Metallothionein transgenic yeast construction and heavy metal adsorbing material preparation method by utilizing metallothionein transgenic yeast

A technology of metallothionein and biosorption, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of high operating costs, high energy consumption, and serious secondary pollution, and achieve heavy metal tolerance Improvement, good adsorption capacity, and broad application prospects

Inactive Publication Date: 2013-07-17
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the disadvantages of high operating cost, high energy consumption, and serious secondary pollution in the existing physical and chemica

Method used

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  • Metallothionein transgenic yeast construction and heavy metal adsorbing material preparation method by utilizing metallothionein transgenic yeast
  • Metallothionein transgenic yeast construction and heavy metal adsorbing material preparation method by utilizing metallothionein transgenic yeast
  • Metallothionein transgenic yeast construction and heavy metal adsorbing material preparation method by utilizing metallothionein transgenic yeast

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Construction of Metallothionein Multi-copy Integrated Expression Vector

[0021] The source of the nucleotide sequence used in the present invention is as follows: the Saccharomyces cerevisiae metallothionein gene (cup1) sequence is derived from S.cerevisiae288c, and the electrophoresis results of PCR cloned products are as follows: figure 1 ; 3-phosphoglycerate kinase gene (PGK1) promoter was derived from S. cerevisiae288c; the homology arm rDNA gene sequence was derived from S. cerevisiae288c; plasmid pFA6a was donated by Mr. Chen Guoqiang from Tsinghua University.

[0022] 1. Saccharomyces cerevisiae Total DNA Extraction

[0023] 1) Cultivation and harvesting of bacteria: Pick a single colony from a yeast plate cultured at 30°C for 2 days, insert 10mL of YPD liquid medium, culture at 30°C for 16 hours, and centrifuge at 12000r / min for 5min to collect bacteria .

[0024] 2) Lysis of the cells: Wash the cells twice with sterile water, add 500 μL of lysate t...

Embodiment 2

[0057] Example 2 The acquisition of metallothionein transgenic yeast S.cerevisiae4126

[0058] The present invention uses the method of electric shock transformation to transfer the target vector into S.cerevisiae4126 cells, and the specific operation method is as follows:

[0059] 1. Preparation of yeast competent cells

[0060] 1) Inoculate S. cerevisiae4126 cryopreserved in the laboratory into YPD medium, and activate it in a shaker at 30°C and 150r / min for 16h.

[0061] 2) Take out the yeast culture solution and put it in an ice bath for 15 minutes to stop the cell growth, then transfer the bacterial solution to two sterile 50mL centrifuge tubes, centrifuge at 4°C for 5 minutes, and carefully discard the supernatant.

[0062] 3) Place the centrifuge tubes containing the cells on ice, and add 35 mL of pre-cooled sterile 1M sorbitol to suspend the cells therein. Centrifuge at 4°C for 5 min and carefully discard the supernatant. Repeat this operation.

[0063] 4) The cell...

Embodiment 3

[0073] Example 3 Tolerance test and adsorption capacity test of heavy metal biosorbent materials prepared by using metallothionein transgenic yeast in the present invention to heavy metals

[0074] 1. Heavy metal tolerance test

[0075]In the present invention, the constructed metallothionein transgenic yeast S.cerevisiae4126-cup1 and the starting bacteria S.cerevisiae4126 are cultured overnight in the same YPD medium, and then inoculated into 100mL fresh medium according to the ratio of 1:100 and cultivated until the OD600 is 1.5 . Configure YPD plates containing Cu(II) (copper sulfate), Cr(VI) (potassium dichromate), and Cd(II) (cadmium chloride) respectively, and set 2 concentration gradients for each heavy metal. Then, the bacterial solution was diluted step by step with a 10-fold gradient, and 2.5 μL of different concentrations of the bacterial solution were sucked up with a pipette gun, and then spotted on the plate containing heavy metals. Set the same plate without ad...

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Abstract

The invention belongs to the environmental engineering heavy metal pollution regulation field, and relates to metallothionein transgenic yeast construction and a heavy metal adsorbing material preparation method by utilizing the metallothionein transgenic yeast. According to the charactristics that rDNA has 100 to 200 repetitive units in a yeast cell, the rDNA is taken as a multi-copy integrated expressive vector of a consanguinity integrated site to establish yeast metallothionein, and an S.cerevisiae288c3 phosphoglyceric acid kinase gene (PGK1) promoter is utilized by the rDNA in S.cerevisiae4126 to express, so that yeast metallothionein multi-copy expression can be realized, the metallothionein transgenic yeast with high tolerance and adsorption to heavy metal is obtained, and the economic and safe heavy metal adsorbing material preparation method by utilizing the metallothionein transgenic yeast is provided.

Description

technical field [0001] The invention belongs to the field of heavy metal pollution control in environmental engineering, and relates to a transgenic yeast transformed by metallothionein and a method for preparing a heavy metal biosorbent. Background technique [0002] Heavy metal pollution is one of the most serious environmental problems in the world today. Heavy metal pollution is mainly caused by the development of modern industry and agriculture and some bad treatment methods of human beings. It poses a serious threat to the ecological environment, public health and human health. Unlike other pollution, heavy metal pollution cannot be biodegraded, thus making the treatment of heavy metal pollution more difficult. At present, the treatment of heavy metal pollution mainly uses physical or chemical methods, including chemical precipitation, ion exchange, membrane separation, reverse osmosis, etc. However, these methods have problems such as high energy consumption, high op...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C02F1/58C12R1/865C02F101/20
Inventor 陈丽杰王晓珊袁文杰
Owner DALIAN UNIV OF TECH
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