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Mutated mycobacterium smegmatis capable of secreting niacin and construction method of mutated mycobacterium smegmatis

A technology of Mycobacterium smegmatis and a construction method is applied in the field of bioengineering to achieve the effects of low cost, easy amplification and simple production conditions

Active Publication Date: 2018-08-21
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report to directly use microbial strains to prepare niacin without relying on the addition of 3-cyanopyridine and enzyme catalysis

Method used

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  • Mutated mycobacterium smegmatis capable of secreting niacin and construction method of mutated mycobacterium smegmatis
  • Mutated mycobacterium smegmatis capable of secreting niacin and construction method of mutated mycobacterium smegmatis
  • Mutated mycobacterium smegmatis capable of secreting niacin and construction method of mutated mycobacterium smegmatis

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Construction of plasmid pMHS-NrtR ms

[0033] 1) Take Mycobacterium smegmatis mc 2 155 genomic DNAs were used as templates, and the primer pairs shown in SEQ ID No. 1-4 were used for PCR amplification. like figure 1 As shown, the PCR product nucleic acid gel electrophoresis was used to separate the target DNA fragments, and the Omega company gel back kit was used to recover the target DNA fragments shown in SEQ ID No. 5 and 6. The recovered DNA was digested with Van91I restriction endonuclease, and the digested DNA was recovered by Omega's Cycle-pure recovery kit for use;

[0034] 2) Escherichia coli DH5α strain containing the pMHS plasmid was inoculated with LB liquid medium (adding 150 μg / mL hygromycin B) and cultured overnight at 37°C, and then the bacteria were collected to extract the plasmid using the Omega plasmid extraction kit to obtain the pMHS plasmid Carry out nucleic acid gel electrophoresis after Van91I restriction endonuclease digestion and...

Embodiment 2

[0037] Example 2: Construction of plasmid pHAGE-NrtR ms

[0038] 1) LB were cultured with pHAGE (amphicillin resistance) and pMHS-NrtR ms (Hygromycin B resistance) Escherichia coli DH5α strain of the plasmid and use the plasmid extraction kit of Omega company to extract the plasmid, and the two obtained plasmids are linearized with PacI restriction endonuclease and then reacted with T4DNA ligase at 16°C overnight. The ligation product was packaged and transformed into Escherichia coli HB101 competent cells using a packaging kit (EPICENTREBiotechnologies: MaxPlax Lambda Packaging Extracts), and then coated on an LB solid plate (containing 150 μg / mL hygromycin B), and the plate was placed in a 37°C constant temperature incubator After culturing overnight, single clones grown on the plate were picked, inoculated with LB liquid medium (containing 150 μg / mL hygromycin B) and cultured overnight on a shaker at 200 rpm at 37°C, and the plasmid was extracted using Omega’s plasmid ext...

Embodiment 3

[0039] Embodiment 3: Preparation of recombinant TM4 phage

[0040] 1) Preparation of Mycobacterium smegmatis mc 2 155 competent cells: Mycobacterium smegmatis mc 2 155 was inoculated in 5ml 7H9 liquid medium, cultured at 37°C with shaking at 200rpm until the logarithmic growth phase (OD0.5-1.0); the culture was inoculated in fresh 100mL 7H9 liquid medium at a ratio of 1:100, overnight at 37°C Cultivate until the OD600 reaches about 0.6, collect the bacteria by centrifugation at 5000rpm at 4°C for 10 minutes, wash the bacteria with pre-cooled 10% sterile glycerol at least twice, and finally add 10 mL (appropriate amount) of pre-cooled 10% glycerol, weigh After suspending the bacteria, freeze them in separate devices at -80°C for later use;

[0041] 2) will construct the correct positive plasmid pHAGE-NrtR ms Add DNA to 200 μL of M. smegmatis mc 2 155 electroporation-competent cells were incubated on ice for 10 minutes, then transferred to a 2mm BTX electroporation cup, wip...

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Abstract

The invention provides a construction method of mutated mycobacterium smegmatis capable of secreting niacin. The construction method comprises the following steps: 1), constructing a plasma pMHS-NrtRms; 2) constructing a plasma pHAGE-NrtRms; 3) preparing a recombination TM4 phage; and 4) constructing an nrtRms gene mutant strain mycobacterium smegmatis mc2100. The construction method has the beneficial effects that: by constructing the nrtRms gene mutant strain mycobacterium smegmatis, the strain directly prepares niacin without depending on adding 3-cyanopyridine and catalyzing through an enzyme, the various defects of the existing chemical synthesis process of the niacin are avoided, the production condition is simple, pollution-free, convenient and feasible, is easy to amplify, is low in cost, is suitable for large-scale industrial production and application, and has huge promotion and application values.

Description

technical field [0001] The invention relates to the technical field of bioengineering. Background technique [0002] Niacin is 3-picolinic acid, also known as vitamin B3, a water-soluble vitamin, belonging to the vitamin B family, one of the 13 vitamins necessary for the human body. Niacin is an important hydrogen transporter in body tissues and an anti-pellagra factor. It can maintain skin and nerve health and promote digestion. If it is lacking, pellagra can occur, manifested as symptoms such as dermatitis, glossitis, oropharynx, diarrhea, irritability, and insomnia. Together with nicotinamide, it is called vitamin PP, which is used for anti-pellagra and can also be used as a blood dilator. As a pharmaceutical intermediate, it is used in the production of isoniazid, nicotinamide, nicosyl and inositol nicotinate, etc. Niacin is also used in food products, meat additives and feed additives to prevent pellagra. In particular, the addition of nicotinic acid in the feed can...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/66C12R1/34
CPCC07K14/35C12N15/66C12N15/74
Inventor 王绪德周亚凤毕利军周盈张晓丽
Owner FOSHAN UNIVERSITY
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