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Polycyclic aromatic hydrocarbon degradation gene engineering strain, and construction method and application thereof

A technology of genetically engineered strains and genetically engineered bacteria, applied in the field of polycyclic aromatic hydrocarbons degrading genetically engineered strains, can solve problems such as inability to guarantee lethality, and achieve the effects of reducing biosafety risks and improving controllability

Active Publication Date: 2017-05-31
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the function of this system will be affected by various factors such as the exposure of inducing substances and the expression of intracellular proteins in bacteria, and cannot guarantee a 100% lethal rate, this system is very important for artificially controlling the growth of GEB in the environment and preventing other diseases. Diffusion still has great application potential

Method used

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  • Polycyclic aromatic hydrocarbon degradation gene engineering strain, and construction method and application thereof
  • Polycyclic aromatic hydrocarbon degradation gene engineering strain, and construction method and application thereof
  • Polycyclic aromatic hydrocarbon degradation gene engineering strain, and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Fusion of lac promoter with nuclease gene nuc

[0045] Amplify the nuc fragment with primers nuc-f and nuc-r; PCR reaction system (50 μl): template DNA 1.0 μl; HS Premix (Bao Sheng Gong, Dalian) 25.0 μl; 50 μM primer 0.5 μl; ddH 2 O 23.0 μl; PCR reaction program: pre-denaturation at 94°C for 3 min; (pre-denaturation at 94°C for 30 s—annealing at 57°C for 5 s—extension at 72°C for 90 s)×30 cycles; extension at 72°C for 10 min. Amplification results see figure 1 ( figure 1 : PCR amplification of nuc gene, wherein M: 5000bp DNA Marker, 1: nuc gene fragment). Gel electrophoresis imaging showed specific amplification bands, and the fragment length was expected (801bp). Use Nde I and HindIII to digest the vector pUC-18 and the amplified product of the nuc fragment. After ligation, the plasmid pUC-nuc is transformed into E.coli DH5α to amplify and extract the plasmid, and then use it as a template to amplify the nuc fragment. Digest the visible pUC-18 vector a...

Embodiment 2

[0046] Example 2: Construction of recombinant transposon vector pUT-lacnuc

[0047] The lacnuc gene and the vector pUT / mini-Tn5Km were digested with Not I restriction endonuclease, and the lacnuc fragment was recovered after purification and ligated with pUT / mini-Tn5Km, and the ligated product was transformed into E.coli DH5αλpir. The positive clones on the pick plate were verified by colony PCR using primers lacnuc-f and lacnuc-r, and the verification results were positive (see Figure 4 : PCR verification of pUT-lacnuc vector, where M: 10000bp DNA Marker, 1: lacnuc gene fragment); at the same time, the extracted plasmid was digested with Not I, and the results of gel electrophoresis showed that there were two lines near 7.1kb and 1kb. strip (see Figure 5 : Enzyme digestion identification of pUT-lacnuc vector, where M: 5000bp DNA Marker, 1: Not I digestion product), corresponding to pUT / mini-Tn5Km plasmid and lacnuc gene respectively, proving that the pUT-lacnuc recombinant...

Embodiment 3

[0048] Embodiment 3: the construction of genetic engineering bacterial strain Pseudomonas putida GLEB3

[0049] The fusion gene lacnuc 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-lacnuc)). The mixing ratio of each strain is 1:2:1, and the mixed system uses 10mM MgSO 4 Solution, the plate medium used should be the LB plate medium containing kanamycin (25mg / L) and chloramphenicol (25mg / L). After culturing until the colonies were visible, the colonies were picked, and the colony PCR verification of the lacnuc gene was performed using primers lacnuc-f and lacnuc-r. Image 6 It shows that the lacnuc gene has been successfully transformed into the recipient bacterium Pseudomonas putida GLB3, and the recombinant bacterium is named Pseudomonas putida GL...

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Abstract

The invention relates to a polycyclic aromatic hydrocarbon degradation gene engineering strain. 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 controllability of the gene engineering strain can be effectively enhanced in the growth process, and the biosafety risk is lowered in the microbe-reinforced restoration process.

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 the past ten years, the research on the application of genetically engineered bacteria (GEB) for environmental restoration has been relatively slow. From a technical point of view, researchers have discovered a large number of genetic systems that can be used for bioremediation. Through genetic engineering, the relevant metabolic systems of microorganisms can greatly improve their degradation efficiency for pollutants. Therefore, GEBs often have good specific degradation ability. However, the factors that really limit the application of GEB in environmental restoration are often its unknown and uncontrollable ecological risks. Once the exogenous resistance genes carried by these strains enter other organisms through horizontal transfer, it may cause serious resistance gene pollution....

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12N15/66B09C1/10C12Q1/02C12R1/40
CPCB09C1/10B09C2101/00C12N9/22C12N15/66C12N15/78C12Q1/02
Inventor 管运涛刘梁
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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