M.Sc. Biotechnology
http://hdl.handle.net/10464/3047
2024-03-18T12:39:30ZThe Linkage of Yeast Metabolites, Produced Under Hyperosmotic Stress, to Cellular Cofactor Systems During Icewine Fermentation.
http://hdl.handle.net/10464/16392
The Linkage of Yeast Metabolites, Produced Under Hyperosmotic Stress, to Cellular Cofactor Systems During Icewine Fermentation.
Allie, Robert
Icewine is a dessert wine of critical importance to the Canadian wine industry. The Icewine grapes are frozen on the vine, creating ice crystals, and subsequently concentrating the solutes in the juice. Icewine juice places yeast under increased osmotic stress, resulting in altered metabolism. This includes increased glycerol production, an internal osmolyte, and higher acetic acid production as they are linked to the cytosolic NAD+ and NADP+ cofactor systems. The yeast glycerol transporter Stl1p allows for glycerol uptake, lowering the production of glycerol and therefore acetic acid. Here we compare two Saccharomyces cerevisiae wine yeast strains, K1-V1116 wild type and K1-V1116 Δstl1, with Saccharomyces uvarum CN1, and relate the differences in metabolite production to the cofactor systems.
To that end, starter cultures of each strain were established Icewine juice with samples collected at fixed intervals and assayed for acetic acid, glycerol, ethanol, acetaldehyde, sugar, and the NAD+/NADH and NADP+/NADPH cofactor systems.
K1-V1116 wild-type, K1-V1116 Δstl1 knockout, and CN1 showed different kinetics of glycerol and acetic acid production. Although glycerol production per unit time did not vary among the three yeast strains, per unit sugar consumed, K1V1116 Δstl1 produced the most glycerol followed by CN1 and then K1-V1116. K1-V1116 Δstl1 was found to produce the highest amount of acetic acid as a function of sugar consumed compared to the wildtype. CN1 produced the lowest amount of acetic acid as a function of sugar despite producing higher glycerol than the K1 V1116 wild-type.
While there was no statistical difference in the NAD(H) redox system ratios between the three yeast to account for the differences in glycerol and acetic acid production, S. uvarum CN1 showed statistically lower amounts of oxidized NADP+ to total NADP(H) compared to both of the S. cerevisiae K1 strains. These findings provide further insight about yeast metabolism under hyperosmotic stress.
Re-evaluation of analytical chemistry techniques in studying DNA structures
http://hdl.handle.net/10464/16389
Re-evaluation of analytical chemistry techniques in studying DNA structures
Vanloon, Jesse
This work describes the use of analytical chemistry techniques to examine the structural changes that DNA adopts when subjected to a number of external/internal factors. A self-complementary sequence, d(CG)9, and a non-self-complementary sequence (mixed sequence) were used to study the conformational effects displayed by each type of oligonucleotide sequence. The structural changes adopted by DNA was examined using a variety of analytical techniques, such as: nuclear magnetic resonance imaging (NMR), differential scanning calorimetry (DSC), ultra violet visible (UV-Vis) spectroscopy, circular dichroism (CD) spectroscopy, and high-performance liquid chromatography (HPLC). 1) d(CG)9 and a mixed sequence in the B- and Z-DNA conformation was examined by CD and UV-Vis at a concentration of 1mM using a home-made cuvette called a Flexicell with a minimum pathlength of 0.129± 0.015 mm. The CD and UV-Vis spectra’s produced were found to be reliable when compared to commercial cuvettes with a pathlength of 1 cm and sample concentration of 10 µM. 2) d(CG)9 was lyophilized and reconstituted using either water or buffer to determine if d(CG)9 adopts a different structure when reconstituted using different conditions. It was determined that lyophilized d(CG)9 adopts a hairpin conformation when reconstituted with water, and a B-DNA duplex when reconstituted with a buffer containing NaCl. 3) d(CG)9 was thermally denatured using DSC to determine if DSC can be a viable method to study oligonucleotides. It was determined that d(CG)9 undergoes a two-state unfolding pathway. 4) Nuclear Overhauser Effect spectroscopy (NOESY) and correlation spectroscopy (COSY) were used to examine the conformational differences of 2’-deoxyadenosine when incubated in water. From the distance and torsion angle constraints obtained from NOESY and COSY respectively, and from existing crystal structures, it was found the structures that were determined by NMR spectroscopy were misleading because of spectral artifacts. 5) A mixed sequence was treated with organic modifying agents to determine the minimal condition required for DNA denaturation when different modifiers were used. It was determined that urea at a concentration of 8 M and at a pH of 12.5 is sufficient to denature the mixed sequence duplex.
Analysis of the effect of genetic heterogeneity on de novo genome assembly using Xylocopa virginica as a model.
http://hdl.handle.net/10464/15030
Analysis of the effect of genetic heterogeneity on de novo genome assembly using Xylocopa virginica as a model.
Tang, Haimeng
Next generation sequencing (NGS) technology has revolutionized genomic and genetic research, and as a result, de novo genome sequencing and assembly for non-model organisms has now become a common task in genome research. However, the integral properties of a genome such as ploidy, mutations, and repeat content impose issues for current genome assemblers. In this work, we used Xylocopa virginica (Eastern carpenter bees) as a unique model organism for examining on the effect of sequence heterozygosity on quality of de novo genome assembly. Using two de Bruijn graph genome assemblers, we assembled four bee genomes representing different sex and age (unworn male, worn male, unworn female, worn female) using standard Illumina sequencing and one genome using 10X linked-reads library for an unworn female. We discovered that there is a noticeable difference in a variety of genome assembly quality metrics, with the haploid unworn male genome having the highest quality and the worn diploid female genome having the lowest quality. In fact, the N50 value of the unworn male genome was >100 times higher than that of the worn female genome. The genome quality pattern supports the hypothesis that sequence heterozygosity resulting both from ploidy and somatic variants can affect the result of an assembly with former shown to be a much bigger player than the latter. Furthermore, we observed that the density of variants was moderately correlated to the density of breakpoints in the genome assemblies. Overall, our results indicate that increased ploidy and accumulation of somatic variants both negatively affect the quality of the resulting assembly with the former being much more significant than the latter. When considering a de novo assembly project for a non-model organism, whenever possible, haploid samples at the youngest possible age are to be recommended. Furthermore, use of a long-read platform can lead to better genome quality. However, at least for the 10x linked reads, having too much sequencing data does not necessarily lead to a better genome assembly.
Hard Pressed to Find A Difference: Evaluating the total tannin content of Cabernet franc L cv. wines, made using pre- and post-fermentation pressing treatments
http://hdl.handle.net/10464/15010
Hard Pressed to Find A Difference: Evaluating the total tannin content of Cabernet franc L cv. wines, made using pre- and post-fermentation pressing treatments
de Felice Renton, Leah Isobel
Total tannin concentrations were monitored in Cabernet franc grapes in 2018 and 2019 from two vineyard areas in the Niagara Peninsula, Ontario. Total tannins were measured using the methyl cellulose precipitation (MCP) assay. In 2018, post-fermentation pressing treatments of 100kPA, 150kPA and 200kPA were applied with a control (no press treatment). In 2019, pre-fermentation pressing treatments in combination with juice removal (saignée) were applied with a control (no pre-fermentation treatment). Free sorting and consumer preference testing of the 2018 wines were evaluated using trained panellists and a consumer panel. Pre- and post-fermentation press treatments had little impact on the tannin concentrations in wines post fermentation. Observed trends over time suggest treatments have an influence in the behavior of tannin polymerization and stability, but these results are specific to vineyard site. This suggests that Cabernet franc varietal wines can be produced using low press-treatments without compromising the concentration of total tannins which may contribute to improved wine quality.