Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Construction and Application of the Transgenic Yeast Strain Expressing Metallothionein

Author LvPin
Tutor YiHuiLan
School Shanxi University
Course Cell Biology
Keywords Metallothionein Green fluorescent protein Transgenic yeast Bio-detection Heavy metal tolerance
Type Master's thesis
Year 2011
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With the rapid development of modern industry, water pollution becomes a serious problem. Heavy metals contamination is one kind of water pollution with significant harmful effects to human health. In the present study, we constructed three metallothionein (MT) recombinant expression vectors and transformed into yeast cells to further understand how transgenic yeast respond to environmental heavy metals.The metallothionein gene CUP1 promoter was amplified from S. cerevisiae genome and used to replace the AOX1 promoter on pPIC9K, thus the recombinant vector pCUP9K was constructed. The green fluorescent protein (GFP) gene was amplified from plasmid pYX112-GFP and inserted into vector pCUP9K to construct a recombinant expression vector pCUP9K-GFP. The AsMT2b gene from plasmid pGEM-T-AsMT2b and GFP gene from plasmid pYX112-GFP were cloned, modified and fused by gene splicing by overlap extension PCR, then the fusion gene was inserted into the vector pCUP9K or pPIC9K to get two recombinant expression vectors pCUP9K-AsMT2b-GFP and pPIC9K-AsMT2b-GFP.These recombinant expression vectors were linearized by restriction enzyme digestion and transformed into P. pastoris GS115, thus three yeast engineering stains GS115-1, GS115-2 and GS115-3 were constructed. The engineering yeast cells could emit green fluorescence under blue light through the fluorescence microscope. The fermentation supernatant showed the excitation wavelength at 475 nm and the emission wavelength at 508 nm. These results indicated that the engineering yeast cells could express GFP and secrete the GFP to extracellular.The fluorescence intensity of GS115-1 was measured when cells incubated with heavy metal ions (copper, chrome, cadmium and arsenic) respectively, and showed a positive response to the concentration of copper ion (0-1000μM), suggesting a novel copper ion detection method to be established. GS115-2 and GS115-3 was induced by copper ion and methanol, SDS-PAGE analysis of medium supernatants detected the protein AsMT2b-GFP, the result indicated that the engineering yeast cells could express foreign protein and secrete it to extracellular. The results of heavy metal tolerance analysis showed that GS115-2 and GS115-3 cells could growth and division in the medium existing certain heavy metals (copper, chrome and cadmium), but control cells can not, demonstrating that AsMT2b expression in engineering yeast cells increased the tolerance to environmental heavy metal ions.

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