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
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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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