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Activating and fermenting method of escherichia coli

A technology of Escherichia coli and fermentation method, which is applied in the field of microorganisms and can solve the problems of unstable fermentation and low expression of target products.

Pending Publication Date: 2020-02-18
ENZYMASTER NINGBO BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the embodiments of the present invention is to provide an activation fermentation method for Escherichia coli, which aims to solve the technical problems of unstable subsequent fermentation and low expression of the target product in the existing strain activation process

Method used

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  • Activating and fermenting method of escherichia coli

Examples

Experimental program
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Embodiment 1

[0035] Take out the Escherichia coli BL21 (DE3) strain in the cryopreservation tube and inoculate it on the first LB plate culture medium at 37°C for 16 hours to obtain the primary plate;

[0036] Take a single colony of Escherichia coli BL21 (DE3) on the primary plate and inoculate it on the medium of the second LB plate, and culture it at 37°C for 16 hours to obtain the secondary plate;

[0037] Inoculate 5 single-colony Escherichia coli BL21 (DE3) strains on the secondary plate into 10 mL of the first LB liquid medium, and rotate at 200 rpm for 10 hours at 25°C to obtain primary seed liquid;

[0038] Take 0.5 mL of the primary seed solution and inoculate it into 50 mL of the second LB liquid medium, and incubate at 25°C at a speed of 200 rpm for 20 hours to obtain the secondary seed solution;

[0039] Take 1 mL of the secondary seed liquid and inoculate it in 400 mL of fermentation bottom liquid for fermentation. During the fermentation process, whenever the dissolved oxyg...

Embodiment 2

[0043] Take out the Escherichia coli BL21 (DE3) strain in the cryopreservation tube and inoculate it on the first LB plate medium, and cultivate it at 37°C for 24 hours to obtain the primary plate;

[0044] Take a single colony of Escherichia coli BL21 (DE3) on the primary plate and inoculate it on the medium of the second LB plate, and culture it at 37°C for 24 hours to obtain the secondary plate;

[0045] Inoculate 10 single-colony Escherichia coli BL21 (DE3) strains on the secondary plate into 20 mL of the first TB liquid medium, and rotate at 300 rpm for 6 hours at 37°C to obtain primary seed liquid;

[0046] Take 1 mL of the primary seed liquid and inoculate it into 100 mL of the second TB liquid medium, and incubate at 37°C for 12 hours at a rotational speed of 300 rpm to obtain the secondary seed liquid;

[0047] Inoculate 2 mL of the secondary seed liquid into 400 mL of fermentation bottom liquid for fermentation. During the fermentation process, whenever the dissolved ...

Embodiment 3

[0051] Take out the Escherichia coli BL21 (DE3) strain in the cryopreservation tube and inoculate it on the first LB plate medium, and cultivate it at 37°C for 20 hours to obtain the primary plate;

[0052] Take a single colony of Escherichia coli BL21 (DE3) on the primary plate and inoculate it on the medium of the second LB plate, and culture it at 37°C for 20 hours to obtain the secondary plate;

[0053] Take 8 single-colony Escherichia coli BL21 (DE3) strains on the secondary plate and inoculate them in 20mL of the first 2×YT liquid medium, and incubate at 30°C for 8 hours at a speed of 250 rpm to obtain first-class seed liquid;

[0054] Take 1 mL of the primary seed liquid and inoculate it into 100 mL of the second 2×YT liquid medium, and incubate at 30°C at a speed of 250 rpm for 16 hours to obtain the secondary seed liquid;

[0055] Inoculate 2 mL of secondary seed liquid into 400 mL of fermentation bottom liquid for fermentation. During the fermentation process, whene...

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Abstract

The invention is suitable for the technical field of microorganisms, and provides an activating and fermenting method of escherichia coli. The activating and fermenting method comprises the steps of taking escherichia coli, and performing inoculating in a culture medium to obtain a first-class flat; taking single colonies, continuing performing inoculating on the culture medium to obtain a second-class flat; taking many single colonies, performing inoculating on a liquid culture medium, and performing rotary culturing to obtain first-class seed liquid; continuing inoculating the liquid culturemedium with the first-class seed liquid, and performing rotary culturing to obtain second-class seed liquid; inoculating a fermentation base solution with the second-class seed liquid, performing fermentation, and supplementing foods in an oxygen dissolving regeneration manner; and adding an induction culture medium, inducing protein expression, taking constant-pH food supplementing for supplementing the foods, and continuing performing fermentation. According to the activating and fermenting method of escherichia coli provided by the embodiment of the invention, through flat culture twice and liquid culture twice, the second-class seed liquid is obtained, so that rejuvenation of strains can be effectively realized, the stability of a subsequent fermentation process can be effectively improved, and the expression level of target products can be increased effectively.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a method for activating and fermenting Escherichia coli. Background technique [0002] Fermentation engineering is an important part of biotechnology and an important link in the industrialization of biotechnology. Fermentation technology has a long history. As a modern scientific concept, microbial fermentation industry is based on traditional fermentation technology, combined with modern new technologies such as genetic engineering and cell engineering. The fermentation process is often accompanied by protein expression. In the process of microbial fermentation, the activation of strains is a prerequisite to ensure the stability of fermentation and also affects the expression of target proteins, which is a crucial link. [0003] However, the existing literature reports on the activation of strains often adopt the method of rejuvenating the strains, cultivating t...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N1/20C12R1/19
CPCC12P21/00C12N1/20
Inventor 周丽孙磊
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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