Team:TU-Delft/Yeast

Yeast for dummies

Since this is the first time that TU Delfts iGEM team is working with yeast, we faced a lot of small yeast-related 'challenges'. With this page we want to inform you about the basics and the pitfalls of working with yeast.

I'm yeast, and I am an alcoholic

Yeast, Saccharomyces serevisiae, is a simple, unicellular eukaryotic organism. This organism has been used for fermentation and baking for over 4000 years and it is probably the oldest domesticated micro-organism in the human history. An important one too, can you imagine a life without bread and beer? (Even if you can imagine it, it would most definitely be less fun!) Whereas these are a few positive traits, yeast can also cause unpleasant infections, which only shows how diverse this organism is.

Auxotrophy

The main advantage from an engineering perspective is that yeast has Auxotrophic markers. In specific strains genes are knocked out which synthesize essential enzymes in the amino acid synthesis routes. By complementing these deficiencies by adding the necessary gene on your DNA this provides a nice selection procedure.

Shuttle vectors

Today, most commonly encountered S. cerevisiae shuttle vectors belong to one of three classes:
- Integrating plasmids YIp
- Centromere plasmids YCp
- Yeast episomal plasmids YEp
We choose to work with the pRS vectors because.
The name pRS415 gives an indication on the presence of a CEN/ARS replication origin. 0 means yeast integrative plasmid, 1 means that it also can be used to maintain the plasmid in circular form. pRS415 Gives an indication of the auxotrophic marker used. pRS415 Version number… not really different.

Chromosomal integration

We encountered a lot of problems with plasmids. Because we wanted our constructs to be universal (with the idea to make it suitable for ‘fast checking’) we tried maintaining a plasmid. As it turned out, yeast cells are not eager to maintain a plasmid and with our construct we suspect homologous recombination occurred. After transformation, a PCR on the transformed plasmid, obtained by isolation, showed two bands instead of the suspected single band, one being ! Integration of the plasmid is therefore advised! Checking of this can be quite gruesome optimizing the necessary PCR reactions on your transformed yeast colonies. Chromosomal isolation can therefore improve the steps.

Knock-out strains

European iGEM teams have the advantage to have Euroscarf available to order strains with knocked out ORFs. The typical nomenclature is also explained here Euroscarf explanation.