Search for: All records

Abstract The budding yeastSaccharomyces cerevisiaeis an excellent model organism for studying a variety of critical cellular processes. Traditional methods to knock in or ‐out at specific yeast loci utilize polymerase chain reaction‐based techniques, in which marker cassettes with gene‐specific homologies are integrated into the genome via homologous recombination. While simple and cost‐effective, these methods are limited by marker availability when multiple edits are desired. More recently, CRISPR‐Cas9 technology has introduced methods to edit the yeast genome without the need for selectable markers. Although efficient, this method is hindered by additional reagents and lengthy protocols to design and test unique guide RNAs and donor templates for each desired edit. In this study, we have combined these two approaches and have developed a highly efficient economical method to edit the yeast genome marker‐free. We have designed two universal donor templates that efficiently repair commonly used selectable markers when targeted by a novel guideRNA‐Cas9 designed to promoter regions inAshbya gossypiifound in most integration modules. Furthermore, we find our newly designed guideRNA‐Cas9 successfully multiplexes when multiple markers are present. Using these new tools, we have significantly improved the cost and efficiency to generate single or multiple marker‐free genetic modifications. In this study, we demonstrate the effectiveness of these new tools by marker‐free ablatingPRC1,PEP4, andPRB1vacuolar proteases typically inactivated before many biochemical and membrane‐trafficking studies using budding yeast. 

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function. 

While understanding macromolecular structural elements and their roles in dictating cellular function is critical to grasp basic concepts in biology, it can be challenging for students to master this content—these elements naturally exist at the nanoscale and are not observable with the naked eye. Oftentimes this understanding is catalyzed by impactful illustrations and animations found online and in textbooks. In recent years, 3D printing technology has become readily accessible as an additional way to generate models and visualize entities of interest. In this report, we describe and discuss the efficacy of an approach using 3D-printed models in combination with online open-source molecular modeling analyses of the macromolecular structure of p53 to engage students with molecular concepts in cancer cell biology and human health. This pedagogy strategy has been successfully integrated into an upper-level undergraduate course at a primarily undergraduate institution and a graduate biology course at a public research university. We describe the potential benefits while providing tools for others to integrate this strategy into their teaching.