I unfroze some new cells from these strains in order to have fresh colonies to start some new Y2H screens with. However, these cells have lost the redness that the original strains had. I plated the cells on regular YPD media. If anyone has any insight as to the implications of this phenotypic change and its cause, I would be very thankful!
The redness of the strains depends on a lot of factors, from growth medium to respiratory state. The deepness of the red depends on limiting adenine in the media, which is usually the case with YPD, but not with SC medium that contains adenine (adenine inhibits this, since they don't need to produce it themselves). YPD, like all complex media, have batch to batch variations that result in differences in adenine composition. Autoclave time also impacts it. In addition, it depends on how long the cells are grown, and at what temperature. In general the longer the redder, and plates left on the bench get the darkest. The final thing is the mitochondrial state. Petite mutants, which have respiratory defects, will not make the red pigment, but will instead look very white. You can identify these by their inability to grow on a carbon source that requires respiration (e.g., glycerol, acetate, ethanol). If they're petite, it's likely a mixture of petite and non petite, and it's worth streaking for singles to identify the correct non-petites. Aside from that, if the cells are bonefide ade auxotrophs (by testing for failure to grow on medium lacking adenine), it'll likely be fine for your screen.
Thank you! I tried growing the cells again, this time without using parafilm. What you said made a lot of sense! I saw the red phenotype, and so will be going forward with my transformations. I am also curious about whether the cream colored strains are auxotrophic for ADE, so I might try to test their growth on medium lacking ADE as a side project. Thanks again for your expert insight!
Quoting James et al. " In pJ694A, ADE2 gene under the control of the GAL2 promoter. ADE2 was chosen as a reporter because ade2 mutants form red colonies, while wild-type cells form white colonies. Intermediate expression of ADE2 results in colonies in various shades of pink; thus colony color can be used as an initial indication of the strength of an interaction or to identify plasmid loss events"
James P, Halladay J, Craig EA. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 1996;144(4):1425-1436.
I use pJ694A as well, our colonies are more of a shade of pink rather than red.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating