I have a yeast strain containing a LYS2 marked plasmid, which makes it prototrophic for lysine. Without the plasmid, it would be auxotrophic for lysine. I would like to know if there is any method by which I can make the yeast lose this plasmid.
It is easy small plasmids are unstable in yeast. Grow it in YEPD lequid plate it on YEPD (dilution platting) and replica plate on YC-lys select colonies which are growing on yEPD but not in YC-lys plateasy
This plasmid also expresses ubiquitin, and in the genomic DNA of the strain, all the four ubiquitin genes have been deleted. Since ubiquitin is an essential gene, I will transform the strain with a URA3 marked plasmid which also expresses ubiquitin, before I attempt to take the LYS2 marked plasmid out of the strain.
So it think that after transformation with URA3 marked plasmid, I should plate the strain on SD containing lysine but no uracil, and then replica plate it on SD containing no lysine, and select colonies that do not grow without lysine. Do you think this would work?
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