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Method for eliminating organic pollutant residues in soil by utilizing genetically engineering modified methane-oxidizing bacteria

A technology of genetically engineered bacteria and methane oxidizing bacteria, applied in the field of eliminating residual organic pollutants in soil, can solve problems such as unusable, and achieve the effect of promoting survival and reproduction

Active Publication Date: 2020-07-24
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no effective solution to this problem
[0005] Methanotrophs are a group of bacteria with unique metabolic methods. They can only grow on one-carbon compounds such as methane or methanol as carbon sources and energy sources, but cannot use compounds with more than one carbon atom in their molecules

Method used

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  • Method for eliminating organic pollutant residues in soil by utilizing genetically engineering modified methane-oxidizing bacteria
  • Method for eliminating organic pollutant residues in soil by utilizing genetically engineering modified methane-oxidizing bacteria
  • Method for eliminating organic pollutant residues in soil by utilizing genetically engineering modified methane-oxidizing bacteria

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Experimental program
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Embodiment 1

[0025] The construction of embodiment 1 genetically engineered bacteria

[0026] In order to integrate the sulfonylurea herbicide hydrolase gene sulE into the chromosome of strain Methylomonas sp.LW13, first, Hansschlegelia zhihuaiae S113 (DSM 18984 T ) genomic DNA as a template with primers sule-F and sule-R to amplify fragment 1 carrying sulE, and use plasmid pAWP89 (Puri A W et al., Applied and Environmental Microbiology, 2015, 81(5): 1775-1781.doi : 10.1128 / AEM.03795-14) as a template with primers km-F and km-R to amplify fragment 2 carrying the kanamycin resistance gene, with the genomic DNA of bacterial strain Methylomonassp.LW13 as a template, with primers LF- Fragment 3 was amplified with F and LF-R, and fragment 4 was amplified with primers RF-F and RF-R; then, by fusion PCR method with primers LF-F and RF-R (Shevchuk NA et al., Nucleic Acids Research ,2004,32(2),e19.doi:10.1093 / nar / gnh014) connected these four fragments into a fragment according to the sequence of 3...

Embodiment 2

[0027] The fermentation culture of embodiment 2 engineering bacteria LW13-sulE

[0028] In order to obtain the cells of the engineering bacteria LW13-sulE, first, the bacteria were streaked on the solid NMS medium, and the streaked plate was placed in a sealable jar, filled with 30% methane (methane to air ratio) Culture at 30°C for 3 days; then transfer the cells on the solid NMS medium to the liquid NMS medium, place the liquid culture in a sealable culture bottle, and fill with 30% methane (the ratio of methane to air) Then culture at 30° C. for 3 days; finally collect the cells by centrifugation (5000 rpm, 10 minutes).

Embodiment 3

[0029] Embodiment 3 repairs the soil polluted by bensulfuron-methyl with engineering bacteria LW13-sulE

[0030] Bensulfuron-methyl is a typical sulfonylurea herbicide. Three concentrations of benzazine were added to the soil, 0mg / kg, 0.4mg / kg (the maximum recommended dosage in the field) and 4mg / kg (10 times the maximum recommended dosage in the field), to obtain three kinds of soil samples. Then, each soil sample was treated with three treatments: no inoculation, inoculation with strain LW13 and inoculation with engineering bacteria LW13-sulE. In the treatment of inoculation, the inoculation amount is 10 5CFU / g soil. Place the inoculated soil in a sealable tank, fill it with 30% methane (the ratio of methane to air) and cultivate it at room temperature for 10 days. During this period, shake the device once every other day to ensure that the soil is well ventilated, and change it every two days Gas, to ensure sufficient gas volume. After remediation, bioassay (planting pl...

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Abstract

The invention discloses a method for eliminating organic pollutant residues in soil by utilizing genetically engineering modified methane-oxidizing bacteria. According to a genetically engineered bacterium, the methane-oxidizing bacteria are used as host, and an organic pollutant degrading enzyme gene is introduced through a genetically engineering means. The gene coding the pollutant degrading enzyme is introduced into the strains by means of the genetically engineering means and is functionally expressed through optical transcription and translation elements, so that the performance of degrading target pollutants is given to the strains. According to the method, the gene coding the pollutant degrading enzyme is introduced into methane-oxidizing bacteria by utilizing the genetically engineering means, and the performance of degrading organic pollutants is given to the strains; and a certain amount of engineering bacteria are mixed into polluted soil, and survival and reproduction of the engineering bacteria are promoted by providing sufficient methane, so that pollutants can be effectively degraded.

Description

technical field [0001] The invention belongs to the field of biological high technology and discloses a method for eliminating organic pollutant residues in soil. [0002] technical background [0003] Soil pollution is a hot issue of global concern. Common organic pollutants in soil include pesticides (insecticides, herbicides and fungicides, etc.), antibiotics, polycyclic aromatic hydrocarbons and chemical intermediates. These pollutants cause serious environmental and health problems. In addition, herbicide residues may also poison crops, resulting in reduced or even crop failure. In response to this problem, a variety of soil remediation methods have been developed, including physical, chemical and biological methods. Although physical and chemical methods have quick results, they are costly and have secondary pollution risks. [0004] The biological method mainly refers to the decomposition of organic pollutants in the soil by adding functional microorganisms. The a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52B09C1/10C12R1/26
CPCC12N9/14B09C1/10
Inventor 闫新刘永闯
Owner NANJING AGRICULTURAL UNIVERSITY
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