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Method for screening self-transmissible broad host range plasmid carrying petroleum hydrocarbon degrading gene by utilizing triparental pairing conjugation and single carbon source of petroleum hydrocarbon

A technology of petroleum hydrocarbons and plasmids, applied in the field of biological technology and bioremediation of polluted soil

Inactive Publication Date: 2012-06-27
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitation of this method is that the isolated plasmid must have specific phenotypic characteristics in order to be used as a marker for screening zygotes

Method used

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  • Method for screening self-transmissible broad host range plasmid carrying petroleum hydrocarbon degrading gene by utilizing triparental pairing conjugation and single carbon source of petroleum hydrocarbon
  • Method for screening self-transmissible broad host range plasmid carrying petroleum hydrocarbon degrading gene by utilizing triparental pairing conjugation and single carbon source of petroleum hydrocarbon
  • Method for screening self-transmissible broad host range plasmid carrying petroleum hydrocarbon degrading gene by utilizing triparental pairing conjugation and single carbon source of petroleum hydrocarbon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The surface layer (0-20cm) of paddy field soil (0-20cm) in the Shenfu irrigation area irrigated with petroleum sewage for more than 50 years was collected, passed through a 1mm sieve, and stored in a refrigerator at 4°C. Weigh 5g (dry weight) of the soil sample and add it to a 100ml Erlenmeyer flask filled with 25ml of normal saline (0.85% NaCl solution), shake it on a shaker for 1 hour; let the suspension stand for about 30min, and pour the supernatant into a 30ml sterile Centrifuge at 10,000rpm at 4°C for 10min in a centrifuge tube; resuspend the pellet in 3ml 1 / 10LB liquid medium, and vortex thoroughly.

[0056] Select a suitable combination of donor bacteria and recipient bacteria to ensure that the two have different genetic selection markers, and preferably come from different Proteobacteria subclasses. The donor bacteria and recipient strains frozen in glycerol tubes were taken out from the -70°C refrigerator, streaked on the LB solid plate containing the corresp...

Embodiment 2

[0061] Scrape the control group grown on the LB plate in Example 1 and the bacterial lawn of the matching group, resuspend the culture in 2ml sterile physiological saline, and dilute to 10% under sterile conditions with sterile physiological saline. -4 , take 0.1ml to dilute to 10 -3 -10 -4 The control group was coated with LB solid plates containing corresponding antibiotic selection markers, and 0.1ml was diluted to 10 0 -10 -1 The paired group was coated with LB solid plates containing corresponding double antibiotics (antibiotic resistance of recipient bacteria and antibiotic resistance of donor bacteria plasmid) markers, placed in a constant temperature incubator at 30°C, and cultured overnight.

[0062] Pick a single colony of the zygote, insert it into 5ml of LB liquid medium containing the corresponding antibiotic, and cultivate overnight on a shaker at 30°C. SDS-alkaline hydrolysis was used to extract the plasmids of each zygote, and the 1% agarose gel electrophore...

Embodiment 3

[0064] Select zygote strains containing different plasmids in Example 2, inoculate in LB liquid medium, and cultivate to OD 600 =0.6-0.8, according to the mass percentage 10% inoculum size access to a certain petroleum hydrocarbon components (n-octane, benzene, toluene) and its main metabolic intermediates (salicylic acid, catechol) as the only substrate In the liquid inorganic salt medium of carbon source, place it on a 30° C. constant temperature shaker at 180 rpm for 5-10 days (cultivation time depends on different petroleum hydrocarbon components). Observe whether the strain containing the zygote can grow with these petroleum hydrocarbon components (measure the OD600 value of the cell density). Preparation method of liquid inorganic salt medium with petroleum hydrocarbon as the only carbon source: take 5g / L petroleum hydrocarbon component mother liquor (prepared with acetone or water), sterilize with 0.22μm filter membrane, measure 50-200μl mother liquor, add 50ml to steri...

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Abstract

The invention relates to an intersecting field of biotechnology and bioremediation technology of polluted soil and specifically provides a method for screening self-transmissible broad host range plasmids carrying a petroleum hydrocarbon degrading gene by utilizing triparental pairing conjugation and a single carbon source of petroleum hydrocarbon. Different Proteobacteria subclass receptor bacteria with selected marker on chromosome, a donor bacterium carrying non-self-transmissible (Tra-Mob+) plasmid with selected marker and a sample are treated with triparental pairing conjugation; plasmids of the donor bacterium enter receptor bacteria with assistance of self-transmissible (Tra+Mob+) broad host range plasmids in the sample; zygosperms are screened through receptor bacteria and selected markers carried by the donor bacterium plasmids; the screened zygosperms containing self-transmissible broad host range plasmids are cultured in a medium using the single carbon source of petroleum hydrocarbon; and broad host range plasmids capable of degrading petroleum hydrocarbon are screened out. The triparental pairing conjugation provided by the invention can directly separate broad host range plasmids capable of degrading normal octane, polycyclic aromatic hydrocarbon naphthalene, fluorenes and phenanthrene from petroleum polluted soil, so as to provide heredity material for furtherly employing the plasmids to carry out bioremediation on polluted soil.

Description

technical field [0001] The invention relates to the interdisciplinary field of biological technology and contaminated soil bioremediation technology, specifically a method for screening self-transferable host plasmids carrying petroleum hydrocarbon degradation genes by using triparental mating and using petroleum hydrocarbons as the only carbon source. 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 chemical substance for 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 hydr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李慧王亚菲张颖徐慧韩斯琴史荣久杨伟超陈冠雄
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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