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Project 1
Project 2
Image:Project overview
Another project is to engineer a Yeast Two-hybrid system aimed at Microcystins(MCs, induce liver cancer) detection in waters. The threat of MCs in seas, rivers, lakes and Reservoirs has become more and more severe worldwide due to eutrophication in pulltion waters. This MCs detection device takes the advatage of Gal4 promoter, which consists of two domains, one is AD(Activation domain), the other BD(Binding domain). When AD and BD are close enough to each other, the Report gene transcription LacZ willed trigued. We selected and modified two peptides that have specific interation with MCs, and engineered them into two vectors to construct a Yeast Two-hybrid system. In the presence of MCs at different concentrations, blue dots in different shades of colours could be seen with naked eyes. As we know, there are far more nitrogen and Phosphor elements in polluted waters, which is called eutrophication, resulting in algal blooms and red tides. Fishes, prowns and almost all the living creatures in these waters are killed by the high concentration of MCs that are produced by algea. It is also a severe threat to drinking water and water in daily use, for MCs induce liver cancer. What's more, the existed detecting technologies are complicated, expensive, time-consuming and require an expert to handle the devices.
Overall design
We designed MCs test papers, similar to PH test papers, they chage colours corresponding to concentrations of MCs in water. We put the same amount of genetically engineered yeast culture on a piece of paper or plass which are suggeste dto be available to everyone, low cost, portable and simple in detecting process. We put it in the water, after some time, if there are MCs, or the concentrations exceed a certain threshold, the yeast culture will turn blue. With higher concentration, the blue thicker.
two-hybrid system
- In a two-hybrid system, takes the advatage of Gal4 promoter, which consists of two domains, one is AD(Activation domain), the other BD(Binding domain). When AD and BD are close enough to each other, the Report gene transcription LacZ willed trigued. We selected and modified two peptides that have specific interation with MCs, and engineered them into two vectors to construct a Yeast Two-hybrid system.
- In our design, the two peptides we selected and modified are pp1 and GSH:
- MCs: In our experiment, we choose the most important and common type, MC-LR, which is a cyclic heptapeptide.
- PP1(Protein phosphatase type 1): it binds on the inner surface cell membrane in organisms. When a MCs moleclue across cell membrane and thus into a cell, it anchors its Adda at the binding sites of PP1, then its Mdha binds to PP1 through covalent bond, and thus inactivating PP1 in living cells.
- Glutathione: a tripeptide, can binds to the Mdha site of MCs, occupying it and thus detoxify.
So we take the advantage of their specific binding sites of high affinity and competitive interaction withMCs.
