Major
Biological Sciences
Advisor
Dr. Kathryn Ramsey
Advisor Department
Biomedical and Pharmaceutical Sciences
Date
5-2022
Keywords
Pathogenic Bacteria; Antibiotic Resistance; Genetics
Abstract
Staphylococcus aureus is a Gram-positive pathogenic bacterium commonly known for being the causative organism of staph infections in clinical settings. In previous literature, it has been observed that mutations in certain genes in S. aureus can result in resistance to commonly used antibiotics. Specifically, mutations in the gene rpsU, encoding the ribosomal protein bS21, have been present in strains of S. aureus resistant to the antibiotic vancomycin. The aim of my project is to validate these findings by creating a strain of S. aureus without bS21 and performing an antibiotic assay to determine the level of resistance to vancomycin in this strain. To do this, I have been working to create a plasmid that can be introduced into wild-type S. aureus and knock out the gene rpsU via homologous recombination. To create the desired plasmid, I am modifying the pIMAY-Z plasmid which contains the gene for chloramphenicol resistance; I began by amplifying two desired sequences (also referred to as flanking regions) via PCR that will be inserted into the plasmid. I then performed a restriction enzyme digest to cut the plasmid at the insertion sites, along with cutting the amplified sequences at their ends so that they can be added to the plasmid. I continued by performing a ligation to join the flanking regions to the ends of the pIMAY-Z backbone and effectively “glue” the entire plasmid together. I used this ligation to transform chemically competent IMO8B E. coli cells on Luria-Bertani agar plates containing 25 ug/ml chloramphenicol, which amplified the desired plasmid for use in further steps. Although I have not confirmed whether this plasmid has been successfully produced through the digests, ligations, and transformations I have performed so far, the positive and negative controls have been successful, and I am now just waiting on official confirmation through sequencing.