I have recently transformed yeast (S. cerevisiae W303) with a pRS315-based plasmid, which gives the transformants the ability to grow in media lacking leucine. I don't know how stable it will be within the cell so, can I grow my transformed yeast in rich media such as YPD or will my cells lose the plasmid as they will not need it?
Thank you very much!
Dear Iago,
when growth in YPD, teast transformed strains can lose the plasmids. Keep in mind that centromeric (monocopy) plasmid, like pRS315, are more stable that episomal (multicopy) plasmids. However, depending on the plasmid, the loss per generation is around 5-8% in non-selective medium, that is at each generation 5-8% of the cells loss the plasmid. After n generations, the % of cells which have the plasmid depends on tree factors essentially:
- the stability of the plasmid
- the gene you have cloned. If the gene gives a selective advantage to the cell, a higher number of cells will keep it. On the contrary, if the gene decreases the fitness, a higher number of cells will lose it
- the growth condition: stress (like high temeprature, low glucose) can induce a loss of plasmid, since mantaining the plasmid is an energetic cost for the cell.
To check the % of cells with (or without) plasmid you can plate, after growth, 150-200 cells on YPD (or synthetic medium with leucine) and replicate the colonies on synthetic medium without leucine, on which only colonies deriving from a cell with the plasmid will grow.
Best,
Enrico
If the content of the plasmid isn't essential to the cell survival then the plasmid will be lost. The kinetics of plasmid loss will depend on plasmid properties.
I agree with both Enrico and Dominique that you should keep the yeast strains on selective media.
Cheers,
Evelyn
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