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A genetically engineered bacteria, its constructing method and use thereof

A technology of genetically engineered bacteria and construction methods, applied in the field of genetically engineered bacteria, can solve problems such as increasing production costs

Active Publication Date: 2005-09-28
CHENGDU NEWSUN CROPSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It also greatly increases the cost of production

Method used

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  • A genetically engineered bacteria, its constructing method and use thereof
  • A genetically engineered bacteria, its constructing method and use thereof

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Experimental program
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Effect test

Embodiment 1

[0064] The plasmid vector pRL1063a and the starting strain were respectively cultured on LB medium containing 10 μg / L kanamycin; the plasmid vector pRL1063a in the logarithmic phase of growth was mixed with the starting strain at a volume ratio of 1:1.2, and mixed at a temperature of 30 ℃, act on a shaker at 200rpm / min for 30 minutes, centrifuge at 12000r / min for 10min, wash twice with normal saline, discard the supernatant, and inoculate the bacterial pellet on LB solid medium, and inoculate at 30℃ Incubate for 24 hours. The obtained culture was a mixture of the plasmid vector pRL1063a, the starting strain, and the mutant strain inserted into the transposon S17-1.

[0065] Scrape off the cultured bacterial pellet with a toothpick, suspend it with MG liquid medium, and spread it on the plate of MG solid medium (the concentration of kanamycin is 10 μg / L, and the concentration of streptomycin is 30 μg / L). Cultivate at a temperature of 30°C for 7-8 days, transfer the grown singl...

Embodiment 2

[0069] The plasmid vector pRL1063a and the starting strain were respectively cultured on LB medium containing 5 μg / L kanamycin; the plasmid vector pRL1063a in the logarithmic phase of growth was mixed with the starting strain at a volume ratio of 1:1, and mixed at a temperature of 26 ℃, act on a shaker at 150rpm / min for 20 minutes, centrifuge at 20000r / min for 5min, wash once with normal saline, discard the supernatant, and inoculate the bacterial pellet on LB solid medium. Incubate for 12 hours. The obtained culture was a mixture of the plasmid vector pRL1063a, the starting strain, and the mutant strain inserted into the transposon S17-1.

[0070] Scrape off the cultured bacterial pellet with a toothpick, suspend it with MG liquid medium, and spread it on the plate of MG solid medium (the concentration of kanamycin is 5 μg / L, and the concentration of streptomycin is 20 μg / L). Cultivate at 26°C for 5 days, transfer the grown single colony to a new MG solid medium (the concent...

Embodiment 3

[0074] The plasmid vector pRL1063a and the starting strain were cultured on LB medium containing 15 μg / L kanamycin respectively; the plasmid vector pRL1063a in the logarithmic phase of growth was mixed with the starting strain at a volume ratio of 1:2, and mixed at a temperature of 32 ℃, 300rpm / min shaker for 40 minutes, centrifuged at 10000r / min for 15min, discarded the supernatant, inoculated the bacterial pellet on LB solid medium, and cultivated at 32℃ for 48 hours. The obtained culture was a mixture of the plasmid vector pRL1063a, the starting strain, and the mutant strain inserted into the transposon S17-1.

[0075] Scrape off the cultivated bacterial precipitate with a toothpick, suspend MG with liquid medium, and spread it on the plate of MG solid medium (concentration of kanamycin is 15 μg / L, concentration of streptomycin is 40 μg / L). Cultivate at 32°C for 10 days, and transfer the grown single colony to a new MG solid medium (with a concentration of 15 μg / L kanamycin...

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Abstract

The present invention relates to one kind of genetic engineering bacteria, one kind of soybean disease causing variety of clove pseudomonads with preservation number of CGMCC 1276, and its construction process and use. The genetic engineering bacteria is obtained through inserting Tn5 transposon S17-1 into the genome of the initial strain to deactivate its temperature controlling gene. The strain may be used in industrial fermentation production of crown bacterin at 26-28 deg.c, in the yield near the initial bacteria and up to 35-40 mg / L.

Description

technical field [0001] The invention belongs to the technical field of microorganisms. Specifically, the present invention relates to a genetically engineered bacterium, its construction method and its use. Background technique [0002] coronatin C 18 h 25 NO 4 , with a molecular weight of 319, is composed of two components, an amino acid-containing coronamic acid and a polyketide-structured coronafacic acid, linked by amide bonds. It was first reported in the literature as a phytotoxin. However, recent studies have found that coronatin has similar functions to the plant growth regulator substances abscisic acid and jasmonic acid, and its activity is thousands of times that of the latter two. substitution. Coronatine is a pure natural product, and its application in production will not cause pollution to the environment. It is an environmentally friendly biological regulator. But the current research on coronatin has been stuck in the laboratory stage. The reason is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/78C12P1/04
Inventor 李召虎张家常段留生李建民田晓莉王保民焦睿吴惠玲何钟佩翟志席奚勤
Owner CHENGDU NEWSUN CROPSCI
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