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Ribotide sequence of 2-naphthanic acid degradation bacteria DNA segment and its preparation method and application

A technology of nucleotide sequence and naphthalene acid-degrading bacteria, which is applied in the field of nucleotide sequence of DNA fragments of 2-naphthoic acid-degrading bacteria, and can solve the problems that the effect has never been reported.

Inactive Publication Date: 2005-05-25
GUANGDONG INST OF MICROORGANISM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the study of microbial degradation of aromatic environmental pollutants, there are few reports on the metabolism of 2-naphthoic acid. Only in 1997, Birgit Morawski et al. reported that 2-naphthoic acid degrading bacteria (Burkholderia sp.JT1500) degraded 2-naphthoic acid Metabolic pathway, its specific gene and gene regulation mechanism have not been reported
The 2-naphthoic acid monooxygenase gene and the coenzyme A (CoA) ligase gene have never been reported, let alone their role in the degradation of 2-naphthoic acid

Method used

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  • Ribotide sequence of 2-naphthanic acid degradation bacteria DNA segment and its preparation method and application
  • Ribotide sequence of 2-naphthanic acid degradation bacteria DNA segment and its preparation method and application
  • Ribotide sequence of 2-naphthanic acid degradation bacteria DNA segment and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Extraction process of 2-naphthoic acid degrading bacteria total DNA

[0020] Inoculate 2-naphthoic acid-degrading bacteria (Burkholderia sp. JT1500) into 5ml LB medium, culture overnight at 30°C on a 140rpm shaker, centrifuge at 6000g for 10min, and collect the bacteria. Add 2.7ml DNA extraction buffer, fully suspend; add 20μl 20mg / ml proteinase K, shake at 200rpm in a 37°C water bath shaker for 30min, then add 0.3ml 20% SDS, gently invert several times to mix well, put it in a constant temperature water bath and let it stand , incubate at 65°C for 2 hours, during which time, mix up and down several times every 15-20min until clear, centrifuge at room temperature at 6000rpm for 10min, and transfer the supernatant to a new centrifuge tube. Add an equal volume of chloroform-isoamyl alcohol (24:1 v / v) for extraction twice, transfer the supernatant to a new centrifuge tube, add 0.6 times the volume of isopropanol, mix well, and let stand at room temperature for 1...

Embodiment 2

[0021] Example 2: Cloning process and sequencing

[0022] Take 10 μl of the above-mentioned total DNA solution, partially hydrolyze it with the restriction endonuclease EcoR I, and perform electrophoresis on agarose gel to obtain the digested fragments. Take 6 μl (5 μg) of enzymatically digested DNA fragments and 3 μl (2 μg) of EcoRI-digested pUC18 plasmid DNA, add 9ml TaKaRa DNA Ligation Kit Ver2.0 solution I to construct a ligation reaction system, and react at 16°C for 30 min; Competent cells of Escherichia coli HB101 prepared by calcium addition method. Transformants were plated on LB solid medium containing 75 μg / ml Amp (ampicillin). After culturing, several blue colonies grew on the transformant plate, and a recombinant strain capable of accumulating indigo in the transformant cells of Escherichia coli was obtained. Pick one of the single colonies for culture, extract the plasmid by alkaline lysis, hydrolyze the recombinant plasmid with restriction endonuclease, and co...

Embodiment 3

[0024] Embodiment 3: Verification test

[0025] (1) The oxygenation activity of the gene product was verified by the indigo generation experiment: indigo was dissolved in dimethylformamide (DMF), and at OD 610 There are specific absorption peaks. Observe OD 610 The amount of indigo produced can be calculated by the increase of light absorption value. The indigo molar absorptivity coefficient of the ultraviolet spectrophotometer is 17200 liters / mol.cm. In the M9 liquid medium containing 0.2% glucose (Amp 100mg / L), add indole 60umol / L as the substrate, and use the recombinant Escherichia coli to do the indigo production experiment. Take a certain amount of culture solution every 30 minutes, dissolve the indigo produced by oxygenase oxindole in DMF, and measure the OD 610 The absorbance value at the place can be used to measure the activity of oxygenase in recombinant cells. Experiments proved that the recombinant strains containing the above genes completely transformed mos...

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Abstract

A DNA fragment (8050 bp) for the 2-naphthalene acid degradating bacterium (Burkholderia sp. JT1500) and its preparing process are disclosed. Its prokaryotic expression plasmid is configured. Its recombinant bacterium strain containing said DNA fragment is obtained by transferring to cobacillus, which can effectively degradating 2-naphthalene acid and sodium benzoate, so degradating the environmental pollutants.

Description

Technical field [0001] The invention relates to a nucleotide sequence of a DNA fragment of a 2-naphthoic acid degrading bacterium, and relates to a preparation method of a recombinant plasmid containing the DNA fragment and a recombinant bacterial strain. Specifically, the present invention relates to a DNA fragment of 2-naphthoic acid degrading bacteria (Burkholderia sp. JT1500) having the activity of degrading 2-naphthoic acid, and this DNA fragment contains two key genes: 2-naphthoic acid monooxygenase gene and The coenzyme A (CoA) ligase gene relates to a recombinant plasmid containing the DNA fragment and a recombinant strain expressing the corresponding enzyme. Background technique [0002] Azoaromatic compounds occur widely in nature and are produced in millions of tons every year. Especially the widespread use of azo dyes in the printing and dyeing industry produces a large amount of printing and dyeing wastewater, which poses serious pollution and harm to the envir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N1/21C12N15/52C12P19/34
Inventor 孙国萍李小波郭俊方向平许玫英任随周曾国驱
Owner GUANGDONG INST OF MICROORGANISM
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