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Promoter from corynebacterium glutamicum and application thereof

A technology of promoter and coryneform bacteria, which is applied in the biological field and can solve the problems of promoter leakage, limited application, difficult for inducer to pass through coryneform bacteria, etc.

Inactive Publication Date: 2010-04-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, because the promoter of E. coli has leaky repression in Corynebacterium, and the related inducer is not easy to pass through the cell wall of Corynebacterium at a lower concentration, its application in actual production is still very limited.

Method used

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  • Promoter from corynebacterium glutamicum and application thereof
  • Promoter from corynebacterium glutamicum and application thereof
  • Promoter from corynebacterium glutamicum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, obtaining the DNA fragment with promoter activity

[0034] 1. Cultivate Corynebacterium glutamicum 10147 and extract genomic DNA

[0035] 1. Culture of bacteria

[0036] Pick Corynebacterium glutamicum 10147 (influence of multiple conditions on transformation efficiency in coryneform bacteria electric shock transformation Shen Tianxiang et al. Bioengineering Journal 11 (3): 245-2791995) (this bacterial strain can be obtained in Institute of Microbiology, Chinese Academy of Sciences, See the letter of guarantee for details) A single colony was inoculated in a test tube of 3ml of LB medium, placed on a shaker at 30°C at 300rpm for overnight culture, and the culture solution was used as the seed solution. Take 400 μL of the seed solution and inoculate it into a Erlenmeyer flask with 30 ml of LB medium, and place it on a shaker at 30°C at 300 rpm for overnight culture.

[0037] 2. Extraction of genomic DNA of Corynebacterium glutamicum 10147

[0038]Centrif...

Embodiment 2

[0042] Example 2, detection and analysis of promoter fragments

[0043] 1. DNA sequence determination of promoter active fragment

[0044] The plasmid DNAs of the 30 active fragments with promoter function in Example 1 (two) were extracted, and the DNA sequences of the promoter active fragments were determined using the artificially synthesized primer SEQ ID NO: 3 respectively, and 30 DNA sequences were obtained. The promoter sequence involved in this patent is SEQ ID NO:1.

[0045] 2. Determination of total protein content

[0046] 1) Preparation of crude enzyme solution

[0047] The DNA fragment shown by the sequence SEQ ID NO:1 was prepared.

[0048] The fragment was ligated with the promoter detection vector pAKC6 treated with endonuclease Bgl II, and the calcium chloride method was used (Sambrook J, Fritsch E, Maniatis T Molecular Cloning Experiment Guide. Jin Dongyan, Li Mengfeng, etc. translation. Second Edition . Beijing: Science Press, 1989) into Escherichia coli ...

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Abstract

The invention discloses a promoter with a nucleotide sequence as follows: 1) a nucleotide sequence shown in sequence 1 in a sequence table; 2) a nucleotide sequence which hybridizes with the nucleotide sequence in 1) under strict conditions and has promoter activity; or 3) a nucleotide sequence which has homology of more than 70% with the nucleotide sequence in 1) and has promoter activity. The promoter provided by the invention can be applied to the biotechnology field with corynebacterium or escherichia bacteria as industrial microbes. Experiments prove a fragment with promoter activity from SEQ ID NO:1 has the activity higher than that of a trc promoter (SEQ ID NO:2).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to nucleotide fragments with promoter activity and applications thereof. Background technique [0002] Corynebacteria are Gram-positive bacteria. Some corynebacteria are widely used in the field of bioengineering as industrial microorganisms, and can be used to produce various chemical substances, such as amino acids and nucleotides, L-glutamic acid, L-lysine, L-threonine, L- Tryptophan, inosinic acid, etc. Amino acids and nucleotides can be used as medicines, food, food additives, animal feed. Corynebacterium mutants obtained through physical and / or chemical mutagenesis have a strong ability to synthesize the above-mentioned useful substances, and through genetic engineering and / or metabolic engineering, wild strains or mutants of Corynebacteria can be obtained. Higher productive strains. The basic operation of this genetic engineering is the expression of genes involved in various...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/00C12N1/21C12N15/70C12N15/77C12N15/74C12R1/185C12R1/15
Inventor 丁久元刘桂明李开赵智王宇张英姿
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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