59. Title: FACs experiment with the same conditions as the microscopy done to quantify the cell sizes for the pGAl1-Cdc42-sfGFP strains (II).#

59.1. Date#

28012020-30012020

59.2. Objective#

To be able to compare the results of the FACs with the microscopy conditions. And also to see if we get a difference in expression when increasing the incubation temperature to 36C.

59.3. Method#

Insights from the Gal1 promoter

  • The location in the genome at which the gal promoter was inserted can have a strong effect on the expression pattern of the gal promoter (Ramon feedback). Hence we should not compare different studies of the Gal1 promoter with ours if the integration in the genome is in a different location and also if it is a plasmid or not.

  • We should compare systematically the Gal1p expression pattern of the strains that has the sfGFP (Werner strains) and the mneonGreen ones (Ramon/Miranda strains), because they have the same type of genomic integration of the Gal1 promoter.

    • Ask Reza for his data with WT+mneongreen to compare with mine

      • Look for the postprocessing results, in this folder

  • Follow the same protocol as I followed for the microscopy measurements

Experimental Design for the microscopy measurements{#fig:experimental-design}

Planned procedure

  • 15 hours of incubation in 2% Gal +2% Raff (i.e. overnight incubation from 17:00 to 08:00 )

  • Washing step with CSM+2% Raff+0%Gal to the respective Galactose (at 08:00-09:00) concentrations. Incubate

  • Measure FACs after 24 hours of incubation . (next day of the washing step)

    • Use the references cdc42-GFP ywkd038 and ywkd001 in 2% dextrose +2% Raff, in CSM-met and CSM respectively.

  • In order to have the same conditions as the microscopy done in December 2018 , where we quantify the cell sizes after 24 hours of incubation in X% galactose (after a washing step from a firsto overnight incubation in 2% galactose), the media has to have 4x the normal amount of aminoacids. This maybe has an impact on the regulation of the promoter.

  • The new plate design for this is shown in @fig:plate-design

Plate design for this FACS experiment{#fig:plate-design width=50% height=50% }

  • The strain ywkd024 will be measured to still compared with previous measurements done by Marit, in 2017.

  • Strains :

    • 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))

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

    • ywkd069 : New YWKD055c a W303 bem1\(\Delta\)::KanmX URA-Gal1pr-sfGFP-Cdc42 sandwich (pWKD011 integrated) MFAprHIS3 3,112 11,15

    • ywkd070 : YWKD070a,b,c New YWKD055c a W303 bem1\(\Delta\)::KanmX bem3\(\Delta\)::clonNAT URA-Gal1pr-sfGFP-Cdc42 sandwich (pWKD011 integrated) MFAprHIS3 3,112 11,15

    • 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)

  • Settings of the FACs experiment

    • Equipment-Model: BDFACSCelesta

    • Lasers: Alexa Fluor 488 at 495V

    • Flow Rate: 2ul/sec

    • Sample volume: 130ul

    • Plate: 96 well plate with flat bottom

    • # of events per well: 10000

    • FSC threshold:20000

    • FSC voltage: 407V

    • SSC volatge: 275V

    • Mixing volume: 65ul

    • Mixing speed: 200ul/sec

    • Nr. of mixes: 5

Actual procedure

  • 1st Incubation , at 14:00 in 28012020 in 2% Gal.

    • Any tube was dense after 24 hours of incubation in 36C .

    • The most dense for ywkd065a.

    • Note: I did the washing step with CSM for ALL strains, including ywkd024 and ywkd038, by mistake. I hope this do not cause their future death.

  • 2nd incubation , at 14:20 in 29012020 in the respective galactose concentrations.

    • I added to each 5ml tube , 10 ul of culture, already resuspended down to 80ul , 320ul and 1.2mL. The different volumes are to account for differences in densities (size of the pellet). The 1.2mL was used for ywkd065, and the 80ul was used for ywkd024, ywkd038 and ywkd001. The 320ul for ywkd069 and ywkd070.

  • At 13:30 the experiment started.

59.4. Results#

59.4.1. Plate data#

Overview of the data for the whole plate{#fig:plate width=50X}

59.4.2. Box plots#

Boxplots of all the dataset per strain{#fig:Boxplots width=50X}

59.4.3. Measure per strain#

The measure per strain consist on computing the geometric mean of each technical replicate, and then compute the average of these values to give an estimate per strain per condition.

Arithmetic mean of the geometric means per technical replicate.{#fig:means width=50X}

To know if these drops were real or due to the some weird behaviour of the FACs machine, I measure the OD of the cells (on 31012020 at 13:30) that luckily they were still in their incubation tubes, on the lab bench , at room temperature, without shaking.

I expected to see also a drop in their optical density in the same galactose concentrations were the drop in expression is perceived, if these drops are real, that is, due to the little concentration of cells.

Optical density of the same cell cultures after 24 hours of FACs measurement. They were in room temperature without shaking, that is, on the lab bench. {#fig:OD width=50X}

Indeed :), there were also drop in the optical density on those galactose concentrations, namely, 0.02% , 0.08% and 2% for the WT+Gal strain. I also see that the mutants dbem1 and dbem1dbem3 were really miserable like I saw in the expression.

Maybe something weird happened from the incubation… (??)

59.4.4. Cdc42 relative expression#

Normalization of each value to the value of ywkd038 to see the overexpression/underexpression pattern.{#fig:relative-to-cdc42 width=50X}

59.4.5. Total counts#

Total counts measured by the FACs for one technical replicate of each strain per condition{#fig:total-counts width=50X}

59.4.6. Correlation between the GFP and sfGFP fluorophuores#

Out of range for comparing the GFP with the sfGFP expression at 2% between ywkd024 and ywkd065. The middle shaded area represents the lines y=1.5x , y=x and y=0.6x{#fig:correlation-fluorophuores width=50X}

Decoupled visualization of the same dataset of @fig:correlation-fluorophuores

Correlation of ywkd065 at 0.1% galactose against ywkd024 in the same galactose concentration. Notice that here some points fall inside the shaded area, in contrast with 2% galactose.{#fig:correlation-in-0.1 width=50X}

59.5. Conclusion#

  • This dataset had some weird/unexpected behaviour:

    • The wild type expression is not steadily increasing with galactose concentrations. At 0.08% and 2% there are “drops” of expression.(See @fig:means)

    • The mutants dbem1 and dbem1dbem3 have extremely low expression this time that never surpasses the native level. (See @fig:relative-to-cdc42)