73. Title : 07082019-Crossing of yEK7a with ylic133_1 :broken_heart: :pensive:#

73.1. Date#

07082019-09082019

73.2. Objective#

In order to do SATAY on the specific genotypes, I have to get a diploid heterozygous on bem1::KanMx , bem3::NAT, nrp1::Hygro , bem2::leu2, and homozygous on ade2- and ura3-

73.3. Method#

  • 07082019- Growth in a YPD plate yEK7a and ylic133_1 separately.

  • 08082019-Mix equal small amounts (1/5 of a typical sized colony is sufficient) of each strain together on an area of fresh YPD agar. Take care to ensure mixing is thorough, to enhance the frequency of mating. Patch the two parent strains separately on a different area of the plate.

  • In this case it is not possible to use auxotrophic selection to obtain the diploid because ylic133_1 doesnt have aminoacid production neither antibiotic resistance .

  • After 5–8 h mating check under the microscope for the formation of shmoos (elongated, pear-shaped cells), mating figures (dumb-bell figures) and zygotes (3-pointed dumb-bells formed by mating of an a and an α cell followed by emergence of a bud as the diploid cell begins to proliferate).

  • Zygotes typically develop by 8–10 h after mixing. Spread the mating mixture along one side of a YPD agar plate, identify zygotes by their characteristic 3-point dumb-bell morphology and micromanipulate them to a clear area of the plate.

  • After germination, verify that they are diploid by absence of expression of a mating type and/or ability to sporulate .

  • 09082019- I selected 4 patches and grow them in liquid YPD media from 9:30am until they are dense (should be around 4 hours)

  • Then I will do the sporulation protocol from 14:00 until 21:00 and put it in KAc over the weekend, to have a quick check if they form spores.

    • Take a bit from each culture an streak onto a new YPD plate to conserve the strains in case it did work.

73.4. Results#

  • 14082019 : After 5 days in incubation in KAc and Miliq for the sporulation protocol I dont see any tetrads :( meaning that they were not diploids yet initially , or the sporulation protocol did not work.

73.5. Next steps#

  • Restreak the mating plate in YPD+G418+Clonat+Hygro because if there is any diploid should grow in that media as well ,due to yEK7a genotype.

  • Evaluate the density compared to just YPD , and do sporulation again and look them in the microscope, to see if we can see some features of diploids cells, e.g. the axial pattern of division.

    I took colony 5,6,7,8 from the YPD + G418+Hygro+NAT to grow in YPD (15082019 10:45am) and incubate until 16082019, to do sporulation again.

  • 16082019 Sporulation protocol of colonies 5,6,7 and 8.

73.6. Conclusion#

  • The sporulation did not work neither :(

  • However I saw few tetrads on tube for colony 8 , meaning that they could mate but not as efficient.

  • Another factor that could influence the sporulation failure was that the KAc used was from a very old stock (2015!!).

  • Next experiment, will be using liquid drops for mating and a new stock of KAc !