We will try to induce sporulation of a yeast strain transformed with a low copy plasmid. Is it possible, that plasmid can be lost during sporulation on media containing yeast extract? Could you recommend the medium that will ensure no possible plasmid loss?
Do you have in mind a cen plasmid? Yes, there would be some loss--a small cen plasmid is lost during meiosis in apprx 1-5% or less of spores. Very long cen plasmids (yacs) are more stable.
Yes, we use a centromeric plasmid with YCpLac33 backbone. Seemly, there will be some loss.
Sporulation medium generally has no selection. The cells do not undergo extensive growth such that selection would be necessary. In my experience, whicle some loss is seen, generally speaking it is not a concern. You just need to dissect enough asci to find what you are looking for.
Yes, my YCpLac33 derivative has URA3 as a marker. There is no problem to maintain plasmid before sporulation. I am looking for a way to minimize plasmid loss during sporulation. I heard of a minimal sporulation media containing just KOAc with no carbon or nitrogen sources for cells. Does it provide minimal plasmid loss?
Plates containing only KOAc may not work for certain strains...you might get some sporulation but spore viability could be low. But try it. If it does not work on solid KOAc plates, try to do these two variants: (1) Put heavy patches on SD-Ura, grow for 2 days, then make heavy patch on pre-sporulation medium, grow overnight, transfer cells to sporulation medium and incubate until you see spores. I used this procedure successfully (see: http://tinyurl.com/n2fb7mk). (2) Grow heavy patches on SD-Ura, grow for 2 days, then scrape cells into KOAc in liquid S medium (no sugar), shake vigorously until you see sporulation. This did not work for me, because I could not get good proportion of 4-viable spores (this was essential for my work). But they did sporulate.
Dmitry, Our sporulation mix is:
10g KOAc, 1g yeast extract, 0.5g dextrose, 0.2g complete amino acid mix (including uracil), 20g agar per liter. No selection, but carbon source and the yeast provide some nitrogen. But the amount is much less than regular YPD. Like I said, minimal loss of plasmid with this, even though no selection. And we generally sporulate up to 10 days, depending on the strain.
Many thanks, guys! Very helpful suggestions! We will try several sporulation media. There is no need to have all spores viable.
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