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Broad-spectrum anti-phage E. Coli BL21-(DE3)-PR and application thereof

A technology of Escherichia coli, bacteriophage, applied in the field of bioengineering

Active Publication Date: 2019-05-17
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the anti-phage strains currently used in various factories or laboratories can only resist infection by a certain strain or type of phage, and engineering strains that can resist infection by multiple strains of phage are still worth exploring

Method used

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  • Broad-spectrum anti-phage E. Coli BL21-(DE3)-PR and application thereof
  • Broad-spectrum anti-phage E. Coli BL21-(DE3)-PR and application thereof
  • Broad-spectrum anti-phage E. Coli BL21-(DE3)-PR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Screening and preservation of anti-phage strain E.coli BL21(DE3)-PR

[0047] 1. Screening of anti-phage strain E.coli BL21(DE3)-PR

[0048] 1. Preparation of BL21(DE3) bacterial solution

[0049] E.coli BL21(DE3) was inoculated in LB liquid medium and cultured overnight at 37°C and 220rpm to obtain BL21(DE3) bacterial liquid.

[0050] 2. Isolation of phage

[0051] Take sewage (the sewage comes from the waste water of the 307 Hospital of the Chinese People's Liberation Army), centrifuge at 10000r / min for 10min, and collect the supernatant. The supernatant was filtered with a 0.22 μm filter to obtain a sewage filtrate. Then add 200 μL of sewage filtrate and 4 mL of BL21 (DE3) bacterial solution prepared in step 1 into 2 mL of 3×LB liquid medium, and culture at 37° C. and 220 rpm for 6 hours to obtain phage stock solution. Then the phage stock solution was filtered with a 0.22 μm microporous membrane to obtain a phage preservation solution, which was stored...

Embodiment 2

[0071] Embodiment 2, performance detection of anti-phage bacterial strain BL21 (DE3)-PR

[0072] 1. Phage plaque test against phage strain BL21(DE3)-PR

[0073] In order to detect the anti-phage performance of the anti-phage strain Escherichia coli BL21(DE3)-PR (hereinafter abbreviated as E.coli BL21(DE3)-PR), a phage plaque test was carried out. The specific steps are as follows: Take 300 μL of the BL21 bacterial solution prepared in Step 1 of Example 1 and 300 μL of the phage-resistant bacterial strain BL21(DE3)-PR screened in Step 1 of Example 1 and add 5 mL of LB semi-solid medium respectively , mix evenly, spread on the LB solid medium plate containing the lower layer, and dry at room temperature for 5 minutes;8 pfu / mL phage preservation solution (T4, vB_EcoM_IME281, vB_EcoM_IME253, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, T1, vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME347, vB_EcoM_IME341, T1, vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoS_IME347, JMPW1 bacteria-free cultur...

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PUM

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Abstract

The invention discloses a broad-spectrum anti-phage E. Coli BL21-(DE3)-PR and application thereof. A recombinant protein expression strain E. coli BL21(DE3)-PR is successfully screened with an engineering strain E. coli E. coli BL21(DE3) as a parent strain. The strain is resistant to infection of a phage capable of splitting E. coli BL21 (DE3). The broad-spectrum anti-phage E. Coli BL21-(DE3)-PR performs growth curve and protein expression assay on the recombinant protein expression strain E. coli BL21(DE3)-PR, and results indicate that the growth capability and protein expression capability of the stain are basically the same as an original strain E. coli BL21 (DE3).

Description

technical field [0001] The invention relates to a broad-spectrum anti-phage E.coli BL21-(DE3)-PR and its application, in particular to an E.coli BL21-(DE3)-PR which is not susceptible to phage infection during growth and protein expression , belonging to the field of bioengineering. Background technique [0002] Phage is a kind of virus that infects microorganisms such as bacteria, fungi, actinomycetes or spirochetes, and exists widely in nature. Due to the large number, small size, fast reproduction and high plasticity of the genome, the industrial fermentation process is extremely susceptible to phage infection. Bacteriophage infection is pervasive and stubborn. Once a fermentation enterprise is infected by it, the loss is immeasurable, and there is still no good countermeasure. [0003] The pET system uses the T7 promoter, which is the most powerful system for cloning and expressing recombinant proteins in Escherichia coli. E.coli BL21 (DE3) is a common expression host ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/70C12R1/19
CPCC12N15/70C12N1/205C12R2001/19
Inventor 童贻刚李萍
Owner ACADEMY OF MILITARY MEDICAL SCI
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