CN113215073B - Preparation method and genetic transformation method of bacillus natto competent cells - Google Patents
Preparation method and genetic transformation method of bacillus natto competent cells Download PDFInfo
- Publication number
- CN113215073B CN113215073B CN202110658479.8A CN202110658479A CN113215073B CN 113215073 B CN113215073 B CN 113215073B CN 202110658479 A CN202110658479 A CN 202110658479A CN 113215073 B CN113215073 B CN 113215073B
- Authority
- CN
- China
- Prior art keywords
- solution
- bacillus natto
- competent cells
- culturing
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A preparation method of bacillus natto competent cells and genetic transformation method comprise streaking frozen and preserved bacillus natto on LB solid medium, culturing at 37deg.C for 16-20 hr, selecting a single colony on streak plate, inoculating into triangular flask containing LB liquid medium, shake culturing at 37deg.C at 180-250rpm for 12-16 hr; inoculating the culture solution into BN-I culture medium, and shake culturing at 180-250rpm at 37deg.C for 12-16 hr; inoculating the bacterial liquid into BN-I culture medium, and shake culturing at 180-250rpm at 37deg.C for 4-5 hr; inoculating the bacterial liquid into BN-II culture medium, and shake culturing at 180-250rpm at 37deg.C for 1-2 hr; adding transformation promoting liquid into the bacterial liquid, and shake culturing at 80-120rpm at 37 ℃ for 10-30min; after the completion of the culture, a bacterial liquid containing competent cells was obtained. The bacillus natto competent cells prepared by the invention have greatly improved conversion rate, and subsequently provide a foundation for gene expression, silencing, knockout and function research of the bacillus natto.
Description
Technical Field
The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a preparation and genetic transformation method of bacillus natto competent cells.
Background
With the rapid development of bioinformatics and molecular genetics technologies, genetic improvement of excellent strains with potential application value is favored by genetic engineering technology. The cell is able to take up DNA molecules from the surrounding environment and is not easily broken down by restriction endonucleases in the cell in a particular physiological state called competence. Competent cells are cells which are treated to increase the permeability of the cells, so that exogenous genes or vectors enter the competent cells, and pores are repaired by the cells themselves due to the fluidity of cell membranes. One of the key technical bottlenecks in genetic engineering is genetic transformation technology, and the target strain can be modified only after the target gene to be modified is introduced into the target strain.
Bacillus is a gram positive bacterium with a thicker cell wall, so the problems of difficult bacillus transformation and low transformation efficiency are always paid attention to. Sipizizen in 1958 proposed a chemical transformation method for bacillus model strain-bacillus subtilis 168 (Bacillus Subtilis) but the transformation efficiency was not high, and no report of a better method was found. Bacillus natto (Bacillus Subtilis natto) is a subspecies of bacillus subtilis, is a directly edible probiotic, is extremely easy to chain in the bacillus natto culture process and forms a stable multicellular polymer compared with bacillus subtilis 168, and meanwhile, various proteins can be secreted in the bacillus natto production process to promote the bacillus natto to form extracellular polymers, so that the extracellular polymers are easier to fix on a growth substrate (such as soybean, polypropylene and the like), and the stress resistance of the bacillus natto is stronger, so that the transformation difficulty is higher. It was found that the transformation rate of recombinant plasmid into competent cells of Bacillus natto was less than 3 cfu/. Mu.g by strain transformation of Bacillus natto according to the method described by Sipizizen.
Disclosure of Invention
The invention aims to provide a preparation and genetic transformation method of bacillus natto competent cells.
To achieve the above and other related objects, the present invention provides the following technical solutions: a preparation method of bacillus natto competent cells comprises the following steps:
step 1: streaking the frozen and preserved bacillus natto on an LB solid culture medium, culturing for 16-20h at 37 ℃, picking a single colony on a streak plate, inoculating into a triangular flask containing an LB liquid culture medium, and culturing for 12-16h at 180-250rpm in a shaking table at 37 ℃;
step 2: inoculating the culture solution obtained in the step 1 into BN-I culture medium, and shake culturing at 37 ℃ and 180-250rpm for 12-16h;
step 3: inoculating the bacterial liquid obtained in the step 2 into BN-I culture medium, and shake culturing for 4-5h at 37 ℃ and 180-250 rpm;
step 4: inoculating the bacterial liquid obtained in the step 3 into BN-II culture medium, and shake culturing for 1-2h at 37 ℃ and 180-250 rpm;
step 5: adding a transformation promoting solution into the bacterial liquid in the step 4, and carrying out shake cultivation for 10-30min at the temperature of 37 ℃ and at the speed of 80-120 rpm; obtaining bacterial liquid containing competent cells after the culture is finished;
every 100 ml of BN-I medium contains: 1.47ml of BN-A solution, 1.47ml of BN-B solution, 0.03ml of 100 XCAYE solution and 0.03ml of xylose solution with A mass fraction of 50%;
every 100 milliliters of BN-II culture medium contains: 4.8ml of BN-A solution, 4.8ml of BN-B solution, 0.1ml of 100 XCAYE solution, 0.1ml of xylose solution with the mass fraction of 50 percent, 0.1ml of CaCl with the concentration of 50mmol/L 2 Solution and 0.1ml of MgCl with a concentration of 250mmol/L 2 A solution;
the BN-A solution is composed of the following materials in percentage by mass: 0.4% (NH) 4 ) 2 SO 4 2.8% of K 2 HPO 4 ·3H 2 O,1.2% KH 2 PO 4 0.2% trisodium citrate dihydrate, balance water;
the BN-B solution is composed of the following materials in percentage by mass: 0.04% MgSO 4 ·7H 2 O,40% of polyethylene glycol and the balance of water.
The preferable technical scheme is as follows: the conversion promoting liquid is composed of the following materials in percentage by mass: 3.8% 100 XEGTA solution at a concentration of 10mmol/L, 2% glycine and 1% serine, the balance being water.
The preferable technical scheme is as follows: the 100 XCAYE solution is composed of the following materials in mass ratio: 2% of hydrolyzed casein and 10% of yeast extract, and the balance of water.
The preferable technical scheme is as follows: the LB solid culture medium comprises the following materials in percentage by mass: 0.5% yeast extract, 1% tryptone, 1% NaCl and 2% agar.
The preferable technical scheme is as follows: the LB liquid medium contains 0.5% of yeast extract, 1% of tryptone and 1% of NaCl; the pH was 7.0.
To achieve the above and other related objects, the present invention provides the following technical solutions: a genetic transformation method of bacillus natto competent cells comprises adding recombinant plasmid into prepared bacterial liquid containing competent cells, and culturing at 37deg.C in 180-250rpm shaker for 1-2 hr; after the culture is finished, the bacteria are removed by centrifugation, resuspended, spread on a resistance plate, cultured in an incubator at 37 ℃ for 16-24 hours, and the positive transformants are selected.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the invention optimizes the sipizezen transformation method, including optimization of culture medium and culture conditions. The culture medium replaces 50% glucose solution with 50% xylose solution, 40% polyethylene glycol is added into SP-B solution, 2% glycine and 1% serine are added into conversion promoting solution, and the culture condition is changed from 37 ℃ for stationary culture for 2 hours to 37 ℃ for culture for 2 hours at 180 rpm. The bacillus natto competent cells prepared by the method have greatly improved conversion rate, and subsequently provide a foundation for gene expression, silencing, knockout and functional research of the bacillus natto.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Comparative example: preparation of bacillus natto competence by sipizizzenz transformation method and genetic transformation method
Streaking the frozen and preserved bacillus natto on LB solid medium, culturing at 37 ℃ for 16 hours, picking a single colony on a streaking plate, inoculating into a 250mL triangular flask containing 50mL of LB liquid medium, and culturing at 250rpm and 37 ℃ for 12 hours. mu.L of the culture solution was inoculated into 3mL of SP-I medium at 250rpm and 37℃and cultured for 12 hours. 100uL of the bacterial liquid was inoculated into 5mL of SP-I medium at 250rpm and 37℃for 4.5 hours. Then, 1mL of the bacterial liquid was inoculated into 10mL of SP-II medium at 250rpm and 37℃for 1.5 hours. 150. Mu.L of 100 XEGTA was added to the broth, and the mixture was cultured at 150rpm and 37℃for 30 minutes to give competent cells of Bacillus natto. 1. Mu.g of the pHYp 43-menoA recombinant plasmid prepared in advance was added to a 1.5mL sterile centrifuge tube containing 500. Mu.L of Bacillus natto competence, and the mixture was left to stand at 37℃for 2 hours. After the completion of the cultivation, the culture was centrifuged at 4000rpm for 3 minutes, 300uL of the supernatant was discarded, and the cells were resuspended, plated on ampicillin-resistant plates having a final concentration of 50. Mu.g/mL, and cultivated in an incubator at 37℃for 16 hours. The experiment was performed in 3 replicates, and 2,1 colonies were grown on the resistance plates, respectively. To verify the reproducibility of the experiment, 3 independent experiments were performed to further verify that the conversion could reach 2 cfu/. Mu.g.
SP-I medium (10 mL): 4.9mL of SP-A solution, 4.9mL of SP-B solution, 0.1mL of 50% glucose solution, 0.1mL of 100 XCAYE solution.
The SP-A solution comprises the following components: 0.4% (NH) 4 ) 2 SO 4 ，2.8% K 2 HPO 4 ·3H 2 O，1.2% KH 2 PO 4 0.2% trisodium citrate dihydrate; the SP-B solution comprises the following components: 0.04% MgSO 4 ·7H 2 O
SP-II medium (20 mL): 9.8mL SP-I medium, 0.1mL 50mmol/L CaCl 2 Solution, 0.1ml of 250mmol/L MgCl 2 A solution.
Example 1: preparation method and genetic transformation method of bacillus natto competent cells
Streaking the frozen and preserved bacillus natto on LB solid medium, culturing at 37 ℃ for 16 hours, picking a single colony on a streaking plate, inoculating into a 250mL triangular flask containing 50mL of LB liquid medium, and culturing at 250rpm and 37 ℃ for 12 hours. mu.L of the culture medium was inoculated into 3mL of BN-I medium at 250rpm and 37℃and cultured for 12 hours. 100uL of the bacterial liquid was inoculated into 3mL of BN-I medium at 250rpm and 37℃for 4.5 hours. Then, 1mL of the bacterial liquid was inoculated into 10mL of BN-II medium at 250rpm and 37℃for 1.5 hours. 150 mu L of transformation promoting liquid is added into the bacterial liquid, 100rpm and 37 ℃ are used for culturing for 30min, and bacillus natto competent cells are obtained. 1. Mu.g of the pHYp 43-menoA recombinant plasmid prepared in advance was added to a 1.5mL sterile centrifuge tube containing 500. Mu.L of Bacillus natto competence, and the mixture was placed in a shaking table at 37℃and 180rpm for 2 hours. After the completion of the culture, the culture was centrifuged at 4000rpm for 3 minutes, 300. Mu.L of the supernatant was discarded, and the cells were resuspended, plated on ampicillin-resistant plates having a final concentration of 50. Mu.g/mL, and cultured in an incubator at 37℃for 16 hours. The experiment set up 3 replicates, 2,1 colonies each grown on the resistance plates using the sipizezenz transformation and 21,20,22 colonies each grown on the resistance plates using the modified transformation. In order to verify the repeatability of the experiment, 3 independent experiments were performed to further verify that the improved transformation method has higher transformation efficiency, and can reach 20 cfu/. Mu.g.
Example 2: preparation method and genetic transformation method of bacillus natto competent cells
Streaking the frozen and preserved bacillus natto on LB solid medium, culturing at 37 ℃ for 16 hours, picking a single colony on a streaking plate, inoculating into a 250mL triangular flask containing 50mL of LB liquid medium, and culturing at 250rpm and 37 ℃ for 12 hours. mu.L of the culture medium was inoculated into 3mL of BN-I medium at 250rpm and 37℃and cultured for 12 hours. 100uL of the bacterial liquid was inoculated into 3mL of BN-I medium at 250rpm and 37℃for 4.5 hours. Then, 1mL of the bacterial liquid was inoculated into 10mL of BN-II medium at 250rpm and 37℃for 1.5 hours. 150 mu L of transformation promoting liquid is added into the bacterial liquid, 100rpm and 37 ℃ are used for culturing for 30min, and bacillus natto competent cells are obtained. 1. Mu.g of pET28a-menA recombinant plasmid prepared in advance was added to a 1.5mL sterile centrifuge tube containing 500. Mu.L of Bacillus natto competence, and the mixture was placed in a shaking table at 37℃and 180rpm for 2 hours. After the completion of the cultivation, the culture was centrifuged at 4000rpm for 3 minutes, 300uL of the supernatant was discarded, and the cells were resuspended, plated on a kanamycin-resistant plate having a final concentration of 50. Mu.g/mL, and cultured in an incubator at 37℃for 16 hours. The experiment set up 3 replicates, 1,2 colonies each grown on the resistance plates using the sipizezenz transformation and 22,21,24 colonies each grown on the resistance plates using the modified transformation. To verify the reproducibility of the experiment, 3 independent experiments were performed to further verify that the improved transformation method showed a higher transformation efficiency, up to 21 cfu/. Mu.g.
Example 2 compared to example 1, a plasmid pET28a was replaced, the plasmid sequence was smaller than pHYp43, and the effect of plasmid size on conversion was verified, and experimental results showed no effect.
Example 3: preparation method and genetic transformation method of bacillus natto competent cells
Step (1): streaking the frozen and preserved bacillus natto on LB solid medium, culturing at 37 ℃ for 16-20h, picking a single colony on streak plate, inoculating into a 250ml triangular flask containing 50ml LB liquid medium, culturing at 180-250rpm and 37 ℃ for 12-16h.
Step (2): 100. Mu.L of the culture solution obtained in the step (1) was inoculated into 3mL of BN-I medium at 180-250rpm and 37℃for 12-16 hours.
Step (3): inoculating 100 mu L of the bacterial liquid in the step (2) into 3mL of BN-I culture medium, and culturing for 4-5 hours at 180-250rpm and 37 ℃.
Step (4): 1mL of the bacterial liquid obtained in the step (3) is inoculated into 10mL of BN-II culture medium, and the culture is carried out for 1-2h at 180-250rpm and 37 ℃.
Step (5): adding 100-150 mu L of transformation-promoting liquid into the bacterial liquid in the step (4), culturing for 10-30min at 100rpm and 37 ℃, and after the culturing is finished, sub-packaging competent cells, wherein each tube contains 500 mu L of prepared competent cells for preparation.
Step (6): adding 1 mug of the recombinant plasmid prepared in advance into the centrifuge tube containing bacillus natto competent cells in the step (5), and placing the centrifuge tube in a shaking table at 37 ℃ and 180-250rpm for culturing for 1-2 hours. After the completion of the culture, the cells were centrifuged at 4000rpm for 3min, 300uL of the supernatant was discarded, the cells were resuspended, plated on corresponding resistance plates, cultured in an incubator at 37℃for 16-24h, and positive transformants were selected. Colony PCR experiment is carried out on colonies growing on a resistance plate, then agarose gel electrophoresis experiment is carried out on PCR products, colonies with fragments at the positions corresponding to target strips are selected, the colonies are cultured in LB liquid culture medium for 12 hours, bacterial liquid cultured for 12 hours is preserved, and the bacterial liquid is sent to a sequencing company to detect the gene sequence of the bacterial liquid, and the colonies with correct sequences are positive transformants.
The LB solid medium in the step (1) comprises the following components: 0.5% yeast extract, 1% tryptone, 1% NaCl, 2% agar; the LB liquid medium comprises the following components: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.
The BN-I culture medium in the step (2) and the step (3) comprises the following components: 1.47ml BN-A solution, 1.47ml BN-B solution, 0.03ml 100 XCAYE solution, 0.03ml 50% xylose solution. Wherein the BN-A solution comprises the following components: 0.4% (NH) 4 ) 2 SO 4 ，2.8% K 2 HPO 4 ·3H 2 O，1.2% KH 2 PO 4 0.2% trisodium citrate dihydrate; the BN-B solution comprises the following components: 0.04% MgSO 4 ·7H 2 O,40% polyethylene glycol (PEG 4000); the 100 XCAYE solution comprises the following components: 2% hydrolyzed casein, 10% yeast extract.
The BN-II medium in the step (4) comprises 4.8ml BN-A solution, 4.8ml BN-B solution, 0.1ml 100 XCAYE solution, 0.1ml 50% xylose solution and 0.1ml 50mmol/L CaCl 2 Solution, 0.1ml of 250mmol/L MgCl 2 A solution. Wherein the BN-A solution, BN-B solution, 100 XCAYE solution components are as described above.
The conversion promoting liquid in the step (5) comprises the following components: 10 mmol/L100 XEGTA solution, 2% glycine, 1% serine.
The sterilization modes of the BN-A solution, the BN-B solution and the 100 XCAYE solution in the step (2) and the step (3) are 121 ℃ for 20min.
The steps (2), (3) and (4) are 50% xylose solution and 50mmol/L CaCl 2 Solution and 250mmol/L MgCl 2 The sterilization mode of the solution is 115 ℃ for 30min.
In the step (5), 100×EGTA solution, 2% glycine, 1% serine was filtered and sterilized with a 0.22 μm aqueous filter.
The invention optimizes the sipizezen transformation method, including optimization of culture medium and culture conditions. The culture medium replaces 50% glucose solution with 50% xylose solution, 40% polyethylene glycol is added into SP-B solution, 2% glycine and 1% serine are added into conversion promoting solution, and the culture condition is changed from 37 ℃ for stationary culture for 2 hours to 37 ℃ for culture for 2 hours at 180 rpm. The bacillus natto competent cells prepared by the method have greatly improved conversion rate, and subsequently provide a foundation for gene expression, silencing, knockout and functional research of the bacillus natto.
Example 4: preparation method and genetic transformation method of bacillus natto competent cells
A preparation method of bacillus natto competent cells comprises the following steps:
step 1: streaking the frozen and preserved bacillus natto on an LB solid culture medium, culturing for 16 hours at 37 ℃, picking a single colony on a streaking plate, inoculating the single colony into a triangular flask containing an LB liquid culture medium, and culturing for 12 hours at 180rpm in a shaking table at 37 ℃;
step 2: inoculating the culture solution obtained in the step 1 into BN-I culture medium, and shake culturing at 37 ℃ and 180rpm for 12h;
step 3: inoculating the bacterial liquid obtained in the step 2 into BN-I culture medium, and shake culturing for 4-5h at 37 ℃ and 180 rpm;
step 4: inoculating the bacterial liquid obtained in the step 3 into BN-II culture medium, and shake culturing for 1h at 37 ℃ and 180 rpm;
step 5: adding a transformation promoting solution into the bacterial liquid in the step 4, and carrying out shake cultivation for 10min at the temperature of 37 ℃ at 80 rpm; obtaining bacterial liquid containing competent cells after the culture is finished;
every 100 ml of BN-I medium contains: 1.47ml of BN-A solution, 1.47ml of BN-B solution, 0.03ml of 100 XCAYE solution and 0.03ml of xylose solution with A mass fraction of 50%;
every 100 milliliters of BN-II culture medium contains: 4.8ml of BN-A solution, 4.8ml of BN-B solution, 0.1ml of 100 XCAYE solution, 0.1ml of xylose solution with the mass fraction of 50 percent, 0.1ml of CaCl with the concentration of 50mmol/L 2 Solution and 0.1ml of MgCl with a concentration of 250mmol/L 2 A solution;
the BN-A solution is composed of the following materials in percentage by mass: 0.4% (NH) 4 ) 2 SO 4 2.8% of K 2 HPO 4 ·3H 2 O,1.2% KH 2 PO 4 0.2% trisodium citrate dihydrate, balance water;
the BN-B solution is composed of the following materials in percentage by mass: 0.04% MgSO 4 ·7H 2 O,40% of polyethylene glycol and the balance of water.
The preferred embodiments are: the conversion promoting liquid is composed of the following materials in percentage by mass: 3.8% 100 XEGTA solution at a concentration of 10mmol/L, 2% glycine and 1% serine, the balance being water.
The preferred embodiments are: the 100 XCAYE solution is composed of the following materials in mass ratio: 2% of hydrolyzed casein and 10% of yeast extract, and the balance of water.
The preferred embodiments are: the LB solid culture medium comprises the following materials in percentage by mass: 0.5% yeast extract, 1% tryptone, 1% NaCl and 2% agar.
The preferred embodiments are: the LB liquid culture medium contains 0.5% of yeast extract, 1% of tryptone and 1% of NaCl, and the balance of water; the pH was 7.0.
A genetic transformation method of bacillus natto competent cells comprises adding recombinant plasmid into prepared bacterial liquid containing competent cells, and culturing at 37deg.C in 180rpm shaker for 1 hr; after the culture, the cells were discarded by centrifugation, resuspended, spread on a resistance plate, and cultured in an incubator at 37℃for 16 hours, followed by selection of positive transformants.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.
Claims (5)
1. A preparation method of bacillus natto competent cells is characterized by comprising the following steps: comprises the following steps:
step 1: streaking the frozen and preserved bacillus natto on an LB solid culture medium, culturing for 16-20h at 37 ℃, picking a single colony on a streak plate, inoculating into a triangular flask containing an LB liquid culture medium, and culturing for 12-16h at 180-250rpm in a shaking table at 37 ℃;
step 2: inoculating the culture solution obtained in the step 1 into BN-I culture medium, and shake culturing at 37 ℃ and 180-250rpm for 12-16h;
step 3: inoculating the bacterial liquid obtained in the step 2 into BN-I culture medium, and shake culturing for 4-5h at 37 ℃ and 180-250 rpm;
step 4: inoculating the bacterial liquid obtained in the step 3 into BN-II culture medium, and shake culturing for 1-2h at 37 ℃ and 180-250 rpm;
step 5: adding a transformation promoting solution into the bacterial liquid in the step 4, and carrying out shake cultivation for 10-30min at the temperature of 37 ℃ and at the speed of 80-120 rpm; obtaining bacterial liquid containing competent cells after the culture is finished;
every 100 ml of BN-I medium contains: 1.47ml of BN-A solution, 1.47ml of BN-B solution, 0.03ml of 100 XCAYE solution and 0.03ml of xylose solution with A mass fraction of 50%;
every 100 milliliters of BN-II culture medium contains: 4.8ml of BN-A solution, 4.8ml of BN-B solution, 0.1ml of 100 XCAYE solution, 0.1ml of xylose solution with the mass fraction of 50 percent, 0.1ml of CaCl with the concentration of 50mmol/L 2 Solution and 0.1ml of MgCl with a concentration of 250mmol/L 2 A solution;
the BN-A solution is composed of the following materials in percentage by mass: 0.4% (NH) 4 ) 2 SO 4 2.8% of K 2 HPO 4 ·3H 2 O,1.2% KH 2 PO 4 0.2% trisodium citrate dihydrate, balance water;
the BN-B solution is composed of the following materials in percentage by mass: 0.04% MgSO 4 ·7H 2 O,40% of polyethylene glycol and the balance of water;
the conversion promoting liquid is composed of the following materials in percentage by mass: 3.8% 100 XEGTA solution at a concentration of 10mmol/L, 2% glycine and 1% serine, the balance being water.
2. The method for preparing bacillus natto competent cells according to claim 1, wherein: the 100 XCAYE solution is composed of the following materials in mass ratio: 2% of hydrolyzed casein and 10% of yeast extract, and the balance of water.
3. The method for preparing bacillus natto competent cells according to claim 1, wherein: the LB solid culture medium comprises the following materials in percentage by mass: 0.5% yeast extract, 1% tryptone, 1% NaCl and 2% agar.
4. The method for preparing bacillus natto competent cells according to claim 1, wherein: the LB liquid medium contains 0.5% of yeast extract, 1% of tryptone and 1% of NaCl; the pH was 7.0.
5. A genetic transformation method of bacillus natto competent cells is characterized in that: adding the recombinant plasmid into the bacterial liquid containing competent cells prepared by any one of claims 1-4, and placing the bacterial liquid into a shaking table at 37 ℃ and 180-250rpm for culturing for 1-2h; after the culture is finished, the bacteria are removed by centrifugation, resuspended, spread on a resistance plate, cultured in an incubator at 37 ℃ for 16-24 hours, and the positive transformants are selected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110658479.8A CN113215073B (en) | 2021-06-15 | 2021-06-15 | Preparation method and genetic transformation method of bacillus natto competent cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110658479.8A CN113215073B (en) | 2021-06-15 | 2021-06-15 | Preparation method and genetic transformation method of bacillus natto competent cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113215073A CN113215073A (en) | 2021-08-06 |
| CN113215073B true CN113215073B (en) | 2023-05-12 |
Family
ID=77080280
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110658479.8A Active CN113215073B (en) | 2021-06-15 | 2021-06-15 | Preparation method and genetic transformation method of bacillus natto competent cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113215073B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109722427B (en) * | 2019-03-06 | 2023-07-14 | 武汉轻工大学 | Preparation method of nattokinase freeze-dried powder |
| CN110819575A (en) * | 2019-12-11 | 2020-02-21 | 江苏师范大学 | Culture method of bacillus for producing nattokinase |
-
2021
- 2021-06-15 CN CN202110658479.8A patent/CN113215073B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113215073A (en) | 2021-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110157654B (en) | Bacillus natto recombinant strain and construction method and application thereof | |
| JP7475434B2 (en) | Use of transporter genes in Escherichia coli to improve L-tryptophan production efficiency | |
| KR102246356B1 (en) | Method and application to improve production of Bacillus licheniformis fermentation enzyme through spoⅡQ and pcf gene knockout | |
| CN110951667B (en) | Fenogen element high-yield strain LPB-18N and breeding and application thereof | |
| CN112592881A (en) | Engineering bacillus subtilis for high-efficiency exogenous protein expression and high-density culture | |
| CN112522173A (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
| CN111117942B (en) | Genetic engineering bacterium for producing lincomycin and construction method and application thereof | |
| CN111004815B (en) | Method for improving yield of bacillus subtilis surfactant by mutating degU gene | |
| CN113215073B (en) | Preparation method and genetic transformation method of bacillus natto competent cells | |
| CN110484480B (en) | Preparation and transformation method of bacillus subtilis competent cells | |
| CN110295189B (en) | Application of 4-aminobutyric acid aminotransferase in improving fermentation yield of iturin A | |
| CN117070538A (en) | Application of ppt1 gene as screening marker in screening of auxotrophs | |
| CN108588108B (en) | Preparation method and application of bacillus for efficiently metabolizing glycerol | |
| CN110452863B (en) | Application of ornithine cyclohexane enzyme in improving yield of iturin A produced by bacillus amyloliquefaciens | |
| CN109593696B (en) | Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof | |
| CN105002105A (en) | Recombinant bacterium with high biomass and/or high growth speed, and construction method and application thereof | |
| CN112779174B (en) | Saccharomyces cerevisiae genetically engineered bacterium for knocking out Cln3 gene, construction method and application thereof | |
| CN116515724B (en) | Zymomonas mobilis utilizing inorganic nitrogen source, application and nitrogen metabolism regulation gene | |
| CN115895987B (en) | Recombinant strain for improving fructose chondroitin yield and construction method thereof | |
| CN110791509B (en) | Pediococcus acidilactici optimized expression sequence, expression vector, preparation method and strain thereof | |
| CN112877229B (en) | Saccharomyces cerevisiae genetically engineered bacterium for knocking out Sok2, construction method and application thereof | |
| CN107828707A (en) | A kind of genetic engineering bacterium and its construction method of high yield macromolecule γ polyglutamic acids | |
| CN111334445B (en) | Long-chain dicarboxylic acid producing strain and preparation method and application thereof | |
| CN118440172A (en) | Antibacterial peptide cecropin A mutant, recombinant expression vector, bacillus subtilis engineering bacteria and application | |
| CN104962574A (en) | Arthrobacter expression plasmid and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |