Week 7

We have started the process called BioID. BioID exploits the protein biotin ligase. Biotin ligase biotinylates (adds biotin) other proteins based off of transient interactions. We can then selectively isolate proteins that have been biotinylated to see which proteins interact with proteins of interest. We can then take these isolated proteins, and run them through mass spectrometry to identify them. BioID is a decently long process, approximately 1 and a half weeks. For this week, we have started the cell culture and transfected the protein of interest. We have then taken the cells and scaled up, and treated with biotin and tetracycline (to activate the gene of interest). We then will harvest these cells and pellet them before we wash them next week.

Week 6

Canada! We have officially arrived at Mount Sinai Hospital to continue our project. We have started N2A cells to continue optimization with the help of a PhD student who has experience with N2A cells. We have decided to test whether poly-D-lysine helps with adherence of the cells or not. It does look like it is necessary to keep the N2A cells from flying off. We have also learned lab techniques like cloning, which is when you insert a gene of interest into a vector with restriction enzymes, an extremely useful skill for us in biochemistry/molecular biology labs. We have also seen the mass spectrometry room and their 8 mass spectrometry machines which is CRAZY, considering each one costed a fortune plus upkeep money on top of it all.

Week 5

Optimization has gone semi-well. We have seemed to reach a conclusion about RA concentration with 20 micromolar concentration being the most ideal. We have also realized that a major issue with transfection was due to the fact that we did not wait for the cells to become completely confluent. Confluency is when the cells are adhered to the bottom of the flask and are growing happily. If we do not wait for the cells to become confluent, and we have them undergo a toxic process like transfection, it makes sense if they do not grow to be happy. We have decided to add another day to the procedure.

Week 4

During this week, I attempted to optimize transfection. One of our plasmids did not seem to show under fluorescent microscopy, which meant either the plasmid was in the cell and wasn’t showing fluorescence, or the plasmid wasn’t even in the cell to begin with. Because the presence of fluorescence is how we detect the presence of a protein, we needed to make sure transfection was working properly. I attempted to try different amounts of the chemicals we use to transfect DNA into our cells to see which one had the best results. Transfection can be a toxic process for cells, so it was likely that we were using too much chemical or DNA.

Week 3

Optimization trials have been started. As of right now we are trying to optimize transfection and stimulation. We have seeded cells into wells to try and see the proper amount of time to allow the plasmids to transfect into the cells. We are also trying to optimize the amount of retinoic acid to put on cells to allow them to differentiate without dying or getting overly stressed. The results of these experiments will help will my overall experiment.