Cheesy Problems, Yeasty Solutions
Faculty Mentor Name
Der Thor and Geoff Lin-Cereghino
Research or Creativity Area
Natural Sciences
Abstract
Lactose intolerance has been a constant hurdle for those who want to add dairy in their diet. In our lab, we strive to engineer Pichia pastoris into a probiotic and long-term solution to this issue.
Our project goal is to characterize beta-galactosidase (lactase) expression and secretion in mutant strains of P. pastoris, bgs7 and bgs13, which coexpress an adhesion protein on their surface. To increase adhesion of P. pastoris, these mutants have been engineered to integrate plasmids that drive the expression of agglutinin and mucin binding domain (MBD), yielding the JGGA (agglutinin) and JGM10 ( agglutinin + MBD) strains.
We first performed a plate assay on the different strains to visualize the expression levels of lactase. To quantify lactase and agglutinin expression level in these four strains, we then performed western blots and enzyme assays on the yeast pellets (intracellular content) and supernatant (extracellular content). In addition, immunofluorescence microscopy was utilized to determine the expression level and location of agglutinin on the P. pastoris cells.
Our western blot and immunofluorescence microscopy indicated that the expression of lactase and agglutinin were higher in the JGGA strains of bgs 7 and bgs 13 compared to JGM10 strains. These results suggested that the JGGA strains were significantly more successful in secreting lactase and producing agglutinin on their surfaces. Overall, our study revealed that the JGGA strains have potential to be a probiotic for lactose intolerant individuals because of their high expression of lactase and ability to persist in the guts of mammals.
Cheesy Problems, Yeasty Solutions
Lactose intolerance has been a constant hurdle for those who want to add dairy in their diet. In our lab, we strive to engineer Pichia pastoris into a probiotic and long-term solution to this issue.
Our project goal is to characterize beta-galactosidase (lactase) expression and secretion in mutant strains of P. pastoris, bgs7 and bgs13, which coexpress an adhesion protein on their surface. To increase adhesion of P. pastoris, these mutants have been engineered to integrate plasmids that drive the expression of agglutinin and mucin binding domain (MBD), yielding the JGGA (agglutinin) and JGM10 ( agglutinin + MBD) strains.
We first performed a plate assay on the different strains to visualize the expression levels of lactase. To quantify lactase and agglutinin expression level in these four strains, we then performed western blots and enzyme assays on the yeast pellets (intracellular content) and supernatant (extracellular content). In addition, immunofluorescence microscopy was utilized to determine the expression level and location of agglutinin on the P. pastoris cells.
Our western blot and immunofluorescence microscopy indicated that the expression of lactase and agglutinin were higher in the JGGA strains of bgs 7 and bgs 13 compared to JGM10 strains. These results suggested that the JGGA strains were significantly more successful in secreting lactase and producing agglutinin on their surfaces. Overall, our study revealed that the JGGA strains have potential to be a probiotic for lactose intolerant individuals because of their high expression of lactase and ability to persist in the guts of mammals.
