Gene and its application in treatment of quinoline contaminant

A technology of gene and quinoline, which is applied in the application field of gene in the treatment of quinoline pollutants, can solve the problems that the research on quinoline's degradation characteristics and degradation methods is still in the theoretical stage, it is difficult to microbes, and it is difficult to complete.

Active Publication Date: 2011-10-05
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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  • Abstract
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Problems solved by technology

[0004] The degradation pathways of quinoline are mainly divided into aerobic metabolism and anaerobic metabolism. There are various degradation pathways, but the research on the degradation characteristics and pathways of quinoline is still at the theoretical stage.
Since quinoline itself is a refractory organic heterocyclic compound, it is difficult to find microorganisms that can effectively degrade it in nature. Therefore, cloning a gene that can stably and effectively degrade quinoline is a huge and difficult task.
At present, little is known about quinoline degradation genes, and it is impossible to provide sequence references and methods for reference, and it is impossible to predict quinoline degradation genes. It can only be screened and verified with a huge workload, which is actually very difficult to complete.

Method used

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  • Gene and its application in treatment of quinoline contaminant

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Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1, the acquisition of homoquinoline degradation gene purE1

[0023] 1. Establishment of a mutant library of the degrading bacteria Pseudomonas putida KT-ql-116 Tn5:

[0024] Pseudomonas putida KT-ql-116, which the inventor obtained from the activated sludge of Beijing Xiaojiahe Sewage Treatment Plant through primary screening and re-screening, etc., has high degradation activity to quinoline, and uses Tn5 transposition compound The homoquinoline degrading gene purE1 was cloned by constructing bacterial transposition mutant library using in vivo technology. Tn5 Transposome was EZ::TN purchased from Epicentre Tnp Transposome Kit.

[0025] 1. Preparation of degrading bacteria Pseudomonas putida KT-ql-116 electroporation competent

[0026] Pick a single colony of KT-ql-116 and inoculate it into 20ml LB, culture overnight. Inoculate 5ml of culture solution into 250mlL, shake at 210rpm, 30oC. Stop shaking the bacteria when the OD600 of the bacterial soluti...

Embodiment 2

[0042] Embodiment 2, pBBR1MCS-purE1 makes mutant strain E have high quinoline degradation activity

[0043] 1. Construction of pBBR1MCS-purE1 vector

[0044] The cloned purE1 was connected to the T-easy Blunt carrier. After the sequence was correct, it was digested with HindIII and XbaI, electrophoresed on 1% agarose gel, and the purE1 fragment was recovered for use. The broad host plasmid pBBR1MCS-5 was digested with HindIII and XbaI, and then electrophoresed on 1% agarose gel to recover the pBBR1MCS-5 vector fragment. Take 8 μl of purE1 fragment and 1 μl of treated pBBR1MCS-5, add 0.5 μl of ligase, 1 μl of buffer, and ligate overnight at 16oC. The ligation product was transformed into E.coli, colony PCR was used to detect single clones, and the correct clones were selected for sequencing, and the clones with correct sequencing were selected to obtain pBBR1MCS-purE1.

[0045] 2. pBBR1MCS-purE1 transformed mutant strain E

[0046] Mutant strain E is a Pseudomonas ...

Embodiment 3

[0050] Embodiment 3, Ep2 are to the biodegradation experiment of quinoline

[0051] We further studied the biodegradation experiment of quinoline in Ep2. The Ep2 strain was inoculated in MM2 liquid medium containing different concentrations of quinoline, the concentrations of quinoline were 500 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, 850 mg / L, respectively. L, 900mg / L, 25°C, 200rpm, shaking culture. As a result, it was found that, except in the case of 900mg / L, the bacterium did not grow, and the other culture media to be tested all proliferated to varying degrees. The bacteria grew for 40.5h, 64h, 124h, 142h, At 153h and 166h, the OD600 of the bacterial solution could reach 0.7~0.8, and the quinoline in these mediums was completely degraded. The above results show that Ep2 bacteria can grow with quinoline as the only carbon source, nitrogen source and energy source, and can well tolerate and degrade quinoline at a concentration of up to 850mg / L, and can prolifera...

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Abstract

The invention discloses a new gene purE1 which has high degradability on quinoline. Experiment shows that by using the purE1 gene to transform a microbe, the microbe can obtain degradation activity on quinoline, and the maximal degradation concentration of the degradation activity of the microbe provided by the purE1 gene and its coded protein is up to 850 mg/L. The invention has very important practical application value for the treatment of quinoline contaminants.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of a gene in the treatment of quinoline pollutants. Background technique [0002] Quinoline is a typical representative of nitrogen-containing heterocyclic compounds. It has a wide range of uses and is an important raw material and solvent in the production of various medicines, agricultural products, and dyes. However, quinoline and its derivatives are carcinogenic, teratogenic, and mutagenic, and are difficult to biodegrade, so they are serious pollutants in the environment. Quinolines in the environment come primarily from coal gas, fossil fuel processing, coal tar residues, and wood preservation facilities. In addition, quinoline and its derivatives are also refractory organic pollutant components in various industrial wastewater such as coking wastewater, petroleum wastewater, and pharmaceutical wastewater. Some scholars analyzed the organic c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/14C12N15/63C12N1/00C12R1/40
Inventor 夏勉徐海英乔琳
Owner BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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