Curing strains of endogenous 2-micron

Curing strains of endogenous 2-micron plasmid

In order to create a circle-zero derivative from a Cir strain, the method of Rose and Broach (1990) is used. A 2-micron-based plasmid, YEp351-GAL-FLP1 *, that carries the LEU2 selectable marker and also the FLP1 gene under the control of the GAL promoter is transformed into yeast. Leu transformants are grown on medium containing galactose as the carbon source. Under these conditions, the FLP1 gene is overexpressed and causes over-replication of the endogenous and artificial 2-micron plasmids, which is deleterious to cells. Therefore, cells that have lost the plasmids have a significant growth advantage over those that retain them.

1. Transform yeast with YEp351-GAL-FLP1, and select transformants on SC-leu-glu 2% GAL (galactose) medium.
2. Patch transformants onto SC-leu-glu 2% GAL medium.
3. Patches that grow well are candidates for strains that have lost the 2-micron plasmid. Grow cultures from these patches overnight in 5 ml of YPD.
4. Plate cells from the grown cultures at a density of ~200 colonies per YPD plate and allow them to grow into colonies.
5. Replica plate to SC-leu to identify those lacking YEp351-GAL-FLP1. (Cells that have lost YEp351-GAL-FLP1 should not be able to grow).
6. To verify the loss of the 2-micron plasmids, prepare genomic DNA from Leu^- strains (as described in Section VII) and perform Southern analysis (Section VIII) on undigested DNA using DNA of YEp351-GAL-FLP1 as a probe. Include DNA from a Cir strain on the same gel as a positive control.

People always ask us for a map of YEp351-GAL-FLP1, but we don't have one. All we know is, it's YEp351 based. Linearize it with something and label the whole thing.

Errors!

• In previous publications, we have erroneously designated the YEp351-GAL-FLP1 plasmid as pFV17. pFV17 is an integrating plasmid described in the same Rose and Broach paper.
• In previous versions of this web page, we said the plasmid contained GAL-REP1. Whoops!