M sucrose, 55% (volumn concentration) 2 * SMM nutrient solution, 28% (volumn concentration), 4 * PAB nutrient solution) with precooling is washed twice, change the resuspended protoplastis of liquid with the 500ul electricity afterwards, making protoplastis concentration is 10
8Cfu/mL; Protoplastis with every pipe 120 μ L packing, is added 1 μ g pBCJ164.3.Back ice was put 5 minutes; Be transferred to then in the electric shock cup of precooling, 25 μ F electric capacity are set, 400 Ω resistance, with the 0.5kv electric shock once; Adding the 1mL electricity after the electric shock immediately changes liquid, is transferred in the EP pipe, and 37 ℃, the 100rpm shaking table was cultivated 12 hours; Be applied to then on the paraxin 4.0 μ g/ml DM3 substratum and screen.Experiment repeats 3 times.
After the screening, the colony number on the flat board is 30 on the DM3 substratum.
Embodiment 4, Bacillus licheniformis transform
One, the preparation of Bacillus licheniformis protoplastis
Bacillus licheniformis (Bacillus licheniformis) CICC 10181 (Chinese industrial microbial strains preservation administrative center) is inoculated in the PAB nutrient solution 37 ℃ of shaking table overnight incubation.Next day is according to cultivating 4 hours in 2.5ml/100ml switching as the fresh PAB nutrient solution, to logarithmic growth early stage.Centrifugal 10 minutes of 5000rpm collects thalline, and is resuspended with thalline protection liquid, add then N,O-Diacetylmuramidase (sigma company, Lysozyme, code0663) making its final concentration is 10mg/mL, 37 ℃, shaking table was cultivated 60 minutes, promptly formed protoplastis.
PAB nutrient solution: 100ml4 * PAB nutrient solution adds the 300ml aqua sterilisa.
4 * PAB nutrient solution is made up of following material: 6g extractum carnis, 6g yeast extract paste, 20g peptone, 5.5g KH
2PO
4, 15g NaCl, 17.5g K
2HPO
43H
2O and 3g glucose.
Described thalline protection liquid is made up of following material: 20g/L BSA, 2M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution, 50% (volumn concentration), 2 * SMM nutrient solution.
2 * SMM nutrient solution: 4.6g/L toxilic acid, 8.1g/L MgCl
26H
2O, 2M sucrose, pH6.5.
Two, Bacillus licheniformis is that acceptor transforms with the protoplastis
When the cell when 95% becomes protoplastis, 4 ℃, behind centrifugal 5 minutes of the 6000rpm, electricity commentaries on classics liquid (20g/L BSA, 1M sucrose, 50% (volumn concentration) 2 * SMM nutrient solution, 25% (volumn concentration), 4 * PAB nutrient solution) with precooling is washed twice, change the resuspended protoplastis of liquid with the 500ul electricity afterwards, making protoplastis concentration is 10
8Cfu/mL; Protoplastis with every pipe 120 μ L packing, is added 0.3 μ g pAXOI (preserving center, Bacillus Genetic Stock Center (BGSC) The Ohio State University available from Ohio State Univ-Columbus USA's bacterial classification) back ice and put 5 minutes; Be transferred to then in the electric shock cup of precooling, 25 μ F electric capacity are set, 400 Ω resistance, with the 0.6kv electric shock once; Adding the 1mL electricity after the electric shock immediately changes liquid, is transferred in the EP pipe, and 37 ℃, the 100rpm shaking table was cultivated 12 hours; Be applied to then on the DM3 substratum that contains 10 μ g/ml erythromycin and screen.Experiment repeats 3 times.
After the screening, the colony number on the flat board is 20 on the DM3 substratum.
Embodiment 5, Bacillus licheniformis transform
One, the preparation of Bacillus licheniformis protoplastis
Bacillus licheniformis (Bacillus licheniformis) CICC 10181 (Chinese industrial microbial strains preservation administrative center) is inoculated in the PAB nutrient solution 37 ℃ of shaking table overnight incubation.Next day is according to cultivating 4 hours in 2.5ml/100ml switching as the fresh PAB nutrient solution, to logarithmic growth early stage.Centrifugal 10 minutes of 5000rpm collects thalline, and is resuspended with thalline protection liquid, add then N,O-Diacetylmuramidase (sigma company, Lysozyme, code0663) making its final concentration is 10mg/mL, 37 ℃, shaking table was cultivated 60 minutes, promptly formed protoplastis.
PAB nutrient solution: 100ml4 * PAB nutrient solution adds the 300ml aqua sterilisa.
4 * PAB nutrient solution is made up of following material: 6g extractum carnis, 6g yeast extract paste, 20g peptone, 5.5g KH
2PO
4, 15g NaCl, 17.5g K
2HPO
43H
2O and 3g glucose.
Described thalline protection liquid is made up of following material: 20g/L BSA, 2M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution, 50% (volumn concentration), 2 * SMM nutrient solution.
2 * SMM nutrient solution: 4.6g/L toxilic acid, 8.1g/L MgCl
26H
2O, 2M sucrose, pH6.5.
Two, Bacillus licheniformis is that acceptor transforms with the protoplastis
When the cell when 95% becomes protoplastis, 4 ℃, behind centrifugal 5 minutes of the 6000rpm, electricity commentaries on classics liquid (20g/L BSA, 1M sucrose, 50% (volumn concentration) 2 * SMM nutrient solution, 25% (volumn concentration), 4 * PAB nutrient solution) with precooling is washed twice, change the resuspended protoplastis of liquid with the 500ul electricity afterwards, making protoplastis concentration is 10
8Cfu/mL; Protoplastis with every pipe 120 μ L packing, is added to ice behind the 0.3 μ g pAXOI and put 5 minutes; Be transferred to then in the electric shock cup of precooling, 25 μ F electric capacity are set, 400 Ω resistance, with the 0.8kv electric shock once; Adding the 1mL electricity after the electric shock immediately changes liquid, is transferred in the EP pipe, and 37 ℃, the 100rpm shaking table was cultivated 12 hours; Be applied to then on the DM3 substratum that contains 10 μ g/ml erythromycin and screen.Experiment repeats 3 times.
After the screening, the colony number on the flat board is 30 on the DM3 substratum.
Claims (7)
1, a kind of method of transforming bacillus is to be transformation receptor with the genus bacillus protoplastis, with electroporation dna molecular is imported in the genus bacillus protoplastis.
2, method according to claim 1, it is characterized in that: described genus bacillus protoplastis is prepared as follows: genus bacillus is cultivated at the PAB nutrient solution, collect described genus bacillus, resuspended with thalline protection liquid, add N,O-Diacetylmuramidase then and obtain the genus bacillus protoplastis; Described PAB nutrient solution is 25% (volumn concentration), 4 * PAB nutrient solution, and described 4 * PAB nutrient solution contains 5-7g/L extractum carnis, 5-7g/L yeast extract paste, 18-22g/L peptone, 4.3-6.5g/L KH
2PO
4, 13-15g/L NaCl, 17.5-20.3g/L K
2HPO
43H
2O, 3-5g glucose; Described thalline protection liquid contains 19-22g/L BSA, 1.8-2.2M sucrose, 25% (volumn concentration) 4 * PAB nutrient solution; 50% (volumn concentration), 2 * SMM nutrient solution, described 2 * SMM nutrient solution contains 3.8-5.0g/L toxilic acid, 7.5-9.0g/L MgCl
26H
2O, 1.8-2.2M sucrose, pH6.2-6.8.
3, method according to claim 1 and 2 is characterized in that: described genus bacillus is a subtilis.
4, method according to claim 1 and 2 is characterized in that: described genus bacillus is a Bacillus licheniformis.
5, method according to claim 3 is characterized in that: described electroporation is at 25 μ F electric capacity, 400 Ω resistance, and 0.3-0.5kV electric shock electricity changes the subtilis protoplastis in the liquid.
6, method according to claim 4 is characterized in that: described electroporation is at 25 μ F electric capacity, 400 Ω resistance, and 0.6-0.8kV electric shock electricity changes the Bacillus licheniformis protoplastis in the liquid.
7, according to claim 5 or 6 described methods, it is characterized in that: described electricity changes liquid and contains 19-22g/L BSA, 1.8-2.2M sucrose, 47%-55% (volumn concentration) 2 * SMM nutrient solution, 20-28% (volumn concentration) 4 * PAB nutrient solution.
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| CN103266127A (en) * | 2013-05-03 | 2013-08-28 | 安徽工程大学 | Method for converting bacillus subtilis by electric shock |
| CN110150321A (en) * | 2019-05-24 | 2019-08-23 | 苏农（广德）生物科技有限公司 | A kind of bacillus subtilis wettable powder and its preparation process |
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| CN101230329A (en) * | 2008-02-02 | 2008-07-30 | 江南大学 | Bacillus licheniformis industrial production strain genetic transformation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103266127A (en) * | 2013-05-03 | 2013-08-28 | 安徽工程大学 | Method for converting bacillus subtilis by electric shock |
| CN103266127B (en) * | 2013-05-03 | 2014-10-01 | 安徽工程大学 | Method for converting bacillus subtilis by electric shock |
| CN110150321A (en) * | 2019-05-24 | 2019-08-23 | 苏农（广德）生物科技有限公司 | A kind of bacillus subtilis wettable powder and its preparation process |
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