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Recombinant escherichia coli and application thereof

A technology for recombining Escherichia coli and microbial strains, applied in bacteria, biochemical equipment and methods, microorganisms, etc., can solve problems such as industrial production of MicrocinJ25, which has not been seen before, and achieve the effect of improving the expression level.

Active Publication Date: 2020-05-29
ZHENGZHOU ZHONGHAO BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

But there is no report on the industrial production of Microcin J25 so far

Method used

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  • Recombinant escherichia coli and application thereof
  • Recombinant escherichia coli and application thereof
  • Recombinant escherichia coli and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 2

[0034] Preparation Example 2 Linking the Target Fragment to the pMD18-T Vector and Transforming the Plasmid

[0035] 1. The enzyme-linked reaction system is shown in Table 3 below.

[0036] table 3

[0037] Element Dosage dd H 2 o

13.5μl 10×T4DNA Ligase Buffer 2μl pMD18-T vector 0.5μl dna 3μl T4DNA Ligase 1μl total 20μl

[0038] Mix the above components and centrifuge briefly, and place the prepared system at 16°C for overnight stability. The connected system was placed in a 4°C refrigerator for later use.

[0039] 2. Heat Shock Plasmid Transformation

[0040] a. Take out 100 μl of the prepared E. coli BL21 competent cells from the -80°C freezer, place them on ice for 10 minutes, and make them enter the 0°C competent state. In the ultra-clean bench, add 10 μl of the corresponding enzyme-ligated product (the pMD18-T vector prepared above to connect the fragment of the target gene mcjABCD) to the competent cells, ...

preparation Embodiment 3

[0047] Preparation of Example 3 double enzyme cut (BamHI and EcoR I) plasmid and expression vector pGEX-6p-1 and connection

[0048] 1. First prepare the enzyme digestion system mixture for restriction plasmid and expression vector, and then distribute it to each 0.6ml EP tube. The final enzyme digestion system (Takara Company) for each tube is shown in Table 4 below.

[0049] Table 4

[0050] Element Dosage wxya 2 o

30μl 10×Proteinase K Buffer 4μl BamHI 1μl EcoR I 1μl plasmid 4μl total 40μl

[0051] Carry out enzyme digestion in a 37°C incubator for no more than 1.5 hours, and add 2 μl of 10×Buffer to stop the enzyme digestion. The results showed that the enzyme digestion effect was good. The plasmid digested in the same step was used to recover the target fragment, and the expression vector after digestion was first placed in a 4°C refrigerator for later use.

[0052] 2. Recover the obtained target fragment an...

preparation Embodiment 4

[0059] Cultivation of Preparation Example 4 Recombinant Escherichia coli Transformant

[0060] Pick 4 single colonies of transformants and 1 single colony of Escherichia coli MC4100 and inoculate them into 25ml liquid medium respectively (32g corn flour, 20g soybean meal, 13g peptone, 15g glucose, 2g KH 2 PO 4 and 1.20 g of ammonium sulfate were dissolved in purified water and adjusted to 1 L with purified water), 37 ° C, 200 rpm shaking culture for 12-18 h, 10000 rpm centrifugation for 10 min, and the supernatant was collected. The concentration of the target polypeptide Microcin J25 in the supernatant was detected by high performance liquid chromatography.

[0061] The high performance liquid chromatography chromatographic conditions are as follows:

[0062] Use octadecylsilane bonded silica gel as filler (high performance liquid chromatography: Agilent1260, C18 chromatographic column: ZORBAX 300SB-C18, 5μm, 4.6×250mm), and trifluoroacetic acid-water-acetonitrile (1:950: ...

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Abstract

The present invention discloses recombinant escherichia coli and an application thereof. The recombinant escherichia coli is deposited on August 28, 2018 in the China General Microbiological Culture Collection Center and has deposit number of CGMCC No.16349. After the recombinant escherichia coli is fermented and cultured, concentration of Microcin J25 in supernatant can reach 4,000 mg / L or more.

Description

technical field [0001] The invention relates to a genetically engineered bacterial strain and its application. Specifically, the invention relates to a high-yield MicrocinJ25 recombinant Escherichia coli and its application. Background technique [0002] The problem of bacterial resistance caused by overuse of antibiotics is becoming more and more prominent, which makes clinical anti-infection treatment difficult. In the breeding industry, the long-term use of antibiotic growth-promoting agents is even more serious. It not only indirectly brings harm to human health due to the large amount of antibiotic residues, but also leads to low immunity of livestock and poultry, increasing the occurrence of infectious diseases and increasing the cost of breeding. Gram-negative bacteria such as Escherichia coli and Salmonella are prone to drug resistance, and the diseases caused by them have caused huge losses to animal husbandry. It is urgent to solve this problem. [0003] Antimicro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/00C12R1/19
CPCC07K14/245C12N1/20C12P21/02C12N1/205C12R2001/19C12P7/46
Inventor 郭良兴
Owner ZHENGZHOU ZHONGHAO BIOLOGICAL TECH CO LTD
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