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Anammox bacterium nitrite reductase expression vector and construction method thereof

A technology of anammox bacteria and nitrite, applied in the field of anammox bacteria nitrite reductase expression vector and its construction, can solve the problem of low survival rate, inability to enrich and cultivate pure anammox bacteria, The growth environment of anaerobic ammonium oxidation bacteria is demanding and other problems, to achieve the effect of optimizing fermentation conditions, improving functional activity, and increasing expression

Inactive Publication Date: 2015-04-08
SHANGHAI UNIV
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Problems solved by technology

Although the anammox reaction has promising prospects for removing nitrite, the anammox bacteria have strict requirements on the growth environment, the survival rate is low, and the pure anammox bacteria cannot be enriched and cultivated, which leads to the stagnation of the application of the anammox reaction Not moving forward

Method used

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  • Anammox bacterium nitrite reductase expression vector and construction method thereof
  • Anammox bacterium nitrite reductase expression vector and construction method thereof
  • Anammox bacterium nitrite reductase expression vector and construction method thereof

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Embodiment 1

[0026] 1. Extraction of total DNA in soil environmental samples: The soil environmental samples were collected from Dongtan Wetland, Chongming Island, Shanghai. The soil samples were stored in a -80°C refrigerator for extraction of total DNA in environmental samples. The total DNA of the soil samples was extracted using the PowerSoil DNA Isolation Kit (MO BIOL laboratories, Inc., Carlsbad, CA), a kit produced by MO BIO, and the extracted DNA was stored in a sterilized 2mL centrifuge tube at -20°C in a refrigerator.

[0027] 2. Construction of the pGEM-T-nirS recombinant vector: amplify the nitrite reductase nirS gene sequence according to the primers obtained in the literature and connect it to the pGEM-T expression vector.

[0028] The primers are as follows: forward primer AnnirS379F: 5 ， -TCTATCGTTGCATCGCATTT-3 ，

[0029] Reverse primer nirS821R: 5 ， - GGATGGGTCTTGATAAACA-3 ，

[0030] 5 of upstream and downstream primers ， A T restriction site was designed at the end ...

Embodiment 2

[0037] Use LB liquid medium to expand (20 times) culture nir S gene recombinant engineering bacteria to OD 600 reached 0.6. Take 30 mL of bacterial liquid, apply it to 100 mL of sodium nitrite-contaminated solution with an initial concentration of 45 mg / L, and shake at 200 rpm for 27 hours at 37°C. Take 5mL of the reaction solution and pass it through a 0.22μm filter membrane, and use ion chromatography to detect the concentration of sodium nitrite remaining in the solution during the reaction process, and the calculated removal rate is 98%. At the same time, take 5mL of the reaction solution and detect it with a UV-4802 spectrophotometer, using OD 600 The value indicates the growth of the bacteria, after testing nir The growth of S gene recombinant engineered bacteria presents a complete S-shaped growth curve, reaching a stable period in 7-8 hours, and the growth rate is fast.

[0038] Shanghai University

[0039] Anammox bacteria nitrite reductase expression vector and ...

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Abstract

The invention relates to an anammox bacterium nitrite reductase expression vector and a construction method thereof. The recombinant engineering strain has a base sequence represented by SEQ ID No. 1. According to the recombinant engineering strain disclosed by the invention, the expression level and functional activity of the gene are greatly improved, so that the application of anammox bacterium nitrite reductase to an anaerobic ammonia oxidation process is realized.

Description

technical field [0001] The invention relates to an anammox bacteria nitrite reductase expression vector and a construction method thereof. Background technique [0002] With the development of society and the improvement of people's living standards, people's dietary structure has changed, and the nitrogen content in the resulting domestic sewage has increased, forming sewage with a low carbon-to-nitrogen ratio. When the traditional nitrification / denitrification process treats sewage with a low carbon-to-nitrogen ratio, the denitrification efficiency is lower due to insufficient carbon sources, and the total nitrogen content of the effluent seriously exceeds the discharge standard. After this kind of sewage is discharged into natural water bodies, plankton such as algae in the water will grow and reproduce in large numbers, destroying the ecological balance of the water body. At the same time, due to insufficient carbon sources, the nitrification / denitrification process is ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N15/66C12R1/19
Inventor 陆永生陈学萍杨兴兴刘冬秀钱光人
Owner SHANGHAI UNIV
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