I have different strains of S.c yeast expressing equimolar amounts of eGFP amd mCherry from a resident plasmid (pDRF1-GW-eGFP-T2A-mCherry). When a cell expresses GFP it also, as expected, expresses mCherry. However, many of the cells don’t appear to express the GFP/mCherry combination at all or do so at very low intensitiy. I’ve tried different ways to generate mid-log growth cells for imaging but does anyone have any tips for producing a culture with more uniform per cell FP expression?
If many cells fail to express the proteins, I’d double-check the selectable marker in the host strain, and ensure you always grow them in selective media.
And also check the cells’ growth rate with empty vector vs the FPs. If the expression is somehow detrimental to growth in your host strain, that may favor cells that don’t express.
Looks like this is a 2µ plasmid, which has high, but variable copy number. For consistency, the best option is to integrate the FPs into the genome. If you have to use a plasmid, try a CEN plasmid which should have more consistent, but lower, copy number.
Hope this helps,
Theresa