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Biology restoration method of petroleum pollution soil by using broad-host-range self-transfer degrading plasmid

A technology for degrading plasmids and oil pollution, applied in the field of molecular biology technology and contaminated soil remediation, to achieve lasting remediation of petroleum hydrocarbon-contaminated soil with obvious effects

Inactive Publication Date: 2015-07-01
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

There are no relatively mature reports on the comprehensive treatment of petroleum hydrocarbon-polluted environments using self-transfer plasmids from a wide variety of hosts

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1) Host bacteria activation and expansion culture:

[0029] Take out the host bacteria carrying the broad-host self-transfer plasmid stored in the glycerol tube from the -70°C refrigerator, inoculate it on the LB solid medium containing the corresponding double selection markers (the selection marker of the host bacteria and the selection marker of the plasmid), and set the line. Incubate overnight at 30°C. Use a sterilized inoculation needle to inoculate the above-mentioned cultured colony into 5 ml of LB liquid medium without antibiotics, and cultivate overnight on a shaker at 30°C. The above-mentioned cultivated bacterial solution was inserted into 100ml LB liquid medium according to the inoculum amount of 5% volume to carry out expansion cultivation. Cultivate to the logarithmic growth phase, and the number of bacteria reaches 1.0×10 after fermentation 9 .

[0030] Wherein, the host bacteria carrying the broad host self-transfer plasmids are respectively transfer...

Embodiment 2

[0041] The surface soil of the Shenyang Ecological Station of the Chinese Academy of Sciences that was not polluted by petroleum hydrocarbons was collected, and the 0-20cm surface soil was passed through a 100-mesh sieve to measure the moisture content of the soil (to facilitate the adjustment of the soil moisture in the final follow-up experiment). The phenanthrene is dissolved in acetone and mixed into the soil at a ratio of 5mg / g dry soil, mixed evenly and the acetone is volatilized. The cultivation experiment was carried out in 1000ml jars, and 500g of phenanthrene-contaminated soil samples were placed in each jar.

[0042] Guangshu self-transferred petroleum hydrocarbon degradation plasmid pW22-3G, the size is 77kb; its genetic marker is green fluorescent protein (gfp), and the degradation genes are aromatic ring hydroxylation dioxygenase gene (phdA), benzene dioxygenase gene ( ben), xylene monooxygenase gene (touA), salicylic acid hydroxylase gene (nahG), catechol 2,3-di...

Embodiment 3

[0047] The in-situ oil-contaminated farmland surface (0-20cm) soil in the Shenfu Irrigation Area was collected and passed through a 2mm sieve. The total petroleum hydrocarbon pollution concentration was determined to be 3482mg / kg dry soil, which was severely polluted. Take 500g of contaminated soil samples and place them in a 1000ml glass jar.

[0048] Inoculate broad host self-transferred petroleum hydrocarbon degradation plasmid pS3-2G, the size is 106kb; its genetic marker is kanamycin resistance (Km R ), the degradation genes are aromatic ring hydroxylation dioxygenase gene (phdA), benzene dioxygenase gene (ben), xylene monooxygenase gene (touA), salicylic acid hydroxylase gene (nahG), Catechol 2,3-dioxygenase gene); the host bacteria is Agrobacterium tumefaciens C58, which belongs to α-Proteobacteria). The plasmid is transferred to the host strain by the conventional method of the prior art, and is ready for use.

[0049] The above-mentioned host bacterium carrying the ...

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Abstract

The invention belongs to an intersect field of a molecular biology technology and a pollution soil restoration technology, and concretely relates to a biology restoration method of petroleum pollution soil by using broad-host-range self-transfer degrading plasmid. host bacteria of the broad-host-range self-transfer degrading plasmid containing petroleum hydrocarbon degraded gene and genetic selection markers can be inoculated to the polluted soil to be treated, a nutrient solution is added, under conjugal transfer effect among microbes, the degraded gene carried by the broad-host-range self-transfer plasmid can perform high efficiency degrading effect, so that purpose of lasting restoration of petroleum hydrocarbon pollution soil can be reached. The method is different with an inoculation degrading bacteria operation process in a traditional method. Through conjugal transfer after inoculation, the broad-host-range degrading plasmid can be converted to pollution soil indigenous flora, horizontal transfer is happed on degraded gene, and degraded gene can express in indigenous flora having growth competition advantage and can perform degrading function, a bottleneck problem of difficult long-term viability of the inoculation bacterial strain can be avoided, and the purpose of lasting restoration of petroleum hydrocarbon pollution soil can be reached.

Description

technical field [0001] The invention relates to the interdisciplinary field of molecular biology technology and contaminated soil remediation technology, specifically a method for bioremediation of oil-contaminated soil by using a broad host range (BHR) self-transfer degradation plasmid. Background technique [0002] Petroleum is a complex mixture containing a variety of hydrocarbons (normal alkanes, branched alkanes, aromatic hydrocarbons, alicyclic hydrocarbons) and a small amount of other organic compounds (sulfides, nitrides, naphthenic acids, etc.), and is the most important source of human beings. One of energy. However, with the increase in oil extraction and use, a large amount of oil and its processed products enter the environment, among which industrial sewage discharge and oil spill accidents in the process of crude oil extraction and smelting are the main causes of soil petroleum hydrocarbon pollution, not only destroying The ecological environment is damaged, ...

Claims

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Application Information

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IPC IPC(8): B09C1/10C12N1/21
Inventor 李慧李小彬王亚菲张玉革杨山姜勇
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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