Week 1

Hello everyone!

The big news of this week is that the nature of my project has changed. Originally, I was told that my project would involve the creation of a plasmid by combining the gene p21, a green fluorescent protein, and a destabilizing domain using a process called the gateway method. However, when looking over the lab supplies, it became apparent that we do not have the genes in a form that could be utilized for gateway. It would be possible to prepare the genes for the process, but this would take more time than I have in the lab. For these reasons, this project has been abandoned.

My new project involves the plasmid YFP-AID-E2F1, which was previously created in the lab. There are three genes of importance in this plasmid. E2F1 is a gene that is involved in proliferation and apoptosis in cells. When E2F1 is expressed in a cell for a short amount of time, it begins to divide rapidly. However, if E2F1 is expressed for a long period of time, the cell kills itself. The gene YFP is a fluorescent yellow protein that is attached to the gene E2F1, so the expression of E2F1 is visible. AID is a unique gene that regulates the expression of other genes. Normally, if AID is present, the cell expresses the genes that are after it normally. However, if an inducer is added, the expression of the genes following it on the plasmid is halted.

The goal of my experiment is to create a stable cell line of REF52 cells containing the gene YFP-AID-E2F1. The plasmid will be inserted the cells into the cells through a process known as NEON electroporation, and the cells will then be grown out from a 96 well plate to a single well plate. Once we have achieved a stable cell line, we will test the effect of the expression of E2F1 over different time frames, and in different patterns, in order to determine at what point the effect of E2F1 switches from causing proliferation to causing apoptosis.

I have not begun working on this project yet, because this week has been a training week. In order to familiarize myself with the procedures of the lab, I worked on amplifying the stock plasmids that we were running low on. This process involved five major steps. First, the cells were transformed into E. coli by heat shocking them, and they were allowed to grow out in tubes. The E. coli were then spread onto agar plates, where they formed colonies. Single colonies were picked and grown out again in tubes. The desired plasmids were isolated from the cells through a process known as miniprep. Finally, the concentration of the DNA was tested through NanoDrop.

Overall, it has been a very busy week, and I cannot wait to continue to work in the lab!