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Site-specific gene knockout method of vibrio harveyi based on absolute ethyl alcohol or sodium dodecyl sulfate stimulation

A technology of sodium dodecylsulfonate and Vibrio harveylius, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of added compounds to stimulate growth, etc., and can solve the problems of non-directional mutation genes, restriction genetics, Achieving the effects of not being prone to polarity effects, simple operation, and clear purpose due to heavy workload

Pending Publication Date: 2021-01-05
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most researchers such as Teo JW, Bassler BL, and Rattanama P use transposition mutation for the gene knockout work of Vibrio harveylius. The advantage of transposition mutation is that it can randomly mutate any gene site on the genome. However, its disadvantages are randomness, heavy workload, inability to mutate a certain gene in a targeted manner, and easy to produce polar effects
The traditional method of using Escherichia coli (Escherichia coli) host bacteria to carry exogenous genes, and conjugating transfer with recipient bacteria to realize exogenous vectors into cells, and further homologous recombination for site-directed gene knockout methods, in Harvey Vibrio is hampered by the inability of most strains to successfully conjugate transfer, which limits the study of the genetics of this bacterium

Method used

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  • Site-specific gene knockout method of vibrio harveyi based on absolute ethyl alcohol or sodium dodecyl sulfate stimulation
  • Site-specific gene knockout method of vibrio harveyi based on absolute ethyl alcohol or sodium dodecyl sulfate stimulation
  • Site-specific gene knockout method of vibrio harveyi based on absolute ethyl alcohol or sodium dodecyl sulfate stimulation

Examples

Experimental program
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Effect test

Embodiment 1

[0063] Taking the gene hsdM (type I restriction enzyme, M subunit) as an example to illustrate the steps of targeted gene knockout in Vibrio harveyli based on the stimulation of organic substance absolute ethanol ( figure 1 ).

[0064] The ORF of the hsdM gene (SEQ ID NO.1) is 1500 bp in full length and can encode the type I restriction modification system M subunit HsdM consisting of 499 amino acids. HsdM is one of the host specificity determinants (hsd) of the type I restriction modification system, which is responsible for the methylation modification enzyme cleavage (Modification subunit, M) of non-methylated nucleotide sequences. Metastasis has a certain regulatory effect.

[0065] The hsdM gene knockout strain obtained in this study provides a premise for in-depth study of the function of the hsdM gene, and the elucidation of the gene function will help to further analyze the regulatory mechanism of horizontal gene transfer in Vibrio harveylius, and may serve as a way t...

Embodiment 2

[0112] Taking the gene hsdR (type I restriction enzyme, R subunit) as an example to illustrate the steps of realizing targeted gene knockout in Vibrio harvelis based on the stimulation of the organic substance sodium dodecyl sulfate ( figure 1 ).

[0113] The ORF of the hsdR gene (SEQ ID NO.3) is 3063bp in full length, and can encode the type I restriction modification system R subunit HsdR consisting of 1020 amino acids. HsdR is one of the host specificity determinants (hsd) of the type I restriction modification system, responsible for the restriction subunit (R) of non-methylated and mismethylated nucleotide sequences, and has been reported to be effective at the bacterial level Gene transfer has a certain regulatory effect.

[0114] The hsdR gene knockout strain obtained in this study provides a premise for in-depth research on the function of the hsdR gene, and the elucidation of the gene function will help to further analyze the regulatory mechanism of horizontal gene t...

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Abstract

The invention discloses a site-specific gene knockout method of vibrio harveyi based on absolute ethyl alcohol or sodium dodecyl sulfate stimulation. The vibrio harveyi serves as a receptor bacteriumand is subjected to conjugal transfer with a donor bacterium; gene knockout of the vibrio harveyi is achieved through homologous recombination; and the vibrio harveyi is vibrio harveyi subjected to stimulation treatment of absolute ethyl alcohol or sodium dodecyl sulfate. According to the site-specific gene knockout method of the vibrio harveyi based on organic matter absolute ethyl alcohol or sodium dodecyl sulfate stimulation in the invention, on the technical basis of a traditional conjugation transfer method, organic matter absolute ethyl alcohol or sodium dodecyl sulfate stimulation treatment on the conjugation recipient bacterium vibrio harveyi is carried out; therefore, the conjugation transformation efficiency of the vibrio harveyi is changed from nothing; and the vibrio harveyi issuccessfully subjected to gene knockout.

Description

Technical field: [0001] The invention belongs to the technical field of Vibrio harvelii, and in particular relates to a method for targeted gene knockout of Vibrio harvelius stimulated by organic matter absolute ethanol or sodium dodecyl sulfate. Background technique: [0002] Vibrio harveyi (Vibrio harveyi) is an important conditional pathogenic bacteria of mariculture organisms, which has caused great harm to the mariculture industry. In recent years, in the investigation of the disease of southern cultured marine fish, it was found that about 90% of the vibriosis of marine fish was caused by Vibrio harvei, and the damage range and degree of Vibrio harvei had gradually replaced Vibrio alginolyticus and traumatic bacteria. Vibrio, becoming the main pathogenic Vibrio. Therefore, it is imminent to elucidate its pathogenic mechanism and prepare related vaccines for immune prevention and control. [0003] The study of pathogenic mechanism and the development of attenuated liv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/03C12N1/38C12R1/63
CPCC12N15/74C12N1/38C12N15/03Y02A50/30
Inventor 邓益琴冯娟郭志勋程长洪马红玲
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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