Project Category

Perspectives on Research in Science and Mathematics (PRISM)

Presentation Type

Presentation

Description

Yeast fermentation produces a number of commercially important products, most typically represented by alcoholic beverages. In craft breweries that do not use expensive filtration systems, selecting a yeast variety for fermentation can determine the clarity of the product from hazy Belgian pilsner to a crisp, clear American lager. Flocculation refers to a stress response behavior in yeast to group together (flocculate) in conditions like low sugar or high alcohol content as experienced at the end of fermentation. Higher levels of flocculation increase the clarity. The process of flocculation is genetically controlled through a series of complicated interactions and depends on a set of extremely similar, redundant, extracellular proteins called Flocculins. The flocculin family of proteins are long lectin-like structures that will link the cell wall of one yeast cell to the sugar molecules (typically mannose) present on the cell wall of another yeast cell. To better understand the flocculation characteristic of select brewing strains, we want to determine sequence variation in the sugar binding domain of the yeast flocculin proteins, FLO1, FLO5, and FLO9 as well characterized determinants of flocculation. While the most efficient strategies for DNA sequencing is to directly sequence a PCR produced fragment of DNA, the incredibly high homology between these genes complicated PCR primer design and challenged the reliable production of amplicons from a single gene. Instead we redesigned the PCR approach to first use cloning techniques to isolate amplicons from each of the FLO1, FLO5, and FLO9 genes before sequencing the N-terminal region containing the sugar-binding site. Now that a novel primer set has been designed, we can utilize standard molecular biology techniques to sequence these genes from select brewing strains and complete gene comparison.

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Genetic Variance in Flocculin Protein Family

Yeast fermentation produces a number of commercially important products, most typically represented by alcoholic beverages. In craft breweries that do not use expensive filtration systems, selecting a yeast variety for fermentation can determine the clarity of the product from hazy Belgian pilsner to a crisp, clear American lager. Flocculation refers to a stress response behavior in yeast to group together (flocculate) in conditions like low sugar or high alcohol content as experienced at the end of fermentation. Higher levels of flocculation increase the clarity. The process of flocculation is genetically controlled through a series of complicated interactions and depends on a set of extremely similar, redundant, extracellular proteins called Flocculins. The flocculin family of proteins are long lectin-like structures that will link the cell wall of one yeast cell to the sugar molecules (typically mannose) present on the cell wall of another yeast cell. To better understand the flocculation characteristic of select brewing strains, we want to determine sequence variation in the sugar binding domain of the yeast flocculin proteins, FLO1, FLO5, and FLO9 as well characterized determinants of flocculation. While the most efficient strategies for DNA sequencing is to directly sequence a PCR produced fragment of DNA, the incredibly high homology between these genes complicated PCR primer design and challenged the reliable production of amplicons from a single gene. Instead we redesigned the PCR approach to first use cloning techniques to isolate amplicons from each of the FLO1, FLO5, and FLO9 genes before sequencing the N-terminal region containing the sugar-binding site. Now that a novel primer set has been designed, we can utilize standard molecular biology techniques to sequence these genes from select brewing strains and complete gene comparison.