The aim of this laboratory hour was to determine the yeast`s genotype and to discern the yeast`s auxotrophy factors.
Yeast (Saccaromyches Cerevisiae) consists of only 1 eukaryotic cell and only 6000 genes. It is used a lot in research studies, studies of protein-protein interactions by yeast 2-hybrid screening system and drug tests, because of the 20 % disease ` equivalency with humans, that serves as a proof that these diseases arise because of malfunction in the repair of DNA, division of the cell or genetic expression. Having its genome completed, rapid growth, existence in both haploid and diploid cells, non-pathogenicity, having its cells scattered and easiness in being handled in on the lab either by plasmids or integration methods, makes this organism one of the most used model organisms. Yeasts consist of vector that have their origin of replication and are helpful about selecting antibiotic markers. Yeasts divide by budding, an asexual mode of reproduction where the number of bud signs in the mother`s body determine here age. This process is realized in sub-stages that include the growth of the daughter cell from the mother`s body. Later the nuclear division occurs and immediately cell wall of the new cell begins to form. Finally the new yeasts are formed. They also can undergo diploid phase cycle and meiosis when two different mating types fuse. This is the sexual mode of reproduction is made possible as the yeasts are found in 2 forms: Mat a and Mat α. The yeasts exhibit homothalicity that is basically the ability to shift the mode of reproduction from haploid to diploid .
AUXOTROPHY & PROTOTROPHY
Auxotroph needs one or more factors of growth different from the wild type organisms, while prototroph-s exhibit the same features as the wild type-s .
This originates from S288C that it causes deletion of selectable marker genes that had diminished homology with the mostly used vectors. Its genotype is MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0  and usually strains the yellow color.
W303-1A STRAIN (MAT a)
It originates from ade2-1 and contains a mutation in ybp1-1, which makes the organism more vulnerable toward oxidative stress. It enables yeast to resist toxic effect of sodium and lithium cations, but not the high concentration of the potassium in the contrary of BY4741.It enables the pink color`s release due to ribosylaminioimidazole by causing a mutation in the pathway of using adenine .
MATERIALS & METHODS
Firstly different selective media containing nitrogen base were prepared, specifically: one lacking Histidine, one lacking Methionine, one lacking Uracyl and the one lacking Leucine. In 2 different Petri dishes unspecified types of wild types yeasts were placed. In the beginning of the reaction Bunsen burner was opened in order to reduce the risk of contamination. Procedure was realized in a short distance from Bunsen burner. A piece of yeast from the first type is taken by stirring rod, and it is rubbed gently and carefully to avoid breakage across the other Petri dish containing the selective media. This process was repeated for every type of the selective media. After this process ends, it is repeated for the second type of yeast. The Petri dishes are taken and placed in the incubator in a temperature of 30oC for 4 days.
In our results it is seen that BY4741 exhibit growth in different rates, that can be caused due to different conditions that affected them, such as the way we did the growth. The fact that they all can grow in the absence of these materials, specifically; Histidine, Uracyl, Lecucine or Methionine, shows they are prototroph for all of them. Also they can grow when all the elements are present in the Petri dish demonstrating that the rate their presence or absence affects them is low. On the other side, W303-1A has not grown in any of the mediums, indicating that the lack in any of the mediums negatively affects the growth. This means this yeast is auxotroph for each one of the elements. Additionally there is a result that seems to controverse the results that were expected from this yeast, the last one, where the W303-1A has not grown even in the presence of all those elements it need to normally grow . Probably this has occurred, because of a problem during the procedure. Probably the stain is kept over fire more than the heat it can usually deal with.
- http://what-when-how.com/molecular-biology/auxotroph-molecular-biology/retrieved 05.12.2015 from
- Getting Started with Yeast By Fred Sherman Modified from: F. Sherman, Getting started with yeast, Methods Enzymol. 350, 3-41 (2002)