Pathfinder Report 2 - The professor\'s name was Stephanie Capaldi. This was an assignment for module PDF

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The professor's name was Stephanie Capaldi. This was an assignment for module two....

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Dana Woods Stephanie Capaldi MCB 422 3 October 2017 Pathfinder Report 2 Aim In this exercise, we are given four stock strains of known mating type and auxotrophic properties. We are also given one unknown strain, of which we do not know mating type of auxotrophic properties. The goal of this exercise is to create a haploid strain that has the same auxotrophic properties and opposite mating type as the original unknown strain. Approach The first step was to determine the auxotrophic properties and temperature sensitivity of the unknown strain. The strain was placed on several dropout plates as well as on ALL plates incubated at different temperatures to determine these features. Next, mating type was determined by mating the unknown strain with a known strain (Table 1). Diploids were selected for by replica plating the strains on a double dropout plate (Table 2). They were then plated on sporulation media and incubated at 30˚C. Cells were placed on a microscope slide to visualize the tetrads that formed. Each tetrad was selected and they were all put on an ALL plate at 30˚C. Three replica plates were then created: one for the auxotrophic property of the known strain, one for the auxotrophic property of the unknown strain, and one for the temperature sensitivity of the unknown strain. Because part of the goal is to create a haploid with the same auxotrophic properties as the unknown, we want the haploid to die on the replica plates with the features of the unknown strain and live on the replica plate with the feature of the known strain. Five

haploids with these properties were placed in the fridge. The next part of the goal was to find one that was the opposite mating type of the original unknown. These unknowns were mated with the known strains to determine mating type in the same way as before. When one of the strains was determined to be the opposite mating type as the unknown, the goals were met.

Cross

Determining Mating Type Haploid Strain Genotypes Diploid Selection 1 MATa leu- x unk his- ts Leu-, His2 MAT⍺ leu- x unk his- ts Leu-, His-

Temperature 30˚C 30˚C

Result No Growth Growth

Table 1. Strains mated to determine mating type of unknown strain. The table shows the unknown strain crossed with two known strains of different mating types. There was no growth for cross 1 and growth for cross 2. The unknown strain is MAT a.

Cross Haploid Strain Genotypes 1 MATa leu- x MATa his- ts 2 MAT⍺ leu- x MATa his- ts

Creating Diploids Diploid Strain Genotypes Diploid Strain Phenotypes N/A N/A leu-/LEU, his-/HIS, ts/TS Will grow on leu-, his-, 37˚C

Table 2. Strains mated to create diploids. The table shows the unknown strain crossed with two known strains of different mating types. No diploids were created in cross 1. The diploids created in cross 2 were wild type for all conditions.

Results After plating the unknown strain on four different dropout plates at 30˚C, it was found that it could not produce histidine. When incubated at various temperatures, it was found that the strain was temperature sensitive for heat. The strain was then mated with both a MATa and a MAT⍺ strain, each with leu-, and diploids were selected for. Cells grew when mated with MAT⍺, but not when mated with MATa. Therefore, the unknown strain’s genotype was MATa his- ts. This made the diploid genotype leu-/LEU, his-/HIS, ts/TS. The diploids were replica plated onto sporulation media and placed on a microscope slide to view the tetrads. The tetrads were then moved to an ALL plate at 30˚C. The replica plates made from this were on a his- plate at 30˚C, an ALL plate at 37˚C, and a leu- plate at 30˚C. The haploids wanted are ones that die on the his- and 37˚C plate, but live on the leu- plate, which are unknowns 6-10 in the fridge. A MAT⍺ was desired, so the strains were mated with MATa leu- to determine if they grow. When I

found one, I stopped mating. I found that unknown 8 had the MAT⍺ mating type, so its genotype was MAT⍺ his- ts (Table 3).

Diploid Strain Genotype Sporulated leu-/LEU, his-/HIS, ts/TS

Sporulation and Selection Haploid Replica Plates for Haploid Genotype Screening Selected leu- @ 30˚C, his- @ 30˚C, ALL @ 37˚C his-, ts, LEU

Haploid Mating Type Selected MAT⍺

Table 3. Diploid strain used for sporulation and haploid selection. The table shows the diploid strain genotype that was sporulated as well as the replica plates used to select for the properties desired. The haploid selected dies on his- and at 37˚C but lives on leu-. Selected diploids were mated to find one that was MAT⍺.

Conclusion When spores are created in this scenario, there are nine possibilities of haploid genotypes when considering auxotrophic properties/temperature sensitivity, and we only wanted one, making replica plating essential. The mating type was determined by mating with a known strain and observing growth. The goal of the experiment was met because I was able to identify a haploid strain with the same auxotrophic properties/temperature sensitivity but opposite mating type as the unknown strain....