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Transformation method for bacillus amyloliquefaciens

A technology for dissolving amyloid spores and bacilli, applied in the field of genetic engineering, can solve problems such as difficult transformation of bacillus amyloliquefaciens, and achieve the effects of strong secretion capacity, abundant metabolites, and expanding application prospects.

Inactive Publication Date: 2019-11-08
INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that Bacillus amyloliquefaciens is difficult to transform, the invention provides a transformation method for Bacillus amyloliquefaciens

Method used

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  • Transformation method for bacillus amyloliquefaciens

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Embodiment 1

[0027] A transformation method for bacillus amyloliquefaciens, comprising the steps of:

[0028] Step 1, cultivating Bacillus amyloliquefaciens:

[0029] Take a single colony of Bacillus amyloliquefaciens that has been activated and stably cultured and inoculate it in LB+0.5M sorbitol liquid medium for overnight culture, and inoculate the overnight culture of Bacillus amyloliquefaciens at 4% of the medium volume in LB+0.5M sorbitol In the alcohol liquid medium, culture at a certain temperature and rotation speed for 3-5 hours, add glycine to the culture medium to make it reach a certain final concentration, and then continue to cultivate until the OD600 of the culture medium is 0.9. Bacteria were collected by centrifugation;

[0030] Step 2, preparing Bacillus amyloliquefaciens competent cells:

[0031] Rinse the cells with the pre-cooled electroporation medium, blow and suspend the washed bacteria in 1mL electroporation medium + 14% polyethylene glycol 6000, and distribute ...

Embodiment 2

[0035] A transformation method for bacillus amyloliquefaciens, comprising the steps of:

[0036] Step 1, cultivating Bacillus amyloliquefaciens:

[0037] Pick a single colony of Bacillus amyloliquefaciens that has been activated and stably cultured and inoculate it in LB+0.5M sorbitol liquid medium for overnight culture at 37°C, and inoculate the overnight culture of Bacillus amyloliquefaciens at 4% of the medium volume in LB+ In 0.5M sorbitol liquid medium, culture at 37°C and 200rpm for 3-5 hours, add glycine to the culture solution to make the final concentration reach 7-8 mg / mL, then continue to cultivate for 1.5 hours until the OD600 of the culture solution is 0.9, and the culture solution Cool in ice for 10 minutes, then centrifuge at 5000 g for 15 minutes at 4°C to collect bacteria.

[0038] Step 2, preparing Bacillus amyloliquefaciens competent cells:

[0039] Rinse the cells with the pre-cooled electroporation medium, blow and suspend the washed bacteria in 1mL elec...

Embodiment 3

[0044] A transformation method for bacillus amyloliquefaciens, comprising the steps of:

[0045] Step 1, cultivating Bacillus amyloliquefaciens:

[0046] Pick a single colony of Bacillus amyloliquefaciens that has been activated and stably cultured and inoculate it in LB+0.5M sorbitol liquid medium for overnight culture at 37°C, and inoculate the overnight culture of Bacillus amyloliquefaciens at 4% of the medium volume in LB+ In 0.5M sorbitol liquid medium, culture at 37°C and 200rpm for 3-5 hours, add glycine to the culture solution to make the final concentration reach 7-8 mg / mL, then continue to cultivate for 1.5 hours until the OD600 of the culture solution is 0.9, and the culture solution Cool in ice for 10 minutes, then centrifuge at 5000 g for 15 minutes at 4°C to collect bacteria.

[0047] Step 2, preparing Bacillus amyloliquefaciens competent cells:

[0048] Rinse the cells three times with pre-cooled electroporation medium containing 0.5M sorbitol, 0.5M mannitol a...

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Abstract

The invention relates to a transformation method for bacillus amyloliquefaciens, and belongs to the technical field of gene engineering. In order to solve the problem of difficult transformation of the bacillus amyloliquefaciens, the invention provides a transformation method for the bacillus amyloliquefaciens. The transformation method includes the following steps that the bacillus amyloliquefaciens are cultured by an LB+0.5 M sorbitol liquid medium, glycine is added to make the final concentration reach 7-8 mg / mL when culturing is carried out for 3-5 h, the bacillus amyloliquefaciens are continuously cultured to OD 600 to be 0.9, thallus is collected centrifugally, the thallus is placed into an electrotransfer medium to obtain efficient competent cells after rinsing, plasmids are conducted into the competent cells through electrotransformation, and after the electrotransformation, positive transformants can be obtained rapidly and efficiently by screening LB plates containing corresponding antibiotics. The invention provides a method for transformation in the bacillus amyloliquefaciens. According to the method, more positive transformants can be obtained rapidly, and the positive rate can reach about 80%.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a transformation method for bacillus amyloliquefaciens. Background technique [0002] Bacillus amyloliquefaciens widely exists in nature and is an important class of biocontrol resource bacteria. Because of its easy isolation and culture, no pollution to the environment, strong secretion ability, rich metabolites, and broad-spectrum antibacterial properties, it is widely used in plant diseases. It can be widely used in biological control, production of industrial fungicides and in the feed industry. Although the research on Bacillus amyloliquefaciens has gradually increased in recent years, most of them focus on biological control. At present, the research on the expression system of Bacillus amyloliquefaciens is still in its infancy, and it is not perfect and immature. Thereby, the development of utilizing genetic engineering to transform Bacillus amylo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 刘宇帅张淑梅李晶孟利强胡基华陈静宇曹旭姜威
Owner INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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