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A recombinant plasmid, construction method, recombinant engineering bacteria and application for removing mercury pollution from industrial wastewater

A technology of recombining plasmids and industrial wastewater, which is applied in the field of genetic engineering, can solve problems such as human harm, and achieve the effect of improving mercury tolerance and increasing the accumulation of mercury in cells

Inactive Publication Date: 2016-01-20
舒海燕 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this is not the fundamental solution to the problem, because the zero-valent mercury volatilized into the air will re-enter the mercury cycle and continue to cause harm to the human body

Method used

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  • A recombinant plasmid, construction method, recombinant engineering bacteria and application for removing mercury pollution from industrial wastewater
  • A recombinant plasmid, construction method, recombinant engineering bacteria and application for removing mercury pollution from industrial wastewater
  • A recombinant plasmid, construction method, recombinant engineering bacteria and application for removing mercury pollution from industrial wastewater

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Experimental program
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Effect test

Embodiment 1

[0049] According to the plasmids pmr26 (GenBank accession number: D83080.2) and pmr28 (GenBank accession number: AB013925.1) of Pseudomonas K-62 strain, TAKARA Company was commissioned to synthesize the artificial operon merT-merP-merB1-merB2, using NdeI and Plasmid pET28a (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) was digested with NotI, and then recovered; the artificial operon merT-merP-merB1-merB2 was inserted into plasmid pET28a (purchased from From the region between NdeI and NotI of Shanghai Beinuo Biotechnology Co., Ltd., the specific operation process can be found in the In-FusionHDCloningKitUserManual (Clontech, Protocol number: PT5162-1). The ligation product was used to transform Escherichia coli DH5a, and to screen for kanamycin The resistant positive clones were cultured overnight, and the plasmids were extracted, and the plasmids whose results were identified as expected by restriction enzyme digestion and sequencing were named p1.

Embodiment 2

[0051] Enterobactera aerogenes (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) was cultured in ordinary LB medium, and the culture conditions were 37°C and 180rpm shaking culture for 72h.

[0052] According to the ppk gene sequence of Enterobactera aerogenes (GenBank accession number: D14445.1), primers were designed to extract the genomic DNA of Enterobacter aerogenes. For details, please refer to the instruction manual of the Genomic DNA Extraction Kit of Enterobactera erogenes (Cat. No.: DP302-02 ). Using Enterobacter aerogenes genomic DNA as a template, the ppk gene was amplified. Primers are as follows:

[0053] PPPKF: 5'-ATAAGAAT GCGGCCGC ATTTACCACGTCCTGTGATT-3' (the underlined NotI restriction site)

[0054] PPPKR: 5'-CCGATCCG CTCGAG GGTTAATCGGGTTGCTCGAG-3' (underlined is the XhoI restriction site)

[0055] PCR reaction system: 2×PfuMasterMix (Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0686A) 25μl, primer PPPKF1pmol, primer PPPKR1...

Embodiment 3

[0059] 根据烟草叶绿体基因组DNA序列(GenBank登录号:Z00044.2)和大肠杆菌T7噬菌体基因10的5'UTR序列(OlinsandRangwala.AnovelsequenceelementderivedfrombacteriophageT7mRNAactsasanenhanceroftranslationofthelacZgeneinEscherichiacoli.TheJournalofBiologicalChemistry,1989,264(29):16973-16976),委托TAKARA公司合成 primers,

[0060] P1F: 5'-CGA CATATG GCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCGAATACGAAGCGCTTGGATACAGTTGTAGGGAGGGATTTATCTTTTAACTTTAAGAAGGAG TGGCCA AGCGCTATTCGATC GAATTC GGAC-3' (The italic letters indicate the 5' end UTR sequence of E. coli T7 phage gene 10; the underline marks NdeI, BalI and EcoRI restriction sites)

[0061] P1R: 5'-GTCC GAATTC GATCGAATAGCGCT TGGCCA CTCCTTCTTAAAGTTAAAAGATAAATCCCTCCCTACAACTGTATCCAAGCGCTTCGTATTCGCCCGGAGTTCGCTCCCAGAAATATAGCCATCCCTGCCCCCTCACGTCAATCCCACGAGCCTCTTATCCATTCTCATTGAACGACGGCGGGGGAGC CATATG TCG-3' (the italic letters indicate the 5' UTR sequence of Escherichia coli T7 phage gene 10; the underline marks EcoRI, BalI ...

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Abstract

The invention discloses a recombinant plasmid for eliminating industrial wastewater mercury pollution, a construction method, a recombinant engineering bacterium and an application. The recombinant plasmid is established by inserting tobacco chloroplast rrn16 gene strong promoter, ribosome binding site conserved sequence of 5'UTR sequence of escherichia coli T7 phage gene 10, 3'-end non-coding area of tobacco chloroplast rps16 gene, enterobacter aerogenes polyphosphate kinase gene ppk and pseudomonas K-62 strain on the basis of plasmid pET28a and by eliminating mer operon of merA and merG. Organic mercury and inorganic mercury in wastewater are transferred into bacterial cells through merT-merP, the organic mercury is degraded into divalent mercury through merB1 and merB2, the divalent mercury is chelated into the cells through ppk, the toxicity of mercury to the bacteria cells is reduced, mercury in the wastewater is accumulated inside the bacterial cells, and mercury pollution in the waster is eliminated through collecting the recombinant engineering bacteria.

Description

technical field [0001] The invention relates to a recombinant plasmid used for removing mercury pollution from industrial wastewater, and also relates to a construction method of the recombinant plasmid, a recombinant engineering bacterium containing the recombinant plasmid and an application of the recombinant engineering bacterium, belonging to the technical field of genetic engineering. Background technique [0002] Soil is one of the main natural resources that human beings rely on for survival, and it is also an important part of the human ecological environment. With the intensification of industrial and urban pollution and the increase in the types and quantities of agricultural chemicals, soil heavy metal pollution is becoming more and more serious. Mercury is one of the major heavy metal pollutants. Heavy metal pollution in agricultural soil mainly comes from sewage irrigation. According to the national survey of sewage irrigation areas conducted by the Ministry o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N1/21C12N15/66C02F3/34C12R1/19
Inventor 舒海燕常胜合
Owner 舒海燕
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