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Construction and application of anti-bacteriophage escherichia coli chassis cell

A technology of Escherichia coli and cells, applied in the field of genetic engineering and microbial engineering, can solve the problems of strict requirements, unable to fundamentally solve the phage infection, and time-consuming

Active Publication Date: 2020-10-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After Escherichia coli is infected with phages, measures such as formaldehyde fumigation, pipeline sterilization, or strain rotation were usually used in the past to ensure the continuity of production. Solving the problem of phage infection

Method used

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  • Construction and application of anti-bacteriophage escherichia coli chassis cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of yafC overexpression strain BL21 / pET28a(+)-YafC

[0039] Specific steps are as follows:

[0040] (1) Design primers YafC-F and YafC-R according to the nucleotide sequence of Escherichia coli BL21 phage resistance protein YafC coding gene yafC (nucleotide sequence shown in SEQ ID No.2), with Escherichia coli BL21 genomic DNA as The template is amplified to obtain a DNA fragment containing the yafC gene sequence, and the DNA fragment is connected to the pET28a (+) plasmid after digestion with BamHI enzyme and EcoRI enzyme to obtain the recombinant plasmid pET28a (+)-YafC; Plasmid pET28a(+)-YafC was transformed into E. coli (Escherichia coli) BL21, and the transformation solution was spread on LB plates containing 100 μg / mL Kanna resistance, and cultured at 37°C until a single colony grew; pick a single colony to LB liquid medium containing 100 μg / mL of Kanna resistance, cultivated at 37°C and 200 rpm for 8-12 hours, extracted the plasmid in the ...

Embodiment 2

[0043] Example 2: Verification of yafC overexpression strain BL21 / pET28a(+)-YafC tolerance to phage sensitivity

[0044] Specific steps are as follows:

[0045]The wild-type strain BL21 cultivated overnight, and the yafC overexpression strain BL21 / pET28a(+)-YafC obtained in Example 1 were inoculated in fresh liquid LB medium, and cultivated at 37° C. and 180 rpm until the early logarithmic growth phase ( OD 600 = 0.6), the anti-phage ability of the yafC overexpression strain BL21 / pET28a(+)-YafC was verified by spotting assay and phage action on the bacteriostatic curves of the two strains.

[0046] Specific steps for spotting assay: inoculate wild-type Escherichia coli BL21 and yafC overexpression strain BL21 / pET28a(+)-YafC cultured overnight (16h) into fresh liquid LB culture sets, and cultivate to logarithmic growth at 37°C and 180rpm Early period (OD 600 =0.8), and then pipet 200 μL of BL21 and BL21 / pET28a(+)-YafC colonies into 3 mL of semi-solid LB medium, mix well, and...

Embodiment 3

[0049] Example 3: Application of high-efficiency anti-phage chassis cells BL21 / pET28a(+)-YafC in the process of γ-aminobutyric acid whole-cell transformation

[0050] Construction of the vector pET28a(+)-YafC-Gad: the nucleotide sequence shown in SEQ ID NO.5 was digested with SacI and NotI enzymes and connected to the recombinant plasmid pET28a(+)-YafC to obtain the recombinant plasmid pET28a(+ )-YafC-Gad; the recombinant plasmid pET28a(+)-YafC-Gad was transformed into Escherichia coli (Escherichia coli) BL21, and the transformation solution was spread on LB plates containing 100 μg / mL kana resistance, and cultured at 37°C until A single colony grows; pick a single colony to LB liquid medium containing 100 μg / mL of Kanna resistance, culture at 37°C and 200 rpm for 8-12 hours, use the gel recovery kit to extract the plasmid in the bacterial liquid, and pass the plasmid to the enzyme Cutting verification, and sent for sequencing, verification is correct as a positive transforman...

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Abstract

The invention discloses construction and application of an anti-bacteriophage escherichia coli chassis cell, and belongs to the technical field of gene engineering and microbial engineering. Accordingto the invention, the coding gene yafC of the LysR family transcription regulation factor YafC is overexpressed in escherichia coli BL21 so that the anti-bacteriophage capability of escherichia coliis remarkably improved; further, the anti-bacteriophage escherichia coli BL21 obtained through breeding is used for constructing a gamma-aminobutyric acid (GABA) production strain BL21-pET28a (+)-YafC-Gad, it is found that no bacteriophage infection risk exists in the whole fermentation process, the yield of the recombinant strain GABA reaches up to 278.3 +-16.8 gL, and the molar conversion rate reaches 98.4 +-0.3% when the recombinant strain GABA is used for biotransformation production of gamma-aminobutyric acid.

Description

technical field [0001] The invention relates to the construction and application of a bacteriophage-resistant Escherichia coli chassis cell, and belongs to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Fermentation industry is one of the important links of biotechnology industrialization. As my country pays more attention to biotechnology industry and the national demand for biotechnology products is increasing, the total fermentation industry represented by amino acids, organic acids, vitamins and other products The output value has been among the top in the world. However, in the process of traditional fermentation industry and modern industrial bio-manufacturing system, bacteriophages have always accompanied the fermentation industry and threatened the safety of the fermentation process, causing huge economic losses to fermentation enterprises for a long time. Taking the common industrial microorganism Escherichia col...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/70C12P13/00C12R1/19
CPCC07K14/245C12N15/70C12P13/005
Inventor 饶志明尤甲甲潘学玮徐美娟杨套伟张显王雅玲易敢峰付维来
Owner JIANGNAN UNIV
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