I suspect the EBY100 stock I have is contaminated or perhaps not even EBY100 because they are growing in the absence of tryptophan on SD-CAA or minimal dextrose agar. Colony morphology fits the description from the ATCC (round, smooth, cream, butyrous) on YPD agar. I am aware the phenotype is Leu-, Trp-, but wonder if it possible for a mutation to occur. The growth kinetics in YPD broth appears normal (i.e. starting OD600 of 0.2-0.4 takes 3-5 hours to reach OD of 0.6-0.8, so not likely contaminated with Candida). Are there primers for amplifying EBY100 unique regions? Any suggestions on how to confirm identity?
Hi Shan
Looking at genotype:
MATa AGA1::GAL1AGA1::URA3 ura352 trp1 leu2delta200 his3delta200 pep4::HIS3 prb11.6R
You could consider testing Leu and His, as these are deletion mutations. Deletions can't revert, so a better test than Trp phenotype.
If in doubt, chuck it out!
L
Dear Shan Goh,
Well it depends of how sure you want to be of that identification.
Looking at auxotrophies is quite nice but not fully complete. Most of the time it's fully sufficient.
The only full complete identifiation is the full genome sequencing of your strain and compare it to the known sequences. If you have access to recent DNA sequencer, such as IonTorrent from Life Technologies, confirm the identity of a yeats strain by sequencing its geneome is a matter of 2-3 weeks, including the long bioinformatic step.
Best regards,
Philippe
Hello Shan,
Phenotypes are the best way to identify whether your strain is the one you want. When yeast (S. c.) grow on medium that they shouldn't, there are a couple of possibilities. 1) What is the strain background? Strain genetic backgrounds vary and so too do the phenotypes of auxotrophic markers within them. Since I've used this strain before and didn't see any reversion or suppression of the trp1 allele, I don't think the genetic background is necessarily the problem. 2) It's some sort of WT prototroph. Yeast are all around us. The biggest problems I've encountered with contamination from other yeasts is when the weather changes and AC or heating systems are activated. These prototrophs will fail the mating test (in your case, they will not be able to mate with an alpha strain, assuming the strain you want is MATa). 3) If your strain is leu- and TRP+, then potentially whom ever stored it, stored the strain with a transformed plasmid. If then strain is given a chance to lose the plasmid with growth in YEPD, then colonies will arise that are leu-trp- or leu-TRP+. Hope this has helped,
Dave
I woud suggest to reorder the strain from a reliable source and test the auxotrophies upon arrival. Beware that casamino acids may contain some tryptophan, so I would rather rely on minimal media that contain only the amino acids and nucleobases that are required. This is quicker and cheaper than doing a comprehensive analysis of the cells that you have in hand.
Hi there,
As the trp1 is an amber mutation (GAG to TAG), so reversion is not totally excluded...
If contamination of your stock occured, it's easy to spot because your stock will contain both trp+ and trp- cells : isolate clones from your stock and screen them on SD-TRP. The non growing are definitely trp- then test the other available marker (Leu). Another way of screening your stock is to test a streak on media containing 5-FAA (5-fluoroanthranilic acid) which becomes poison when metabolized by TRP1 gene product (in fact it's similar to 5-FOA effect on URA3 cells). The growing cells will be trp-.
Hope it helps.
the EBY100 is a genetically modified strain that has the AGA1(encoding a cell wall protein) under the control of GAL1 promotor. Basically the above method suggested by others cannot give a confirmative result. I would suggest you isolate the genome of the yeast and design a pair of primer one targets the GAL1 promotor, the other targets the AGA1 gene.
- Badges
- Science method