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Curing strains of endog. 2m

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.


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