52. Title : 20200305-FACs experiment with strains ywkd065, ywkd024, ywkd038 and ywkd001#

52.1. Date#

02032020-05032020

52.2. Objective#

  • To measure using the height channel the fluorescence intensities (to understand why the heights and not the area, look here) in different galactose concentrations of the strains:

    • ywkd024, ywkd038,ywkd001,ywkd065

    • ywkd024 : RWS119 Wedlich-Söldner Lab collection a W303 can 1 1-100 his3 11,15 Galpr-myc-GFP-CDC42 YipLac204-MET-CLN2 cln1\(\Delta\)::HisG, cln2\(\Delta\), cln3\(\Delta\)::HisG (strain to compare with ywkd065(sfGFP))

    • ywkd038: RWS1421 Wedlich-Söldner Lab collection a W303 can1 1-100 his3 11,15 CDC42pr-myc-GFP-CDC42 YipLac204-MET-CLN2 cln1\(\Delta\)::HisG, cln2\(\Delta\), cln3\(\Delta\)::HisG (Reference for the native CDC42 expression)

    • ywkd001: \(\alpha\) W303 can 1 1-100 his3 11,15 leu2 3,112 S288C ,yLL3a -Laan Lab collection (Reference for the background fluorescence)

    • ywkd065a New YWKD055c W303 URA-Gal1pr-sfGFP-Cdc42 sandwich (pWKD011 integrated) leu2 3,112 his 3 11,15

52.3. Method#

  • Plate design :

    {width=50%}

  • 1st incubation in 30C in 02032020

    • The cells for ywkd024 and ywkd038 were single colonies from the plates of 30C in raffinose.

      Phenotype comparison of ywkd24 and ywkd038 varying temperature and sugar source. 108 hours after plating{#fig:phenotype-checking-108 width=50%}

  • 2nd incubation in 30C in 04032020 at 10:30

    • all the strains had a good pellet to distribute.

    • I washed two times in CSM, CSM-met accordingly + 2% Raff, then resuspended the cells in 2.5ml of these media, and distribute 2ul in each tube containing 5ml media + x% gal.

  • at 13:30 the FACs started.

    • observations: The ywkd024 and ywkd038 strains were still transparent , so maybe still in lagphase. For next time I should put more cells to inoculate to increase the chances of getting out the lagphase. So I think around 100ul of pellet.

52.4. Results:#

Relative number of Cdc42 molecules to ywkd038 concentration wise, and also without the background from ywkd001 concentration wise.{#fig:relative-cdc42 width=60%}

Stadistics from the plate{#fig:stadistics}

Boxplots of the raw data{#fig:boxplots}

Counts of each well on the plate{#fig:counts}

Strain:ywkd065. FSC from the height channel vs area channel to inspect the data against doublets{#fig:065-doublets}

Strain:ywkd001. FSC from the height channel vs area channel to inspect the data against doublets{#fig:001-doublets}

Strain:ywkd024. FSC from the height channel vs area channel to inspect the data against doublets{#fig:024-doublets}

Strain:ywkd038. FSC from the height channel vs area channel to inspect the data against doublets{#fig:038-doublets}

52.5. Conclusion#

  • One take home message is to grow more amount of cells for ywkd024 and ywkd038 because they have very large lag phase, and it seems by taking very small amount as I did in this experiment I increase the chances of taking slow growers (?)

  • However they grew much better than in the previous experiment. Yet they were not expressing much CDC42 (ywkd24)

  • In 0% gal , the fluorescence the wild type show is the left over after 24 hours in this concentration. Which can be used to estimate the amount of cdc42 we see in the microscopy experiments. So in this case will be that in 0% gal, the wt has around 0.38x the native amount and dbem1 was almost dead and also dbem1dbem3.

Gal concentration

[cdc42-gal]/mean-ref

0% Gal

0.380224

0.01% Gal

0.679128

0.02% Gal

0.729760

0.06% Gal

0.741663

0.08% Gal

1.081503

0.1% Gal

0.917127

0.2% Gal

1.909962

2% Gal

2.913480

52.6. Next steps#

  • Do microscopy on the cells before doing FACs to check the state/ proportion of aggregates from sfGFP during the measurements.