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Yeast strain for displaying metallothionein on cell surface and application of same in heavy metal adsorption

A metallothionein, cell surface technology, applied in the preparation of heavy metal bioadsorption materials, in the field of transgenic yeast, can solve problems such as no obvious effect, and achieve the effects of good fermentation performance, cost saving, and efficiency improvement

Inactive Publication Date: 2015-07-15
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Kuroda, etc. [1] The constructed recombinant strain is only for Cd 2+ The adsorption capacity is improved, but for Cu 2+ and Cr have no apparent effect

Method used

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  • Yeast strain for displaying metallothionein on cell surface and application of same in heavy metal adsorption
  • Yeast strain for displaying metallothionein on cell surface and application of same in heavy metal adsorption
  • Yeast strain for displaying metallothionein on cell surface and application of same in heavy metal adsorption

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of Metallothionein Cell Surface Display Integration Vector

[0057] 1. Saccharomyces cerevisiae S.cerevisiae 288c DNA extraction and PCR amplification of cup1 gene

[0058] Saccharomyces cerevisiae S.cerevisiae 288c was inoculated in YPD medium, cultured overnight, the cells were collected by centrifugation, and the genomic DNA of S.cerevisiae 288c yeast was extracted by glass bead method.

[0059] According to the metallothionein (NCBI Reference Sequence: NM_001179183.1) sequence, the upstream primer cup1-F and the downstream primer cup1-R were designed, and EcoRI and XhoI restriction sites were added to the 5' ends of the two primers (horizontal line part of the enzyme cutting site), using S. cerevisiae 288c genomic DNA as a template, using the PCR enzyme of Dalian Bao Biological Co., Ltd. to amplify the cup1 gene. PCR program: 94°C pre-denaturation for 5 min; 94°C for 1 min, 56.5°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0060]...

Embodiment 2

[0087] Example 2 The acquisition of metallothionein cell surface display yeast (S.cerevisiae HACg)

[0088] 1. Transformation of S.cerevisiae 4126 cells by electric shock method

[0089] (1) Take a ring of S. cerevisiae 4126 yeast from the slope and inoculate it in YPD medium, culture at 30°C and shake at 150 rpm for 18 hours;

[0090] (2) Insert 2% (v / v) of yeast cultured for 18 hours into 100 mL of YPD medium, and culture at 30°C and 150 rpm for 12 hours until the OD is about 1.0;

[0091] (3) Place the yeast culture solution on ice for 15 minutes to stop the growth of yeast cells, then transfer the bacterial solution to two sterile 50mL centrifuge tubes, and centrifuge at 3000rpm at 4°C for 5 minutes to collect the bacterial cells;

[0092] (4) Wash the bacteria twice with ice-precooled ultrapure water, and the centrifugation conditions are the same as above;

[0093] (5) Place the centrifuge tubes containing the bacteria on ice, add 40 mL of ice-precooled sterile 1 mol / L...

Embodiment 3

[0102] Embodiment 3 heavy metal tolerance experiment

[0103] Inoculate the constructed S.cerevisiae HACg and the starting strain S.cerevisiae 4126 into the liquid seed YPD seed medium from the slant, culture it in a shaking flask at 150 rpm at 30°C for 16-18 hours, and then inoculate it into the YPD seed medium with an inoculation amount of 1%. , for secondary activation, when OD 620 reach about 4, with 5% inoculum amount, were inoculated to a certain concentration of Cu 2+ 、Cr 6+ 、Cd 2+ In the YPD liquid medium, the shake flask was cultured for a certain period of time, and the sample was diluted 10 times to measure the OD value at 620nm.

[0104] The experimental results show that S.cerevisiae HACg is more sensitive to heavy metal Cr than the starting strain S.cerevisiae 4126 6+ 、Cu 2+ 、Cd 2+ The endurance has been significantly improved. Such as Figure 7 , S.cerevisiae 4126 contains 25mg / L Cr 6+ The maximum OD of growth in the medium 620 is 5.13, while S.cerevis...

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Abstract

The invention discloses a yeast strain for displaying metallothionein on a cell surface and application of the same in heavy metal adsorption. The yeast strain for displaying metallothionein on the cell surface is obtained by integrating a box gene for displaying metallothionein on the cell surface and a constitutive promoter P<PGK1> gene into the chromosome of a host stain via an integrative vector; and the yeast strain can continuously and stably express metallothionein on the cell surface. The recombinant Saccharomyces cerevisiae strain provided by the invention has good adsorption capability and tolerance to a plurality of heavy metals like Cd<2+>, Cu<2+> and Cr<6+>; e.g., the strain needs 12 h for complete reduction of Cr<6+> with a concentration of 50 mg / L, 6 h faster compared with an original strain, and has a reduction rate 1.5 times of the reduction rate of the original strain; the removal rates of the strain to Cu<2+>, total Cr and Cd<2+> are increased by 13.35%, 10.13% and 19.39% respectively compared with the original strain; so the strain has great application potential in the field of treatment of heavy metals.

Description

technical field [0001] The present invention relates to the field of genetic engineering technology and environmental engineering heavy metal pollution control, in particular to an integrated carrier capable of expressing metallothionein on the cell surface, a method for constructing the carrier, and a method for transforming the integrated carrier capable of expressing metallothionein on the cell surface The transgenic yeast and the application of the yeast in the preparation of heavy metal biosorption materials. Background technique [0002] Environmental pollution from heavy metals from industrial waste has become one of the most important environmental problems in the world today. Heavy metal pollutants mainly come from the electroplating industry, metallurgy industry, mining industry, etc. Heavy metal pollutants cannot be degraded into non-toxic substances through chemical or biological treatment. Not only can they exist permanently in the environment, but also heavy me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/31C12N1/19B01J20/24C12R1/865
Inventor 陈丽杰汤晓燕袁文杰秦配玲
Owner DALIAN UNIV OF TECH
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