Research On The Prairies – Allissa Haney And BphAE

My name is Allissa Haney and my studies originate from Lake Superior State University in Sault Ste Marie Michigan (quite a trip!).  I will be graduating this spring with a Bachelor of Science degree in Forensic Chemistry as well as a minor in Biology.

The biggest decision of my undergraduate career has been choosing a direction in which to further my education. This past year I fell in love with both of my Biochemistry classes (yeah, I know that makes me sound like a total nerd) – so that should be easy enough right, biochemistry? Not quite, now I get to decide what area of biochemistry I want to work in. That is when this 10 week opportunity at North Dakota State University popped up and I decided lets dive in and see what graduate school is all about. The project I got assigned is perfect, biochemistry with an environmental push that fits perfect with my “save the world” streak.

Here at NDSU I work in Dr. Christopher Colbert’s lab on a protein that helps degrade PCBs. So we have all heard of PCBs but what are they exactly? Good question. They are polychlorinated biphenyls, which in simple terms mean they are a 2 ring structure with chlorines attached to them. These PCBs can come with differing degrees of chlorination (meaning some have more chlorine and some less). PCBs were widely used in everything from coolants to coating on electrical wiring starting in the 1920 until they were banned in the late 1970’s. They were banned because there was a link between PCBs and health issues in animals and possibly some cancers in humans. These PCBs entered the environment through use and disposal, and are considered persistent organic pollutants because their breakdown is such a slow process. The short of the story is that there are many places with PCB contaminated soils and a solution is needed.

This is where my project comes into play. There is an enzyme made up of three proteins that can start the degradation of PCBs. This is done by using electrons in the cell that get transferred through two proteins (BphG and F) to the third protein complex that carries out the actual degradation step(BphAE) by inserting two oxygen atoms into the ring structure. This first step must be done by this pathway that only a few bacteria contain. Once this first step is done other ring cleaving enzymes can come in and chop up the PCBs.

I am interested in the structure of BphAE so we can better understand and study how it operates. In order for us to do this I first have to make a lot of protein and this is done by tricking bacteria (Escherichia coli) cells into taking up DNA and producing protein as they replicate and grow. The E.coli cells are grown in a rich media for about 24 total hours then the cells are harvested so the protein can be extracted out of the cells. The cells are opened, which means the protein I am looking for as well as cellular protein and debris are also present in this mixture, so the protein must be purified to get only BphAE, my protein of interest.  My particular project is to go over procedures for this protein that have been done previously to be sure the science can be replicated with the same results.

Once pure protein is gathered we can then grow crystals of the protein in certain mixtures of solutions. Crystallization can be a long process that entails looking for the perfect mixture of solutions. This particular stage of the project is very time consuming because the right mixtures of solutions are crucial to get big enough crystals to be used later.

The crystals can then undergo further testing and the use of software helps show us the structure. From the structural data we can then get an idea of how this protein interacts with the other proteins in the enzyme or the substrate (PCB) it binds. Once it is understood how this protein works, we can then change items within its structure to make it perform on different compounds or perhaps make it degrade PCBs more effectively.

This program has been an excellent way for me to see how research is conducted at a bigger university. This research experience has given me direction in which to further my studies as well as get a sneak peek into the life of a graduate student. I am very glad I took this opportunity to step out of my comfort zone and expand my horizons. A big thanks to Dr. Erika Offerdahl for heading this program as well as all the mentors that took all us newbies in.

1 Response

  1. to see whether the oil is being degraded (broken down) or not. The design and implementation is completely up to your group, and the materials available to you will be those from all of the activities we have done thus far in the unit.

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