25. Title: Supp controls on sfgFP influence on the bulk growth

25.1. Date

25012021-

25.2. Objective

• To discard the influence of the sfGFP on the Cdc42 dependency for growth

25.3. Method

• Strains:

Strain	Name
BEM1 BEM3 CDC42	YWKD062(2)
BEM1 BEM3 GAL1-CDC42	YWKD071aI,YWKD071aII
BEM1 BEM3 GAL1-sfGFP-CDC42	YWKD065a,YWKD065b,YIdB003
\(\Delta\)bem1 BEM3 CDC42	YWKD063a,YWKD068a,YWKD063
\(\Delta\)bem1 BEM3 GAL1-CDC42	YIdB005(2)
\(\Delta\)bem1 BEM3 GAL1-sfGFP-CDC42	YWKD069a(2)
\(\Delta\)bem1 \(\Delta\)bem3 CDC42	YWKD067a(2),YWKD067b
\(\Delta\)bem1 \(\Delta\)bem3 GAL1-CDC42	YWKD073aI,YWKD073aII
\(\Delta\)bem1 \(\Delta\) bem3 GAL1-sfGFP-CDC42	YWKD070a,YWKD070c

• Measure in 0,0.06% and 0.1% Galactose concentrations in 36C , using two technical replicates per plate . (If possible do two plates)

• Booking of the plate reader Biotek on 10022021

  • Incubation on 08022021

  • Layout modified in the moment (because a pipette mistake):

  

  • Experiment started at 11:30

    • Linear 3 mins:30 secs and orbital 3 mins:30 secs shaking before each measurement at 36C

25.3.1. Protocol

• Growth assay protocol (p.66 Werner PhD Thesis)

  • First, a 96 well plate is inoculated with about 5 ul of cells into 100 ul medium per well in a predefined layout. In addition, sterile MiliQ is put in the four reservoirs in the perimeter (4 times 2 mL) and 77 times 100 ul in the reservoirs between wells. The wells are then covered by a sticker and with the lid on, it is placed shaking slowly (100-150 rpm) for 2 days at 30C..

  • Then, using a multi-pipette, 10 ul of cells of this plate are transferred to a new plate which has 90 ul of media in the same layout, which dilutes the cells a factor of ten. This is then repeated for a new plate, where 5 ul of cells are transferred to 95 ul media, to ultimately accomplish a 200x dilution compared to the original plate. When making the dilution, wells are gently mixed before dilution by pipetting up and down. The 200x diluted plate is then sealed by a transparent sticker, and covered with the lid to minimize evaporation (and water is placed in the reservoirs as before).

• Measurement protocol:

  • The first 1000s linearly pre-shakes (amplitude 1 mm) the plate, before shaking just before each measurement round in a 380 second interval. These rounds start with 90” linear shaking (amplitude 2 mm), 90” orbital shaking (amplitude 1.5 mm), 90” linear shaking (amplitude 1 mm), waiting 10” and then OD-600 +/-9 nm measurements (25 flashes, 5ms settle time). Rounds were executed in two consecutive loops of 24 hours, after which OD values were documented in a excel sheet.

25.4. Results

25.4.1. Plate layout to check all WT+pGal biological replicates and mutants+pGal+sfGFP

• Observations from the incubation

  • 2uL of melted glycerol stocks per well

  • started at 10:30

  • Media base: 4xCSM-NF+2%Raff

  

• Measuring at 36C at 11:00

  • 1uL of cells from incubation to 100uL media (easier to pipette)~100X dilution

25.4.2. Checking the strains from the glycerol stocks

25.4.2.1. Plating all glycerol stocks in selection media

• Plates:

  • CSM-URA +2% Raffinose + G418(for dbem1) + NAT(for dbem1dbem3)(for the plates , I autoclaved the agar with MiliQ and then added the SC-URA mixture with the sugar, one note here is that I added the SC-URA without filter sterilize to the 80C liquid agar.)

  • Results of the plating(the plates do not seem to be contaminated):

Strain	Name	Growth plates from 25022021
BEM1 BEM3 CDC42	YWKD062(2)	+
BEM1 BEM3 GAL1-CDC42	YWKD071aI,YWKD071aII	-
BEM1 BEM3 GAL1-sfGFP-CDC42	YWKD065a,YWKD065b,YIdB003	-
\(\Delta\)bem1 BEM3 CDC42	YWKD063a,YWKD068a,YWKD063
\(\Delta\)bem1 BEM3 GAL1-CDC42	YIdB005(2)	-
\(\Delta\)bem1 BEM3 GAL1-CDC42 heterozygous diploid	YWKD054a,b,c	+
\(\Delta\)bem1 BEM3 GAL1-sfGFP-CDC42	YWKD069a(2)	-
\(\Delta\)bem1 \(\Delta\)bem3 CDC42	YWKD067a(2),YWKD067b
\(\Delta\)bem1 \(\Delta\)bem3 GAL1-CDC42	YWKD073aI,YWKD073aII	-
\(\Delta\)bem1 \(\Delta\) bem3 GAL1-sfGFP-CDC42	YWKD070a,YWKD070c	-

25.4.2.2. Inoculating all glycerol stocks in selection media

• To mimic biotek conditions and se if there is a contamination on the stocks , I will :

  • Inoculate the cells to liquid media CSM-URA+2% Raffinose

  • Streak from the liquid culture to a plate of the same kind.

Strain	Name	Growth from liquid culture 26022021
BEM1 BEM3 CDC42	YWKD062(2)	+
BEM1 BEM3 GAL1-CDC42	YWKD071aI,YWKD071aII	-
BEM1 BEM3 GAL1-sfGFP-CDC42	YWKD065a,YWKD065b,YIdB003	-
\(\Delta\)bem1 BEM3 CDC42	YWKD063a,YWKD068a,YWKD063
\(\Delta\)bem1 BEM3 GAL1-CDC42	YIdB005(2)	-
\(\Delta\)bem1 BEM3 GAL1-CDC42 heterozygous diploid	YWKD054a,b,c	+
\(\Delta\)bem1 BEM3 GAL1-sfGFP-CDC42	YWKD069a(2)	-
\(\Delta\)bem1 \(\Delta\)bem3 CDC42	YWKD067a(2),YWKD067b
\(\Delta\)bem1 \(\Delta\)bem3 GAL1-CDC42	YWKD073aI,YWKD073aII	-
\(\Delta\)bem1 \(\Delta\) bem3 GAL1-sfGFP-CDC42	YWKD070a,YWKD070c	-

25.4.3. 2nd round pGal checks BioteK

• Incubation 08032021-10:00

  • 1ul of thawed glycerol stocks (WT+pGal+sfGFP and all pGal strains)

  • The well G9 was not contaminated it was empty, from the beginning, so I made a mistake and did not add any media on it.

  

• 10032021- measurements in 36C

  • Plate the grown up cells from dbem1+pGAl strains in CSM-uRA+0% gal+2% Raff

    • Not visible growth on 12032021

  • 12032021- Results of measurements

  

25.4.4. Cleaning up the dbem1+pGal glycerol stock

• hypothesis: It is contaminated with plain WT

• Strategy : Plating in CSM+0.1% gal and then replica plate in CSM+0% and SC-ura+0.1% gal (right ones)

  • Plating in CSM+0.1% GAL (15032021)

  • CSM autoclaved media

  • 200ul of diluted glycerol stocks , 10ul cells in 500ul MiliQ, to facilitate the efficacy of the replica plate.

  • Replica plating in CSM+0% Gal (wrong colonies should pop out) and CSM+0.1%Gal +G418 (Right colonies should pop out)

  • For the colonies growing in CSM+0.1% Gal+G418 (they were a lot) I replica plated again in CSM+0%Gal + G418 and SC-URA +0.1% Gal+ G418 to filter more the colonies that should be right. (19032021)

  

• prepare CSM-ura+2% Gal + G418 plates and streak there the glycerol stock . Only the right ones should pop out. Perhaps doing a colony PCR to check for the gal promoter and the bem1::KanMX(?)

  • 200ul of diluted glycerol stocks , 10ul cells in 500ul MiliQ.

  

  • perhaps there are not dbem1+pGal anymore in the stock or the gal promoter is lost ?

  • It seems there is not dbem1+pGal anymore in the stock , because there is no growth of any single colony in SC-URA+G418+2% Gal +2% Raffinose

  

25.5. Conclusion

• Unexpected growth of the pGal1 strains in 0% Gal from the bulk growth in the Biotek.

• The phenotype check on liquid and solid media are consistent between each other.

• What is striking is that ywkd062 did grow on SC-URA and it should not be because it is just a plain WT strain. Maybe for next experiment we should use yll3a.

• The dbem1+pGal strain is completely wrong(contaminated?) from 120320221 measurements.