Experimental-Journal-PhD

# Title : 20200810-SATAY for $\Delta$bem2, $\Delta$nrp1 and WT

# Date

10082020 - 18082020

# Objective

  • To test the technique on the mutants and also to have data for the data analysis pipeline from Greg.

# Method

  • 10082020-Preculture in SD-URA+0.2%glucose +2% raffinose at 14:20

    • Scrape from the colonies from each background that passed the test (2 per each background)
    • Inoculate to a final volume of 20mL and to a final OD of 0.2

  • 11082020

    • OD measurement

      • OD really low around 0.3 for all strains which could be due to the fact that the media did not contain extra adenine.

      • SO I RESTARTED THE PRECULTURE ON 11082020

  • 12082020 - Induction in SD-URA+2% gal to a final OD of 0.2 - 50 hours (2 days)

    • OD measurement:
Strain OD 10x dilution ReaL OD Dilution Factor to OD=0.2 Time of preculture
ylic133_1 0.659 6.6 ~30 20h
ylic133_2 0.577 5.7 ~30 20h
ylic135_1 0.073 0.7 ~3 20h
ylic135_2 0.126 1.26 ~6 20h
ylic136_1 0.678 6.7 ~30 20h
ylic136_2 0.626 6.3 ~30 20h

  • Issues :

    • The ylic135 strains grows a bit more slower than the rest which is expected due to the phenotype of not having bem2. But the amount of culture I need to add in order to have a final OD of 0.2 in 150mL is 50 mL and I only had 20mL. Therefore the initial OD of the induction will be around 0.1 for this strain.

  • Measure the background (T=0 of induction)

    • [x] Plate 200ul of the inoculum in SD-ADE+ 2% dextrose (expect 40-80 colonies per 200uL)

    • [x] Dilute 1000x and spread 200ul in SD-URA

  • [x] Prepare 3 flasks of 3L of SD-ADE media for the reseed of three strains

    • Per flask of 3L , add: 2,4L MiliQ to autoclave, dissolve 2.34g of -ade drop out + 20.7g of YNB (005) and filter sterilize it and add it after autoclaving the MiliQ. Add 300mL of 20% dextrose after autoclaving.

  • 13082020

  • [x] Plating in SD-ADE to check induction after T=22h and SD-URA 1000x dilution.

  • [x] Check OD (should be around 4-5) at 10:45am

Strain OD 10x dilution ReaL OD Time
ylic133_1 0.472 4.7 24h
ylic133_2 1.17 11.7 24h
ylic135_1 0.223 2.23 24h
ylic135_2 0.217 2.17 24h
ylic136_1 1.17 11.7 24h
ylic136_2 1.13 11.3 24h

  • Strains ylic133_2 and ylic136 have a very high OD

  • The cell culture of ylic133_1 have turned pink...

  • Still the background at T=0 can not be clearly determined.

  • 14082020 - End of induction/Reseed at 14:00

    • Choose the 3 strains to continue for the reseed that has the least number of ADE+ cells at T=0 and the highest number of ADE+ after induction.
      • It seems that ylic133_2, ylic136_1 and ylic136_2 are the ones that have the least background at T=0 before induction.

  • Background check at 9:30am

Strain ade+ clones/mL ura+ clones/mL Time
ylic133_1 < 5 ~100* 5* E3 = 5*E5 0h Induction
ylic133_2 ~30*5 = 150 5 *E5 0h Induction
ylic135_1 - - 0h Induction
ylic135_2 - - 0h Induction
ylic136_1 ~ 80*5=400 5 *E5 0h Induction
ylic136_2 ~ 80*5=400 5 *E5 0h Induction
  • Summary of the background

  • [x] Plate 200ul from the induction culture in -ade and 1000X in -ura to know the number of ade+ cells before reseeding. At T=51h of induction, at 13:00.
  • [x] OD measurement of the samples at T=51h of induction and T$_r$=0
  • Summary from the Induction
Strain OD start T=0 OD T=24h OD stop T=51h Time-Induction ADE+/ml-start ADE+/ml-24H ADE+/ml-stop
ylic133_1 0.2 4.7 5.4 51h 330 500 7500
ylic133_2 0.2 11.7 13.9 51h 180 3500 5000
ylic135_1 0.1 2.23 6.5 51h 150 200 350
ylic135_2 0.1 2.17 6.3 51h 50 75 100
ylic136_1 0.2 11.7 13.8 51h 600 5000 10000
ylic136_2 0.2 11.3 12.9 51h 500 3000 7500

  • [x] Reseed at 13:30

  • 17082020

  • [x] Check ADE+ cells during induction. Counting colonies from each plate.

    • Image J protocol:
      1. select image of interest: circle tool and edit-> clear outside
      2. Image -> Type -> 16bits
      3. Make sure to capture the maximum number of right colonies: Image -> Adjust -> Threshold . Make sure the colonies are all red.
      4. Process -> Make binary-> Watershed
      5. Analyse -> Analyse Particles

  • 18082020

  • [x] OD measurement for T=92h after reseeding at 12:00

  • [x] Plate 200ul with 1000X dilution in SD-Ade , and 200ul with 40000X dilution in SD-ura to estimate the growth of ade+cells compared with the T=0 of reseeding. Expect that the ade+ cells have grown by a factor of ~1000x.

  • End of reseeding

  • [x] Harvest of the cell culture.

    • 15ml of solid pellet, frozen in -80C. The pellet is pink (?)
Strain OD START OD STOP Time- Reseeding ADE+/ml-stop
ylic133_2 0.25 8.3 90h 3295000
ylic136_1 0.27 10.2 90h 5350000
ylic136_2 0.18 9.5 90h 3070000

# Results

  • For the strain ylic135 it did not work, because it is very miserable to culture with others. It grows much more slower , so maybe growing it more and with less volume , for next time. Also ylic135 gives a relatively high background compared with the growth in -URA.

  • The way of computing the number of colonies in highly dense plates is pretty inaccurate with the best tool to do it which is ImageJ. So I think that is why I get like an order of magnitude lower than the number of colonies expected by Benoit.

  • The pellet from the reseed culture was pink , which could mean that there were many cells ade- that did not have any transposon event. Hence I suspect the sequencing/transposon coverage will be not optimal.

  • The number of ADE+ cells after reseeding is around 1000X times higher than at then of the induction which is what was expected 😃

# Next steps

  • DNA sequencing protocol
  • Repeat the SATAY for ylic135 from the beginning

# Conclusion

  • First round of SATAY 😃 with WT and dnrp1 strains.

← DNA Prep for SATAY samples →