Team:GDSYZX/Design

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Design

Pcs1

Pcs1 is a small molecule peptide that is synthesized in plants by heavy metal ions’ induction. It has the ability to chelation the heavy mental and has the heavy metal detoxification effect. Pcs can complex with heavy metal ions to form non-toxic compounds and reduce the concentration of heavy metal ions in cells when plants absorb excess of heavy metal. Pcs has the strongest chelation ability for heavy metals, so it is important for the plants which containing large amounts of heavy metals to detoxification.

Compared the wild type and the optimum type Pcs1

We used PCR to amplify PCS1, afterwards，we inserted the PCS1 gene through enzyme digestion and linking to the AOX1 fragment of pPIC9K.Then pPIC9K was screened by colony PCR. After successful sequencing, the cloned strains were cultured at 37 degrees Celsius in E. coliDH5α.Then extracted plasmid was transformed into the prepared yeast GS115 receptor and the protein was expressed at 28 degree Celsius. At the same time, the tolerance test of unmodified yeast was carried out to test the growth under which concentration was greatly inhibited. In addition, the yeast transformed with PCS1 was subjected to the tolerance test to test whether the yeast tolerance improved after the modification.

Prokaryotic expression of Pcs1

The Pcs1 gene was inserted into pEASY-E1 through enzyme digestion and ligation. After screening by the PCR of the colony, the sequence was successful and the cloned strain was expanded at 37 degrees Celsius for the expansion culture of E. coli DH5α. Plasmids were extracted, transformed into prepared E. coli BL21 and expressed the protein at 37 degree Celsius.

In the end, we compared heavy metal chelating capacity, tolerance of yeast GS115 and E. coli BL21. The product with strong chelating capacity and good tolerance was selected.

Something about Hma2

Hma2 was first amplified by PCR ，and then inserted into pPICZ after the enzyme digestion and ligation. After successful screening, the cloned strain was cultured at 37 degrees Celsius in DH5α. Plasmids were extracted, transformed into the yeast that we have transformed Pcs1 into it, and cultured to express protein at 28 degrees Celsius. Then finally screened the superior.