Research On The Prairies – Jessa Justen And Beclin1

My name is Jessa Justen and I am 18 years old. I just graduated high school this past spring, and also received my associates degree from Normandale Community College in Bloomington, Minnesota. Three years ago, I never would have imagined myself being where I am now. Nobody in my family really has much of a background in science, and I went through much of my life with a dread for it. That is, until 10th grade biology, or more specifically, the genetics chapter in 10th grade biology. It was the first time in my life that I found myself enjoying science, and I began to pay more attention to all areas. Once I did, I found that I didn’t really hate science after all; I just didn’t pay attention long enough to realize how awesome it was! So starting my junior year of high school, I began PSEO (Post-Secondary Enrollment Option), which is where you take college courses for both high school and college credit. Luck for me, Normandale offered a genetics course, which I eagerly signed up for. It only took me a couple class periods to realize that this is what I want to do for the rest of my life. This fall I will be attending University of Wisconsin- Madison with genetics as my major, with the aspiration to become a geneticist. I decided it would probably be good to get some more lab experience, so I began searching for internships or lab jobs, and was forwarded an application for NDSU’s Research on the Prairies by one of my professors. I applied thinking I’d never get in, but I’ve never been more thrilled to be wrong.

I am working under Dr. Sangita Sinha in the Biochemistry department. She is studying autophagy, a cellular process through which the cell is able to degrade unnecessary/dysfunctional components through the lysosomes for energy. Structures called autophagosomes engulf and deliver the components to the lysosome for degradation. Autophagy is a repair mechanism, and the failure of autophagy is believed to play a large role in aging, neurogenetive diseases such as Alzheimer’s, and tumor growth. More specifically, she is looking at the way the proteins interact with each other to inhibit autophagy. This is where I come in. I am working with a single protein called Beclin 1. It interacts with five other proteins to nucleate autophagosomes. Autophagy will not proceed with a defective Beclin 1 protein. Currently, the underlying goal in the lab is to understand how the amino acid sequence and protein structure of

No Puncta = No Autophagy

Beclin 1 allows it to mediate autophagy. We are looking to see which amino acids within the Beclin 1 sequence are essential for the protein to function properly by introducing mutations. In order to do this, we note amino acids in the sequence that are highly conserved among all Eukaryotes and design primers with the desired mutation, and performing PCR to replicate the DNA. We then transform our plasmid into bacterial cells in order to grow them up for expression. We allow the bacteria to grow on agar plates and inoculate a single colony from each plate to conduct a miniprep. Minipreps are performed to purify the plasmid DNA prior to sequencing through the use of mini columns and collection tubes. We are then able to send our samples off for sequencing. Upon return of our samples, we use a sequencing alignment tool (at McLab) to compare them to the Wild Type or original sequence without the mutation. We look for sequences that are identical to the wild type, except for the codon differences consistent with the mutations we have inserted. When we confirm a successful mutant, we can move on to protein expression in mammalian cells to monitor how the mutation affects autophagy. In future work, cellular assays will be conducted in mammalian cells expressing mutant Beclin 1 as well as another autophagy protein tagged with Green Fluorescent Protein. If the Beclin 1 mutation does not prohibit the nucleation of autophagy, we can expect to see small puncta, or dots, of glowing green light, which represent the autophagosomes. This indicates that autophagy is being carried out in those regions. If the mutation causes autophagy to cease, we will not see any puncta.

Working with Dr. Sinha this summer has been a great learning experience. I’ve had the chance to try my hand at research, and have found that I enjoy it very much. It has reconfirmed my desire to go to grad school and receive my Ph.D, and maybe conduct some research of my own in the future.