I have a very odd problem! I am doing a Yeast 2 Hybrid experiment and I am having troubles with my auxotrophic strains (AH109 and Y187 from Clontech). They should not grow in medium without leucine, but they are growing. Leucine is the auxotrophic marker of a subset of plasmids I need to transform in the yeasts (pGAD), but with this problem I am unable to transform them.
Did anyone ever encountered this problem before?
Many thanks
Hi there,
Make sure you are actually growing the right strains and make sure about the media content. It's weird to observe the same problem with 2 different strains...
Sure, we are going to buy a commercial media without leucine. Nevertheless, it only happens with Leucine, they do not grow without tryptophan, histidine, adenine or methionine.
Dominique's comment is spot on. Also, how are you determining that the strains are growing in medium w/o leucine? If you throw a million cfus onto minimal plates and incubate them forever, something is bound to grow, either via reversion (very common if the auxotrophy is caused by a point mutation) or due to some cells surviving off the leucine/proteins leaking out of dead cells.
The media is YNB (salts and vitamins) without aminoacids. I add the aminoacids they need, except leucine.
About the number of cfus, I tested 2 dilutions and also by streaking. I obtained well separated colonies, so I'm almost sure it's not a matter of excessive cfus and satelite growth. Plus the incubation was not "forever". 1 day incubation was enough to see that something was growing in YNB-Leu.
The reversion of the mutation is something I already though of. That's one of the reasons I put the question here, to see if someone else already had a similar situation.
Do you observe the same phenomenon if you grow them on plates without tryptophan or histidin?
Hi again,
I would suggest to restart experiment from frozen strain masterstocks if possible...
@Alejandro: as I said above, they don't grow without tryptophan (unless they are transformed with pGBK), neither they grow without histidine, methionine or adenine.
@Dominique: Done already, with no differences... It really indicates that it's something with the media, and I'm trying to understand what. When I get my hands on the comercial -Leu media, I will post the result.
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