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Method for improving genetic transformation of Vibrio harveyi

A technology for genetic transformation of Vibrio harveylius, applied in the field of improving the genetic transformation of Vibrio harveylia, can solve the problems of unsuccessful conjugation transformation and low efficiency, and achieve the effect of low cost and simple operation

Active Publication Date: 2018-06-15
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

[0004] The object of the present invention is to provide a method for improving the genetic transformation of Vibrio harveylius. The method aims at the problem of unsuccessful conjugative transformation of traditional Vibrio harvelii or low efficiency. Hit treatment to establish an improved method for highly efficient conjugation transformation

Method used

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  • Method for improving genetic transformation of Vibrio harveyi
  • Method for improving genetic transformation of Vibrio harveyi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Preparation of donor bacteria:

[0045] (1) Preparation of Escherichia coli E.coli GEB883 Competent Cells

[0046] (1) E.coli GEB883 was streaked onto an LB solid plate supplemented with 0.3mM DAP, and cultured overnight at 37°C;

[0047] (2) Pick E.coli GEB883 single clone into LB liquid medium supplemented with 0.3mM DAP, and culture overnight at 37°C and 200rpm;

[0048] (3) Dilute 1 mL of the overnight bacterial solution to 100 mL of LB liquid medium supplemented with 0.3 mM DAP, and culture at 37°C and 200 rpm until OD600nm=0.6-0.8;

[0049] (4) Cool the bacterial solution on ice for 10 minutes, and centrifuge at 4°C and 4000 rpm for 15 minutes;

[0050] (5) Remove the supernatant, and use 50mL sterilized and cooled 50mM CaCl for bacterial precipitation 2 Resuspend and place on ice for 20 min;

[0051] (6) Centrifuge the bacterial solution at 4°C and 4000rpm for 15min;

[0052] (7) Remove the supernatant, and use 2.5 mL of sterilized and cooled 50 mM CaCl con...

Embodiment 2

[0079] Set up metal baths at 37°C, 40°C, and 43°C respectively, and perform heat shock treatment on the recipient bacteria liquid with early logarithmic OD600nm=0.3-0.7, and heat shock for 15 minutes, 30 minutes, 45 minutes, and 60 minutes at each heat shock temperature , and set a group without heat shock as a control group of heat shock 0min; other steps are the same as in Example 1.

[0080] Experimental data proves that the survival rate of the recipient bacteria Vibrio harveii is stable at 90-100% ( figure 1 ). In addition, when the heat shock temperature was 37°C and the heat shock time reached 30 min, the conjugation conversion efficiency increased from scratch, and with the prolongation of the heat shock time, the conjugation conversion efficiency increased significantly, reaching a maximum of 2.29×10 -9 ; When the heat shock temperature is 40°C, the conjugation conversion efficiency increases significantly with the prolongation of the heat shock time, and compared wi...

Embodiment 3

[0082] Set up a metal bath at 46°C, and heat-shock the recipient bacteria liquid with OD600nm=0.3-0.7 in the early logarithmic period. Group as the control group of heat shock 0min; other steps are the same as in Example 1.

[0083] The experimental data proves that when the heat shock temperature is 46°C and the heat shock time is 15 minutes, the bacterial survival rate is stable at 90-100%, but when the heat shock temperature is 46°C and the heat shock time is 30-60 minutes, the survival rate is significantly fall below 10% ( figure 1 ). In addition, when the heat shock temperature is 46 °C and the heat shock time is 15 min, the bonding efficiency is still relatively high, which is 3.35×10 -08 , and when the thermal shock time exceeds 30min, the conjugation conversion efficiency is zero (such as figure 2 ).

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Abstract

The invention discloses a method for improving the genetic transformation of Vibrio harveyi. The method comprises carrying out heat shock treatment on recipient Vibrio harveyi cultured to the early logarithmic phase and bonding donor bacteria carrying expression plasmids in the early logarithmic phase to the recipient Vibrio harveyi. The method solves the problem that the traditional joint transformation method of Vibrio harveyi is unsuccessful or has low efficiency. Through heat shock treatment on recipient Vibrio harveyi for binding, the highly efficient improved method for joint transformation is built.

Description

technical field [0001] The invention belongs to the technical field of Vibrio harvelii, and in particular relates to a method for improving the genetic transformation of Vibrio harvelii. Background technique [0002] Vibrio harveii is widely distributed in the marine environment, and is usually a normal flora in the ocean. When the external conditions of the flora change, or when the flora itself mutates, it reproduces in large numbers and produces toxicity, becoming a pathogenic bacterium, and It infects the host and causes its death. It is widely found in Thailand, Indonesia, Ecuador, Australia, India, China and other countries. It is the main pathogenic bacteria of prawns. It can infect a variety of fish, shrimps, crabs, etc. and cause a large number of deaths. In recent years, the vibriosis pathogens of the main cultured fish in the South China Sea have been replaced by Vibrio alginolyticus and gradually replaced by Vibrio harveylius, which has caused huge economic losse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74
CPCC12N15/74
Inventor 邓益琴吴金军冯娟苏友禄
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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