Chemical VS. Genomic: Does Taxonomy Really Tell All?
Poster Number
17B
Research or Creativity Area
Natural Sciences
Abstract
Fungi are naturally found in all environments with over 600 species interacting daily with humans (Casadevall, American Society for Microbiology, 2017). Fungal natural products are a rich source of antimicrobial anticancer drugs (Jakubczyk, Molecules, 2020). The Carlson Library has isolated 108 unique aquatic fungal species based on observations on collection location, morphology, growth rate, and areas of spore coverage used to distinguish strains. We aim to characterize fungal species based on chemical identity and 18s rRNA sequencing to build a phylogenetic tree to understand the diversity captured by current isolation and culturing methods. Fungi are initially plated on Potato Dextrose Agarose (PDA) media plates and grown until they sporulate 7-10 days (Ezeonuegbu, Sci Rep, 2022). They are then cultured into liquid media and grown on a rotary shaker 110 rpm. Fungi were pelleted, DNA was extracted for PCR, and submitted for Sanger sequencing of the 18s rRNA region using standard primers ITS1 and ITS4. Representative strains from each phylogenetic branch will be grown for chemical characterization. Ethyl acetate is added to the flask to cover the media and hyphae. The ethyl acetate is then filtered and dried in vacuous. All extracts will be profiled by High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectroscopy (LCMS). The chemical fingerprints will be analyzed using the mPACT metabolomics platform to identify “chemical families” (Samples, 2023). Previously unknown metabolites, as identified by comparing to databases, will be prioritized for further characterization. This will determine if genetic or chemical families will be more robust for strain prioritization.
Purpose
Fungi, a group of organisms that include a vast and diverse role crucial to ecosystems worldwide. Beyond their mere presence, they play a pivotal role in active nutrient cycling, organic matter decomposition, symbiotic relationship with a multitude of organisms, and even the production of valuable compounds like antimicrobial anticancer drugs. Within Dr. Skylar Calson’s Laboratory at the University of the Pacific, an exploration of the diversity of fungi is focused on, with a particular focus on characterizing species from both saltwater and freshwater environments.
After careful isolation and characterization of fungal species based on their unique habitats, the students in the laboratory were also able to divide them according to appearance, spore morphology, and adaptability to varying environments. This approach not only unveils the diverse array of the fungal species, but also sets the stage for deeper investigations into their genetic makeup. Utilization of advanced molecular techniques, such as the 18S rRNA sequencing, will allow us to construct phylogenetic trees revealing the evolutionary relationships among these fungi.
Results
Liquid extraction was facilitated using ethyl acetate as the solvent followed by evaporation using a Rotary Evaporator. Evaporation of liquid, allowed for gaining an accurate mass of the sample which was then used to dilute the sample to one milligram over one milliliter. The sample was then rehydrated using high-performance liquid chromatography grade methanol (HPLC Methanol) and placed into HPLC vials. Thirty microliters of the HPLC mixture was placed in another HPLC vial to run a Liquid Chromatography Mass Spectroscopy (LCMS).
Both of these powerful analytic techniques will then be used to identify the compounds and separate the compounds that are present in the solution. This will answer the question and will characterize these species based on their fungal identity and the 18s rRna sequencing and build a phylogenetic tree.
Significance
This work is important because it will determine if genetic or chemical families will be more robust for strain prioritization.
Location
Don and Karen DeRosa University Center (DUC) Poster Hall
Start Date
27-4-2024 10:30 AM
End Date
27-4-2024 12:30 PM
Chemical VS. Genomic: Does Taxonomy Really Tell All?
Don and Karen DeRosa University Center (DUC) Poster Hall
Fungi are naturally found in all environments with over 600 species interacting daily with humans (Casadevall, American Society for Microbiology, 2017). Fungal natural products are a rich source of antimicrobial anticancer drugs (Jakubczyk, Molecules, 2020). The Carlson Library has isolated 108 unique aquatic fungal species based on observations on collection location, morphology, growth rate, and areas of spore coverage used to distinguish strains. We aim to characterize fungal species based on chemical identity and 18s rRNA sequencing to build a phylogenetic tree to understand the diversity captured by current isolation and culturing methods. Fungi are initially plated on Potato Dextrose Agarose (PDA) media plates and grown until they sporulate 7-10 days (Ezeonuegbu, Sci Rep, 2022). They are then cultured into liquid media and grown on a rotary shaker 110 rpm. Fungi were pelleted, DNA was extracted for PCR, and submitted for Sanger sequencing of the 18s rRNA region using standard primers ITS1 and ITS4. Representative strains from each phylogenetic branch will be grown for chemical characterization. Ethyl acetate is added to the flask to cover the media and hyphae. The ethyl acetate is then filtered and dried in vacuous. All extracts will be profiled by High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectroscopy (LCMS). The chemical fingerprints will be analyzed using the mPACT metabolomics platform to identify “chemical families” (Samples, 2023). Previously unknown metabolites, as identified by comparing to databases, will be prioritized for further characterization. This will determine if genetic or chemical families will be more robust for strain prioritization.
