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Plasmid with tracing property and capable of degrading atrazine, gene engineering bacteria and application thereof

A technology of genetically engineered bacteria and atrazine, applied in the field of atrazine-degrading plasmids, engineering bacteria and degradation, can solve problems such as in-situ real-time detection difficulties, to prevent environmental ecological risks, promote formation and scale expansion , The effect of stable biological treatment efficiency

Inactive Publication Date: 2014-04-23
HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for specific strains or specific plasmids, it is still difficult to select the above-mentioned monitoring technology to achieve fast, simple and efficient in situ real-time detection

Method used

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  • Plasmid with tracing property and capable of degrading atrazine, gene engineering bacteria and application thereof
  • Plasmid with tracing property and capable of degrading atrazine, gene engineering bacteria and application thereof
  • Plasmid with tracing property and capable of degrading atrazine, gene engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 pUC18- atzA-gfp Construction of recombinant plasmids.

[0037] Such as figure 1 As shown, the present invention constructs a recombinant plasmid through plasmid pACYC184 and plasmid pUC18. Plasmid pACYC184 contains atzA gene and resistance to chloramphenicol, and its size is about 6500bp. The plasmid map is as follows figure 2 As shown, the gel electrophoresis Figure 4 ; Plasmid pUC18 contains the gfp gene, is resistant to ampicillin, and is about 2600bp in size. The plasmid map is as follows image 3 As shown, the gel electrophoresis Figure 6 shown. Both plasmids contain restriction endonuclease Eco88I restriction sites, therefore, the two plasmids were respectively digested with Eco88I to obtain linear atzA Fragment and linear pUC18- gfp Fragment, then use T 4 Ligase was used for connection, and recombinant plasmids were obtained after screening.

[0038] The electrophoresis pattern of restriction endonuclease Eco88Ⅰ after digestion of plasmid...

Embodiment 2

[0041] Example 2 The acquisition of genetically engineered bacteria

[0042] The recombinant plasmid pUC18- atzA-gfp Transformation into Escherichia coli DH5α to obtain genetically engineered bacteria, and carry out antibiotic resistance, gfp gene expression and atzA DNA Testing.

[0043] 1. Antibiotic resistance detection

[0044] The genetically engineered bacteria were cultured on the medium containing ampicillin and chloramphenicol respectively, and the results were as follows: Figure 9 As shown, only colonies appear on the medium containing ampicillin, but no colony grows on the medium containing chloramphenicol, indicating that the genetically engineered bacteria are only resistant to ampicillin, and have no resistance to chloramphenicol. The results were consistent with the antibiotic resistance of the recombinant plasmid pUC18-atzA-gfp.

[0045] 2. Green fluorescent protein gene ( gfp gene) expression detection

[0046] Cultivate the genetically engineered ba...

Embodiment 3

[0049] Example 3 Degradation of Atrazine

[0050] Under the same conditions, the genetically engineered bacteria obtained in Example 2 (containing the recombinant plasmid pUC18- atzA-gfp ) and the plasmid containing pACYC184- atzA The strain degrades atrazine, the experimental results are as follows Figure 13 shown. It can be seen that the genetically engineered bacteria and the original degradation plasmid (pACYC184) have the same ability to degrade atrazine.

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Abstract

The invention relates to the field of pesticide degradation, and in particular relates to a plasmid for degrading atrazine, gene engineering bacteria and a degradation method. A recombinant plasmid is obtained by inserting an atrazine degradation gene sequence and a green fluorescent protein nucleotide sequence into a plasmid pUC18. A recombinant plasmid pUC18-(i)atzA-gfp( / i) with functions of atrazine degradation, green fluorescent protein tracing and strong horizontal migration is established, and gene engineering bacterial containing the recombinant plasmid are used for performing biological enhancement on atrazine, so that the formation and scale expansion of aboriginal degradation bacteria can be promoted to ensure that the biological treatment effect of atrazine is stabilized around 90%; meanwhile, in situ monitoring can be performed on the recombinant plasmid in a treatment system and an environment to grasp the distribution and loss of the recombinant plasmid in the gene engineering bacteria and aboriginal degradation bacteria so as to facilitate evaluation and prevention of ecological risks of an environment.

Description

technical field [0001] The invention relates to the field of pesticide degradation, in particular to a plasmid for degrading atrazine, engineering bacteria and a degradation method. Background technique [0002] Bioaugmentation technology is considered to be a feasible and effective way to improve the biological removal efficiency of refractory pollutants. Using genetically engineered bacteria to implement bioaugmentation treatment is an effective means to improve the removal efficiency of refractory organic pollutants. Bioaugmentation of genetically engineered bacteria can efficiently remove refractory pollutants, accelerate the process of treatment start-up, improve the impact resistance of the treatment system, and enhance the stability of the microbial community structure and function. [0003] Atrazine is the most used herbicide in the world. Atrazine and its metabolites have certain biological toxicity to aquatic organisms and are a major environmental pollutant. Due...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21A62D3/02C12R1/19A62D101/04A62D101/26
Inventor 刘春刘平李曼杨景亮张静张磊
Owner HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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