Progress 01/01/22 to 12/31/24
Outputs
Target Audience:Progress updates were disseminated quarterly to the members of the Gibney Lab at Cornell University. Excluding myself, the Gibney Lab consists of two PhD students, three undergraduate students, one masters student, and our PI Dr. Patrick Gibney. Progress updates consisted of 30-60 minute PowerPoint presentations which described methods used, my results, and next steps. Four formal presentations were given in academic settings (Gavin Sacks Lab Meeting, Wells College Seminar, two Cornell seminars) and one presentation was given at a conference (NERY in Ithaca). I presented a 30-60 minute PowerPoint presentations which described methods used, my results, and next steps. Additionally, a poster was presented at the Fungal Genomics conference in Monterey, CA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Professional development. Learning new techniques in and out of the lab space. Training others on these techniques and applying the skills to other (small) side projects. Meeting with the bioinformatics facility to discuss useful project approaches, increasing my network and broadening my skillset. Presentation skills in multiple settings with different target audiences. How have the results been disseminated to communities of interest?PowerPoint presentations to my lab (quarterly), four academic presentations, two conference presentations (one oral, one poster). Manuscript in progress for a publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Diastatic strains of Saccharomyces cerevisiae are of particular interest to brewers, yeast distributors, and industrial sectors for their unique ability to release glucose from non-fermentable polysaccharides. While this phenotype can be harnessed to produce distinct beer styles or bioethanol from dextrin-rich substrates, these strains are also considered spoilage risks for brewers. Here, we screened a panel of 96 brewing strains for the presence of STA genes, annotated open reading frame and promoter variants, and measured diastatic functional activity at the strain and allelic levels. Additionally, we compared promoter strengths, induction kinetics, and strain-promoter compatibility between relevant strains. Using high quality genome assemblies derived from combined short (Illumina) and long (Oxford Nanopore) reads, we identified conserved allelic variations, generated phylogenetic trees, and proposed a conceptual model for the evolutionary history of the STA gene family.
Publications
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Progress updates were disseminated quarterly to the members of the Gibney Lab at Cornell University. Excluding myself, the Gibney Lab consists of two PhD students, three undergraduate students, one masters student, and our PI Dr. Patrick Gibney. Progress updates consisted of 30-60 minute PowerPoint presentations which described methods used, my results, and next steps. Two formal presentations were given in academic settings (Gavin Sacks Lab Meeting, Wells College Seminar) and one presentation was given at a conference (NERY in Ithaca). I presented a 30-60 minute PowerPoint presentations which described methods used, my results, and next steps. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Professional development. Learning new techniques in and out of the lab space. Training others on these techniques and applying the skills to other (small) side projects. Meeting with the bioinformatics facility to discuss useful project approaches, increasing my network and broadening my skillset. Presentation skills in multiple settings with different target audiences. How have the results been disseminated to communities of interest?PowerPoint presentations to my lab (quarterly), two academic presentations, one yeast conference presentation. What do you plan to do during the next reporting period to accomplish the goals?Phylogenetic analysis of strains and gene formation history. Work on understanding the kinetics of diastatic spoilage (when are the genes turned on, etc.).
Impacts
What was accomplished under these goals?
BLAST was utilized to identify all STA-containing strains from our panel. In addition to the presence of STA genes, BLAST was utilized to differentiate STA1, STA2, and STA3 strains based on chromosomal location: annotating strains to chromosomes IV, II, and X. Notably, none of the STA variants aligned with chromosome XIV, the published location of STA3 based on CHEF gel analysis, and therefore STA3 has been re-annotated to chromosome X. STA promoter analysis identified strains which reveal a conserved 1,162 nucleotide in the promoter, of which all were annotated as STA3 strains. Further nucleotide analysis identified four conserved STA open reading frame variants which largely clustered by chromosomal location. While differences in diastatic spoilage potential were observed between cloned open reading frame variants, the most significant differences were observed at the strain level, leading to the hypothesis that differences in diastatic activity between strains is mainly controlled by variable gene regulation. To test STA promoter strength and compatibility, a set of mNeonGreen-expressing vectors selectable by drug resistance were designed and tested in a panel of diverse yeast species. As STA promoter function requires the recruitment of transcription factors present in diastatic strain backgrounds, a strongly diastatic strain, French Saison, was used as the host for assessing promoter induction kinetics and individual promoter strengths. Promoters with the conserved 1,162 nucleotide deletion yielded lower fluorescence maxima relative to wild-type promoters, indicating reduced promoter strength. Additionally, it was observed that other host strains were unable to utilize the STA promoter due to missing or mutated transcription factors.
Publications
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Progress updates were disseminated quarterly to the members of the Gibney Lab at Cornell University. Excluding myself, the Gibney Lab consists of three PhD students, three undergraduate students, one masters student, and our PI Dr. Patrick Gibney. Progress updates consisted of 30-60 minute PowerPoint presentations which described methods used, my results, and next steps. Two formal presentations were given to private yeast-research companies (Omega Yeast Labs, Invisible Sentinel) which were interested in the scope of my project.I presented a 30-60 minute PowerPoint presentations which described methods used, my results, and next steps. Changes/Problems:Previously, we were expecting complete genome assemblies for the 95 strains, however this wasn't available. I had to sequence and assemble all of the genomes before progressing towards my aims. What opportunities for training and professional development has the project provided?Professional development. Learning new techniques in and out of the lab space. Training others on these techniques and applying the skills to other (small) side projects. Meeting with the bioinformatics facility to discuss useful project approaches, increasing my network and broadening my skillset. How have the results been disseminated to communities of interest?PowerPoint presentations to my lab (quarterly), and two private yeast companies (once each). What do you plan to do during the next reporting period to accomplish the goals?Further promoter analysis, both phenotypically and bioinformatically. Work on understanding the kinetics of diastatic spoilage (when are the genes turned on, etc.).
Impacts
What was accomplished under these goals?
Genome assemblies for 95 brewing strains were generated and assessed for the presence of STA (formerly referred to as STA1). Strains determined to haveSTA via BLAST were phenotyped for diastatic activityvia the BGM assay. STA gene variants for all STA+ strainswere cloned into a common plamid vector and phenotyped for diastatic activity via the BGM assay. Strains with significant variations between strain and allele diastatic activity levels were further investigated bioinformatically (promoter screening, allellic variation screening). A few key SNPs were identified to play large roles in diastatic activity. Additionally, promoter integrity was a major factor in diastatic activity.
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