Week 3

                This week in the lab we performed our first electroporation on the REF52 cells. Electroporation is a process for getting either linearized DNA or plasmids into cells. Bacterial cells naturally will take up plasmids through a process called transformation, but mammalian cells, such as the REF52 cells we are working with, are unable to do this. Therefore, in order to get our desired plasmid into the cells, we need to open up the cell membrane in such a way that the plasmid can diffuse across. This is done by shocking the cells with a high voltage. This causes the membrane to become porous, and the large DNA molecules are able to easily enter the cell.

                We will be performing two electroporations because the effectiveness of the electroporation can change when the cell is linearized in different ways. Linearized plasmids, or plasmids that are cut so that the DNA is in a straight line instead of a loop, are able to enter electroporated cells more easily than their circular counterparts. The plasmid that we are working with contains some extraneous information encoded into it, and smaller plasmids are able to diffuse into the cells easier than larger ones. We have performed two separate restriction enzyme digest on the plasmid, and each will be electroporated into the cell to see which one is more effective. One cut only uses the enzyme ApaLI, and merely opens up the plasmid without removing any information. The other cut uses both ApaLI and MfeI-HF. This cut removes an unnecessary drug resistance from the plasmid in order to decrease the number of base pairs in the plasmid.

Week 2

Since I previously posted, there has been another small change to my project. The goal of creating and testing a stable cell line is still the same, as are the procedures, but a different plasmid will be used. The new plasmid is YFP-ER-E2F1. Like the previous plasmid, it still has the yellow fluorescent protein and the gene of interest for this experiment, E2F1. However, the gene that controls the “on-demand” mechanism is now ER, an estrogen receptor. In an environment where estrogen is absent, the receptor will prevent E2F1 from entering the nucleus. Since E2F1 is a transcriptional factor (a gene that effects the expression of other genes), it is in a sense inactivated by being kept out in the cytoplasm. When estrogen is present, E2F1 will enter the nucleus and its effects can be observed in the cell.

This week, we received some confusing data while running our first restriction enzyme digest. The purpose of performing the digest was simple: we wanted to double check that the enzymes would make the right cuts before we made a large volume sample to use in NEON electroporation. When we ran the products of our digest on a gel, we saw four bands. This was peculiar, because when we put the sequence for the gel into a restriction enzyme digest calculator, the results indicated that we should have seen only two bands. Eventually, we managed to trace the extra bands back to the fact that one of our enzymes was very inefficient. It left enough uncut, supercoiled, and nicked DNA to cause extra bands to appear. After determining the cause of our problem, we have ordered a new enzyme, and plan to perform another test run with it on Monday.

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!