Thermal shock-based vibrio harveyi homologous recombinant gene knockout method

A technology of Vibrio harveylius and gene knockout, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of low efficiency, unsuccessful conjugation and transformation, etc., and achieve accurate functional analysis and difficulty in polarity Effects, easy-to-operate effects

Active Publication Date: 2018-04-13
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

[0005] Aiming at the problem that it is difficult to knock out the gene due to the unsuccessful or low efficiency of conjugative transformation of Vibrio harveylius, the invention establishes a heat shock-based conjugative transformation method for Vibrio harveylia by performing heat shock treatment on the conjugative recipient bacterium Vibrio harveylius. Bacteria Homologous Recombination Gene Knockout Method

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  • Thermal shock-based vibrio harveyi homologous recombinant gene knockout method
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  • Thermal shock-based vibrio harveyi homologous recombinant gene knockout method

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Embodiment 1

[0070] Taking the gene rbsB (ABC transporter substrate-binding protein) as an example to illustrate a heat shock-based method to achieve homologous recombination gene knockout in Vibrio harveii ( figure 1 ).

[0071] The ORF of rbsB gene (SEQ ID NO.1) has a full length of 879bp, which can encode a ribosome binding protein consisting of 292 amino acids, and its main function is to participate in the binding and transport of D-ribose. In some bacteria, it is speculated that this gene can also serve as AI-2 receptor, participate in quorum sensing, mediate bacterial growth and biofilm formation, but the specific regulatory mechanism is still unclear.

[0072] The knockout strain of rbsB gene obtained in this study provides a premise for in-depth study of rbsB gene function, and the elucidation of the gene function will help to further analyze the pathogenic mechanism of Vibrio harveylius, and may serve as a potential target for vaccine development.

[0073] (1) Determine the upst...

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Abstract

The invention discloses a thermal shock-based vibrio harveyi homologous recombinant gene knockout method. The method comprises the following steps: performing thermal shock treatment on vibrio harveyirecipient bacteria which are cultured to an early logarithmic phase, and uniformly mixing equal volume of the vibrio harveyi recipient bacteria which are cultured to the early logarithmic phase and vibrio harveyi donor bacteria which are cultured to early logarithmic phase; centrifuging at room temperature, removing a supernatant liquid, re-suspending after bacteria are precipitated, dibbling toa fixed flat plate and performing bacterial conjugation overnight; performing the thermal shock treatment on the vibrio harveyi conjugation recipient bacteria to develop the conjugation conversion rate of the vibrio harveyi from nothing, so as to perform gene knockout on the vibrio harveyi. According to the method, the conjugation conversion rate of the vibrio harveyi is developed from nothing byperforming the thermal shock treatment on the vibrio harveyi conjugation recipient bacteria on the technical basis of the conventional conjugational transferring method, so that the vibrio harveyi issubjected to the gene knockout successfully.

Description

technical field [0001] The invention belongs to the technical field of Vibrio harvelii, and in particular relates to a method for homologous recombination gene knockout of Vibrio harveylia based on heat shock. Background technique [0002] Vibrio harveyi is a Gram-negative, luminous, curved, brevibacterium with fermentative capacity and extremely single flagella. Harvey Vibrio exists in large quantities in free water bodies, phytoplankton and seabed sediments in the ocean. It is a common pathogenic bacteria in the marine environment. It can infect a variety of fish, shrimp, crabs, etc. and cause a large number of deaths. , bringing huge losses to the mariculture industry. Therefore, it is imminent to elucidate its pathogenic mechanism and prepare related vaccines for immune prevention and control. [0003] Gene knockout, also known as gene targeting, is a new type of molecular biology technology. This technology uses conjugation transformation to introduce the constructed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/03C12N1/21C12R1/63
CPCC12N15/03Y02A50/30
Inventor 邓益琴冯娟贝蕾苏友禄
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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