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A method for knocking out the catechol dioxygenase gene of the ibuprofenfoss plasmid

A technology of catechol dioxygenase and Foss plasmid, which is applied in the field of microbial degradation of ibuprofen, can solve the problems of inability to promote DNA strand insertion and self-exchange, excessive electric energy, etc.

Inactive Publication Date: 2015-07-29
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] Although material scientists, ecotoxicologists, environmental chemists, and environmental engineers have conducted studies on the release of ibuprofen from electrospinning; the leaching potential of ibuprofen in soil; the ozone degradation of ibuprofen and the Advanced oxidation processes (AOPs) such as photo-Fenton system degradation have been tried, but chemical degradation has its own shortcomings, because many oxidative degradation processes are a combination of oxidants and ultraviolet radiation (O 3 / H 2 o 2 / UV) or a combination of catalyst and UV irradiation (Fe 2+ / H 2 o 2 ;UV / TiO 2 ), its outstanding three disadvantages are: (1) the demand for excessive electric energy; (2) the input of a considerable amount of oxidant and catalyst; (3) the control of pH in the treatment process
The gene α product is a 5′-3′ exonuclease acting on linear ds DNA; the gene β product binds to ss DNA and stimulates the annealing of complementary strands, but cannot promote the insertion and self-exchange of DNA strands

Method used

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  • A method for knocking out the catechol dioxygenase gene of the ibuprofenfoss plasmid
  • A method for knocking out the catechol dioxygenase gene of the ibuprofenfoss plasmid
  • A method for knocking out the catechol dioxygenase gene of the ibuprofenfoss plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Confirmation of 3G7Tn5 mutants G9 and H5 cloned from the Fossmid library

[0081] (1) After using the MilliQ system to prepare pure water for the PCR reaction test, filter it again using a 0.2 μm sterile filter membrane for later use.

[0082] (2) The above sterile ddH 2 O was dispensed into eppendorf centrifuge tubes, sterilized under UV light for 10 min, and used as water for PCR reactions.

[0083] (3) Start PCR Master Mix buffers 10 μL, primer ipf23O F (5'-ATA ATC GCA CGG AAC TCG ACA CCT-3') and R (5'-TCG GTG CGA CCT TCA ATC ATG TCA-3') each 1 μL, make up dd H 2 0 to 20 μL. Add a small amount of single colony (3G7 Tn5 mutant G9 and 3G7 Tn5 mutant H5 respectively) to the PCR reaction tube, the PCR reaction tube without colonies was used as a negative control, and the PCR reaction tube added with EPI3003G7 single colony was used as a positive control.

[0084] (4) After the PCR reaction is over, add 5 μL of 1kb Marker and 10 μL of PCR reacti...

Embodiment 2

[0086] Electric shock transformation of embodiment two recombinant plasmid pKD46

[0087] First prepare the competent cells of the 4F6 strain, the specific operation is as follows:

[0088] (1) Transfer 600 μL of the overnight culture solution of the 4F6 strain to 5.4 mL of LB culture solution supplemented with chloramphenicol (see Table 1 for the amount of antibiotics added), and incubate at 37°C for 3 hours.

[0089] (2) Aliquot the above culture solution into 1.5mL centrifuge tubes, centrifuge at 14,000rpm for 1.5 minutes, discard the supernatant, and repeat the centrifugation twice.

[0090] (3) Wash the above cells 3 times with 1 mL of 300 mM frozen sucrose, and resuspend in 100 μL of frozen sucrose.

[0091] (4) Add 80 μL of competent cells and 1 μL of pKD46 plasmid to a frozen electroshock transformation cup (use an electroshock transformation cup with a width of 1 mm), select 1.8 kV electric shock, quickly add 600 μL of LB culture medium, and recover for 1...

Embodiment 3 G7

[0097] Electroporation Transformation of Linear DNA in Example 3 3G7Tn5 Mutant

[0098] (1) Use ipf23O F and R primers to amplify the linear DNA in the Tn5 mutants G9 and H5 of 3G7 respectively, and the PCR amplification steps are the same as in Example 1.

[0099] (2) Cut glue, Zymoclean TM Gel DNA Recovery Kit purified and recovered samples, and detected the linear DNA above on 1.0% (w / v) agarose gel.

[0100] Agarose gel detection of linear DNA in PCR-amplified 3G7Tn5 mutants G9 and H5 see figure 2 . The bands amplified from the three G9 templates and the four H5 templates were all between 2.0-3.0kb. and figure 1 Same result.

[0101] During the preparation of competent cells of the 4F6-pKD46 strain, 1000× copy number induction solution needs to be added and cultured for 1.5 hours, and the rest of the electroporation transformation method is the same as in Example 2, with 2 repetitions.

[0102] (3) The cells were spread on LB double-antibody plates su...

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Abstract

The invention relates to a method for knocking out relevant genes of a target bacterial strain by utilizing a red regrouping system to obtain a novel mutant. According to the method, Tn5 transposons of an ibuprofen degradation coloned strain 3G7 is inserted in mutant strains 3G7-G9 and 3G7-H5 is used as an original strain, wherein Tn5 insertion sites in the two mutants are arranged in a catechol 2,3-dioxygenase gene; the transposons Tn5 are electrically shocked and converted from a fosmid library positive coloned strain to another coloned strain 4F6 by virtue of the gamma-red homologous regrouping enzyme of pKD46 plasmid, and the catechol 2,3-dioxygenase gene in the 4F6 strain is knocked out to obtain the novel transposons mutants 4F6-G9 and 4F6-H5.

Description

technical field [0001] The invention belongs to the technical field of microbial degradation methods of ibuprofen, focusing on elucidating the microbial degradation mechanism of ibuprofen from the molecular level, and particularly relates to positioning and knocking out catechol-2,3 in the cloned strain of the ibuprofen Fuss plasmid library - Method for dioxygenase genes. Background technique [0002] Ibuprofen is a typical representative of phenylpropionic acid non-steroidal anti-inflammatory drugs (NSAIDs) and belongs to the important class of pharmaceuticals and personal care products (PPCPs). The mass-produced and widely-used ibuprofen has not only alleviated the pain of human beings, but also brought many environmental pollution hazards, and has become one of the important potential environmental pollutants. [0003] Although material scientists, ecotoxicologists, environmental chemists, and environmental engineers have conducted studies on the release of ibuprofen fro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 卫亚红安东尼.海程珂珂曲东马晓慧李震安卫星
Owner NORTHWEST A & F UNIV
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