Discipline: Biological Sciences
Emily S. Gibson - Seward County Community College
Co-Author(s): Stephanie R. Shames PhD, Kansas State University, Manhattan, KS
Legionnaires’ disease, caused by Legionella pneumophila, is an intracellular, accidental human pathogen that naturally replicates in freshwater protozoa. Legionella pneumophila is acquired is from exposure to contaminated water sources, such as an air conditioning unit. For a person to be able to acquire this disease, they must be exposed to the contaminated water source itself, it is unable to be passed person to person. Those with weaker immune systems, are more likely to acquire this disease. This project studies the immune system. To do this we work with an effector called LegC4, and our control is green fluorescent protein (GFP). We know that LegC4 inhibits replication in a mammalian host infected by Legionella pneumophila. We are trying to see if we can use LegC4 to combat other pathogens or find out if it is specific to Legionella. We hypothesize that the delivery of LegC4 to macrophages will inhibit replication. During this project I worked to help purify our protein of interest. To begin the process of protein purification we start with a miniprep to isolate the plasmid DNA from the bacteria, using E. coli. Next, we performed a polymerase-chain reaction (PCR) to amplify the DNA. Then we ran an agarose gel to purify and find the band of DNA we needed. Once we had the band of DNA, we had to perform a DNA gel extraction to turn the gel back into a liquid. After we performed the digestions and ligations on the DNA, which is used to move a piece of double- stranded DNA from one plasmid to another, we performed overnights as well as a boil prep for protein expression the next day. Next, we needed to perform a coomassie stain and destain on a protein gel, as well as a western blot. These were used to identify if the proteins are present. Currently we have GFP purified, and are working towards getting LegC4 as a purified protein as well. Once we have the purified protein, the next stage in this project is to work on getting the protein into the macrophage. When transfected, the macrophages think the protein is a virus, and immediately begin to fight it off. Therefore, we need to trick the macrophage. We completed this task by attaching a well-known cell penetrating peptide (CPP) to help the protein get in. The GFP construct is used to make sure that the CPP works. The plan is to treat different populations of cells with each protein and determine if they can get in. If so, do they restrict the replication? We hypothesize that LegC4 would, but GFP would not. As of right now we only have expected results, as we are still in the process of purifying the protein. However, work has been done to get this the point that we are at, including cloning the construct and overexpressing it. We expect that once we are able to get the protein into the macrophage, that it will inhibit replication in the mammalian host.
Funder Acknowledgement(s): This work was supported by the National Science Foundation, Kansas Louis Stokes Alliance for Minority Participation 1305059 (KS-LASMP).
Faculty Advisor: Stephanie R. Shames, email@example.com
Role: I worked to help purify our protein of interest. To begin the process of protein purification I started with a miniprep to isolate the plasmid DNA from the bacteria, using E. coli. Next, I performed a polymerase-chain reaction (PCR) to amplify the DNA. Then I ran an agarose gel to purify and find the band of DNA we needed. Once we had the band of DNA, I then performed a DNA gel extraction to turn the gel back into a liquid. After I performed the digestions and ligations on the DNA I performed overnights as well as a boil prep for protein expression the next day. Next, I needed to perform a coomassie stain and destain on a protein gel, as well as a western blot.