Discipline: Biological Sciences
Elizabeth Umanah - University of Georgia
Co-Author(s): Dr. Hung-Yueh Yeh , U.S. Department of Agriculture - Poultry Microbiological Safety and Processing Research Unit - Athens, GA
Campylobacter jejuni is a flagellated, Gram-negative spiral-rod bacterium that is the leading biological agent of acute bacterial gastroenteritis (food poisoning) in humans worldwide. Human infection from this microorganism has been linked to the handling and consumption of poultry, in which this microorganism is a commensal in the gut. The overall goal of the research within this lab is to develop an array of recombinant Campylobacter vaccines from gene isolates from the bacterium’s genome in order to combat its proliferation. This was done by first isolating and amplifying 35 campylobacter flagellar genes. The amplified genes were then ligated into pEtite vector plasmids, which were then transformed into Hi-Control 10G chemically competent E. coli cells for cloning. After the competent cells were cultured, the plasmids were then extracted and again transformed, but into BL21 E. coli cells in order to have the isolated gene’s protein expressed. When these cells were grown and cultured, each sample of the cells were then divided into 2 samples in order to begin the screening process for protein expression. One sample had nothing added to it and remained uninduced while the other had IPTG added to it for induction. All of the samples were screened for expression using SDS-PAGE and the positive induced samples along with their uninduced counterparts were then selected to have the protein production scaled up. After that, the proteins were then isolated and then stored. With those proteins an immunoblot analysis will be taken to assess their responses to convalescent sera. The proteins that show a response will again be scaled up and then administered to chickens to examine if any of them provide protection from the Campylobacter bacterium. Results from this study will provide valuable data on which C. jejuni genes code for proteins that play a role in causing food poisoning.Not Submitted
Funder Acknowledgement(s): The National Science Foundation; Louis Stokes Alliances for Minority Participation
Faculty Advisor: Dr. Hung-Yueh Yeh, HungYueh.Yeh@ARS.USDA.GOV
Role: Amplified the Campylobacter gene isolates using PCR; Ligation of amplified genes onto pEtite vector plasmids; Transformation of the pEtite vector plasmids into Bl21 and 10G chemically competent E. coli cells; Performed the protein expression screening process using IPTG and SDS-PAGE; Scaled up protein production of the samples that showed to have protein expression.