Enhanced escherichia coli capable of resisting sundry fungus pollution and bacteriophage infection, and construction method and application of enhanced escherichia coli

A technology of anti-miscellaneous bacteria pollution and Escherichia coli, applied in the field of genetic engineering, can solve problems such as common bacteria

Active Publication Date: 2019-10-08
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the complex surrounding environment and the hidden parts of the fermenta...

Method used

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  • Enhanced escherichia coli capable of resisting sundry fungus pollution and bacteriophage infection, and construction method and application of enhanced escherichia coli
  • Enhanced escherichia coli capable of resisting sundry fungus pollution and bacteriophage infection, and construction method and application of enhanced escherichia coli
  • Enhanced escherichia coli capable of resisting sundry fungus pollution and bacteriophage infection, and construction method and application of enhanced escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Acquisition of Formamidase and Phosphorous Phosphate Dehydrogenase Fusion Gene Containing Constitutive Promoter

[0083] Using the formamidase gene of Paenibacillus pasadenensis.CS0611 genome and the phosphite dehydrogenase gene of Klebsiellapneumonia.OU7 as templates, the Linker sequence was used as a bridge to connect the two genes into a fusion gene. Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to optimize the codons and synthesize the formamidase gene and phosphorous acid dehydrogenase gene.

Embodiment 2

[0085] The construction of anti-miscellaneous bacteria plasmid (pJ23110-fmdA-linker-ptxD-pJ23119-N 20 -sgRNA)

[0086] The amplification conditions were as follows: react at 94°C for 3 min; then react at 94°C for 10 s, 50°C for 5 s, and 72°C for 2 min, and cycle 30 times; finally react at 72°C for 7 min, and finally cool down to 4°C to store the PCR product. Using the amplified pJ23101-fmdA-linker and ptxD fragments as templates, using A1 and A2 as upstream and downstream primers respectively, amplified to obtain HR-pJ23119-N 20 - sgRNA (as shown in SEQ ID NO.3). Connect seamlessly into pETDuet-gfp-pJ23101-fmdA-linker-ptxD after single digestion with Pci I to obtain recombinant plasmid pETDuet-gfp-pJ23101-fmdA-linker-ptxD-pJ23119-N 20 -sgRNA, and transformed into recombinant Escherichia coli DH5α.

[0087] The enzyme reagent used in PCR is TaKaRa company's HS DNA Polymerase with GCbuffer; PCR reaction system and conditions are as follows:

[0088]

Embodiment 3

[0090] Construction of CRISPR / Cas9 system plasmid

[0091] The amplification conditions were as follows: react at 94°C for 4 min; then react at 94°C for 30 s, 50°C for 5 s, and 72°C for 12 min, and cycle 30 times; finally react at 72°C for 7 min, and finally cool down to 4°C to store the PCR product. The plasmid pCas was used as a template, and primer C1 and primer C2 were respectively used as upstream and downstream primers, and the PCR reaction system was as in Example 2. Amplified to obtain the transformed pCas plasmid (pCas-pJ23119-N 20 -sgRNA).

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Abstract

The invention discloses enhanced escherichia coli capable of resisting sundry fungus pollution and bacteriophage infection, and a construction method and application of the enhanced escherichia coli.The construction method comprises the following steps that the escherichia coli BL21 (DE3) is taken as a host, a formamidase gene and a phosphite dehydrogenase gene are amplified into a fused gene, the fused gene and CRISPR/Cas9 plasmid capable of resisting bacteriophage are converted into the escherichia coli, and thus recombined escherichia coli is obtained. The recombined escherichia coli can express formamidase and phosphite dehydrogenase fused proteins, formamide and phosphite oxide can be decomposed simultaneously, and a required nitrogen source and a required phosphorous source are provided for the recombined escherichia coli; other microorganisms cannot grow and breed in an MOPS culture medium containing the formamide and phosphite due to lacking enzymes for metabolizing the formamide and the phosphite, and thus the problem of contamination of the escherichia coli in industrial fermentation can be solved; and secondly, the recombined escherichia coli can shear a bacteriophage genome through a CRISPR/Cas9 system, and infection of the bacteriophage is prevented.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an enhanced Escherichia coli capable of resisting miscellaneous bacteria pollution and phage infection, a construction method and application thereof. Background technique [0002] Escherichia coli is currently one of the most widely used hosts, because the Escherichia coli genome has been thoroughly studied, and it reproduces quickly and has a short fermentation cycle. Therefore, Escherichia coli is closely watched and valued by entrepreneurs in the industrial fermentation industry. However, during the E. coli fermentation process, the problem of bacterial contamination is still the most concerned issue for enterprises. Once contaminated, it will not only cause economic loss, waste of principles and time, but also increase the difficulty of waste disposal. Therefore, if a method can be found to solve the problem of E. coli being contaminated by miscellaneous bacteria during ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/65C12N15/90C12R1/19
CPCC12N9/80C12N9/0004C12N15/70C12N15/65C12N15/902C12Y120/01001C12N2800/22C12N2810/10Y02A50/30
Inventor 娄文勇区晓阳宗敏华吴晓玲
Owner SOUTH CHINA UNIV OF TECH
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