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A polycyclic aromatic hydrocarbon degrading genetic engineering strain, its construction method and application

A technology of genetically engineered strains and polycyclic aromatic hydrocarbons, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of reduced inhibition, preferential utilization of impacts, and reduced efficiency of bioremediation

Active Publication Date: 2019-05-10
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The concentration of PAHs accumulated in the rain garden is 3-5 orders of magnitude lower than that of crude oil-contaminated soil. Due to the existence of a large amount of assimilable organic matter, it directly affects the preferential utilization of PAHs-specific degrading organisms for PAHs; at the same time, the inhibitory effect of PAHs on other microorganisms Decrease, making it difficult for specific degrading organisms to form a growth advantage, thereby reducing the efficiency of bioremediation

Method used

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  • A polycyclic aromatic hydrocarbon degrading genetic engineering strain, its construction method and application
  • A polycyclic aromatic hydrocarbon degrading genetic engineering strain, its construction method and application
  • A polycyclic aromatic hydrocarbon degrading genetic engineering strain, its construction method and application

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Embodiment 1

[0050] Example 1: Screening and cultivation of indigenous dominant strains

[0051] The soil of the rain garden and the control group that did not receive rainwater runoff from the road were collected, and the total DNA was extracted for high-throughput sequencing. According to the relative abundance of each bacterial genera, the top ten dominant bacterial genera in the rain garden soil were identified ( figure 1 : The distribution of the ten prokaryotic microbial genera with the highest relative abundance in the bioretention pond and the soil in the control group); Simultaneously screened and cultivated purebred strains with high colony abundance in soil samples, established by 16S rRNA sequence homology analysis its phylogenetic status. The pure strains belonging to the dominant genus were screened out as recipient bacteria. In the present embodiment, a Pseudomonas putida GLB3 strain Pseudomonas putida GLB3 is selected as the recipient bacteria (GenBank accession No.KT80456...

Embodiment 2

[0052] Embodiment 2: Construction of recombinant plasmid pUT-RHDGfp

[0053] Using the genomic DNA of Mycobacterium vanbaalenii PYR-1 as a template, using primers RHD-f1 and RHD-r1 as primers to amplify the terminator of the iron-sulfur protein β subunit encoded by the RHD gene and connect the 5' end of the Gfp gene17 The sequence of bases, the size of the amplified product is 2010bp; with pGFPuv (purchased from Clontech Company, Japan) plasmid as template, use primers Gfp-f2 and Gfp-r2 to amplify and remove the promoter of the green fluorescent protein encoded by Gfp gene and The sequence of 17 bases at the 3' end of the RHD gene is connected, and the size of the amplified product is 743bp; RHD-f1 and Gfp-r2 are used as primers, and the above two amplified fragments are used as templates to amplify to obtain overlapping target fragments—RHDGfp fusion Gene, the size of the amplified product is 2719bp ( image 3 : Overlap PCR amplified RHDGfp gene, wherein M: 5000bp DNA Marker...

Embodiment 3

[0057] Embodiment 3: the construction of genetic engineering bacterial strain Pseudomonas putida GLEB3

[0058] The fusion gene RHDGfp was recombined into the chromosome of Pseudomonas putida GLB3 by means of triparental combination. The strains required for the operation were as follows: recipient bacteria (Pseudomonas putida GLB3), auxiliary bacteria (E.coli DH5α(pRK2073)), donor bacteria (E. .coli DH5α(pUT-RHDGfp)). The mixing ratio of each strain is 1:2:1, the mixed system adopts 10mM MgSO4 solution, and the plate medium used should be LB plate medium containing kanamycin (25mg / L) and chloramphenicol (25mg / L). After culturing until the colonies were visible, pick the colonies, and use primers RHD-f1 and Gfp-r2 to carry out colony PCR verification of the RHDGfp gene. Validation results are positive ( Image 6 : PCR verification of triparental combination recombinant bacteria, wherein M: 5000bp DNA Marker, 1-3: RHDGfp gene fragment), show that RHDGfp gene has successfully ...

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Abstract

The invention relates to a polycyclic aromatic hydrocarbon degradation gene engineering strain, and a construction method and application thereof. The strain Pseudomonas putida GLEB3 is collected by China General Microbiological Culture Collection Center on September 19th, 2016; the collection number is CGMCC No.13014; and the collection address is Institute of Microbiology, Chinese Academy of Sciences, No.1 Yard 3, Beichenxi Road, Chaoyang District, Beijing City. The strain can enhance the PAHs (polycyclic aromatic hydrocarbons) degradation capacity of the indigenous dominant strain, and solves the problem that the exogenous PAHS degradation bacterium can not form the dominant population in the rainwater garden soil and can not easily display the degradation function.

Description

technical field [0001] The invention relates to a polycyclic aromatic hydrocarbon degrading genetic engineering strain, its construction method and application. Background technique [0002] In recent years, urban non-point source pollution caused by stormwater runoff has received more and more attention. In order to reduce the harm of pollutants in runoff to urban water bodies, rain gardens are commonly used at home and abroad to collect and purify rainwater runoff. However, during long-term operation, some refractory pollutants will be enriched inside the rain garden, aggravating the ecological risks inside and around it. Among such pollutants, Polycyclic Aromatic Hydrocarbons (PAHs) have received more attention because of their strong teratogenic, carcinogenic, and mutagenic effects. [0003] At present, there is no systematic research on the remediation of PAHs pollution in rain garden soil, and the traditional remediation method is generally carried out by inoculating...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/78C12N15/66C12N15/65B09C1/10C12Q1/02C12R1/38
CPCB09C1/10B09C2101/00C07K2319/60C12N9/0069C12N15/65C12N15/66C12N15/78C12Q1/02
Inventor 管运涛刘梁
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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