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In the Wright lab we are currently studying the regulation of endoplasmic reticulum (ER) in yeast. The organization of ER in yeast is sensitive to varying levels of ER proteins. We use the protein HMG-CoA reductase to study the organization of ER in yeast. HMG-CoA reductase is a protein that induces the assembly of karmellae. Karmellae are stacks of endoplasmic reticulum that enclose the nucleus. HMG1 is an ER membrane that regulates sterol biogenesis and signals karmellae production. It is important to study karmellae assembly because if we figure out how karmellae assembles, then we may be able to understand and later control the molecular mechanisms that cells use to increase biogenesis.
My project is to conduct a population study to determine which mutants grow worse when karmellae is induced on galactose. I used four mutants; YALO11W, YOLO12C, YDR137W and YDR136C. I then preformed a plasmid prep, which purified my two plasmids, P3 and P266 in order to transform them into the mutants. Next, I preformed a transformation. I transformed P3 and P266 with my mutants. Both plasmids have a gal promoter. P3 is a control plasmid for P266. P266's gal promoter is attached to HMG1 so it may induce karmellae. I then grew the transformed strains on URA media. Only the strains that had been transformed grew because both plasmids have URA3 (the plasmid has the sequence for the needed uracil). Next, I preformed a serial dilution which grew up the transformants on R*LW glucose and R*LW galactose in four different temperatures (37°C, 30°C, 27°, and 16°C) to see their growth with and without karmellae.
