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Nicotine selectively degrading bacterium construction method and application in waste tobacco water extract of nicotine selectively degrading bacteria

A construction method, selective technology, applied in the field of metabolic engineering

Active Publication Date: 2018-09-14
ZHEJIANG UNIV OF TECH
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Problems solved by technology

So far, there have been no reports on the selective degradation of pollutants when glucose and specific pollutants (such as nicotine) coexist at home and abroad.

Method used

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  • Nicotine selectively degrading bacterium construction method and application in waste tobacco water extract of nicotine selectively degrading bacteria
  • Nicotine selectively degrading bacterium construction method and application in waste tobacco water extract of nicotine selectively degrading bacteria
  • Nicotine selectively degrading bacterium construction method and application in waste tobacco water extract of nicotine selectively degrading bacteria

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Embodiment Construction

[0027] Real-time fluorescent quantitative polymerase chain reaction (RT-PCR): This experiment is based on the whole genome information of Pseudomonas sp.JY-Q, and analyzes the pathways related to glucose metabolism through the existing gene annotation information. We focus on five Glucose metabolism initiation enzyme genes, including one glucokinase gene (AA098_22370) and four glucose dehydrogenase genes (AA098_12490, AA098_22860, AA098_11910 and AA098_05800). Glucose was added as the only carbon source in Basic Mineral Salt Medium (BSM) to cultivate Pseudomonas JY-Q to the logarithmic growth phase, and the total RNA was extracted for fluorescence quantitative experiments. The results are as follows figure 1 shown.

[0028] From figure 1 It shows that all five genes are expressed, and the expression level of gdh-22860 (AA098_22860) gene is the highest, which is about 3 times that of gdh-12490 gene. Followed by gdh-05800 gene, gdh-11910 gene and gck-22370 gene expression leve...

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Abstract

The invention discloses a nicotine selectively degrading bacterium construction method and application of nicotine selectively degrading bacteria in a waste tobacco water extract. Pseudomonas sp.JY-Qis used as an original bacterial strain, through whole genome information analysis of the Pseudomonas sp.JY-Q, the following five target genes are determined, knockout vectors of the target genes areconstructed, by means of a thermal transferring method, the knockout vectors are transferred into an escherichia coli deleted bacterial strain WM3064, and by means of Kanamycin resistance genes on thevectors, recons of first exchange are screened out; after the bacterial strain is subjected to overnight propagation, by means of anti-screen marker SacB genes carried by the vectors, recons, namelythe target genes, of secondary exchange fall off along with plasmids which are integrated into a genome to achieve seamless gene knockout until all the target genes are knocked out. The phenotypic difference between the bacterial strain obtained through the method and a wild type JY-Q mainly comprises growth difference and nicotine and glucose degrading difference.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering. By constructing a Pseudomonas sp. JY-Q (Pseudomonas sp. JY-Q) gene-deleted strain, it can be selected in a coexistence environment such as nicotine and glucose (such as waste tobacco leaf water extract, TWE). Sexual degradation of nicotine. Background technique [0002] Nicotine, also known as nicotine, is composed of three elements C, H and N. It is a nitrogen-containing heterocyclic organic compound with a molecular weight of 162.23Da and a molecular formula of C 10 h 14 N 2 , chemically named 1-methyl-2-(3-pyridyl)pyrrolidine. At room temperature, nicotine is a light yellow oily liquid, which is easily oxidized to dark gray when exposed to light for a long time. It has a strong spicy taste, bitter taste, volatile and highly corrosive. Under normal temperature and pressure, nicotine has a density of 1.01g / mL, a boiling point of 247°C, and a melting point of -79°C. It is easily...

Claims

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Application Information

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IPC IPC(8): C12N15/78C02F3/34C02F101/38
CPCC02F3/34C02F2101/38C12N15/78
Inventor 钟卫鸿张辉李骏金维华黄海婵
Owner ZHEJIANG UNIV OF TECH
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