Investigation of the Role of SNF4 and C4_00610W in the Process of Filamentation in Clinical Strains of C. albicans
Presenter Type
UNO Undergraduate Student
Major/Field of Study
Bioinformatics
Advisor Information
Associate Professor and Chair of Biology
Location
MBSC Ballroom Poster # 1006 - U
Presentation Type
Poster
Start Date
24-3-2023 1:00 PM
End Date
24-3-2023 2:15 PM
Abstract
Candida albicans is the most common fungal species in our microbiome but also is an opportunistic pathogen. It usually takes the form of yeast cells in our gastrointestinal system, but the ability to transition between yeast and filamentous cells is vital for systemic infection. C. albicans causes more than 20,000 systemic infections per year in the United States, 25-40% of which are fatal. This transition is dependent upon environmental factors that have specific gene regulators. In this study, we have mutated clinical strains of C. albicans, targeting 2 genes that are vital for filamentation in the type strain, SC5314. SNF4 encodes a protein-coding AMP-activated protein kinase regulatory subunit that activates the serine/threonine protein kinase Snf1. Snf1 functions as a trigger for filamentation. C4_00610W is a novel gene identified in previous assays involved in filamentation defects. Here we utilized the gene editing technology CRISPR-Cas9 to create homologous deletion mutants. First, we made the knockout construct using a combination of PCR and traditional cloning. Plasmid, pJK1354 was used in this process as a vector. We then synthesized the sgRNA and CAS9 DNA by each using overlap PCR and regular PCR. Prior to transformation, we verified the presence of the knockout construct using specific primers for each gene. We checked the PCR by running gel electrophoresis and observing the bands. Our results demonstrate that we have successfully completed creating the knockout construct. These constructs will be transformed into C. albicans, verified by colony PCR, and then phenotype-tested to determine their role in filamentation.
Scheduling
9:15-10:30 a.m., 1-2:15 p.m., 2:30 -3:45 p.m.
Investigation of the Role of SNF4 and C4_00610W in the Process of Filamentation in Clinical Strains of C. albicans
MBSC Ballroom Poster # 1006 - U
Candida albicans is the most common fungal species in our microbiome but also is an opportunistic pathogen. It usually takes the form of yeast cells in our gastrointestinal system, but the ability to transition between yeast and filamentous cells is vital for systemic infection. C. albicans causes more than 20,000 systemic infections per year in the United States, 25-40% of which are fatal. This transition is dependent upon environmental factors that have specific gene regulators. In this study, we have mutated clinical strains of C. albicans, targeting 2 genes that are vital for filamentation in the type strain, SC5314. SNF4 encodes a protein-coding AMP-activated protein kinase regulatory subunit that activates the serine/threonine protein kinase Snf1. Snf1 functions as a trigger for filamentation. C4_00610W is a novel gene identified in previous assays involved in filamentation defects. Here we utilized the gene editing technology CRISPR-Cas9 to create homologous deletion mutants. First, we made the knockout construct using a combination of PCR and traditional cloning. Plasmid, pJK1354 was used in this process as a vector. We then synthesized the sgRNA and CAS9 DNA by each using overlap PCR and regular PCR. Prior to transformation, we verified the presence of the knockout construct using specific primers for each gene. We checked the PCR by running gel electrophoresis and observing the bands. Our results demonstrate that we have successfully completed creating the knockout construct. These constructs will be transformed into C. albicans, verified by colony PCR, and then phenotype-tested to determine their role in filamentation.