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dimeric FYVE

August 5-10

We started to conduct experiments of the Prevention stage. We acquired the Mus musculus embryonic cDNA from the Institute of Life Science in our school and successfully extracted the FYVE domain. The cloning cycles during these days were to insert the monomeric FYVE into standard backbone pSB1C3. The results were successful.  So for the next step, we tried to construct the dimeric FYVE.

8/11-8/15

In these days, we did lots of paper research about transfection of plant cells. We contacted an investigator of Academia Sinica and discussed about the experimental design of our project. We decided to use pSAT6, which was commonly used for transfection of plant cells, as our vector. We were also granted permission to conduct the experiment in the lab especially for plant transfection in Academia Sinica.

8/16-8/22

This week, we continued to construct the dimeric FYVE on pSB1C3. We constructed the dimeric FYVE with and without stop codon on the pSB1C3 plasmid. After constructing the dimeric FYVE without stop codon, we put the green fluorescence protein after it to create a fusion protein. We also managed to digest pSAT6, and successfully isolated the backbone for ligation. However, we failed to extract the dimeric form of FYVE from pSB1C3, thus we did many trials for troubleshooting and discovered flaws in our primer design.

8/23-8/29

We had lots of discussion this week and designed new primers to extract the dimeric FYVE from pSB1C3. To obtain a more accurate result, we run KOD PCR to proofread the sequence we want, and finally extracted the dimeric FYVE. We ligated dimeric FYVE and pSAT6 afterwards, in anticipation to complete our construct, which would later be transfected into tobacco BY-2 protoplasts.

8/30-9/5

It was confirmed that dimeric FYVE had successfully ligated with pSAT6. We also extracted the recombinant plasmids with the monomeric and dimeric form of FYVE for part submission and for making long-term stocks. We focused on contacting the research lab of Academia Sinica for transfection techniques and searching for the further experimental application of the PI3P inhibitor, Wortmannin.

9/6-9/12

We went to institution of Academia Sinica to do the transfection experiment. We failed to yield tobacco BY2 protoplast at first because the number of the plant cells used was not enough for transfection. After incubating for another day, we cultivated the BY-2 protoplast and transfected the construct into the cell. We also learned how to manipulate the fluorescence microscope. After the observation for a whole day, we got some great experimental results and wrapped up the prevention part!