36. Title : 20200810-SATAY for \(\Delta\)bem2, \(\Delta\)nrp1 and WT#

36.1. Date#

10082020 - 18082020

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

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.
  - Add 6x ADE to the media which means: 44mg in 400mL .
  - SO I RESTARTED THE PRECULTURE ON 11082020
12082020 - Induction in SD-URA+2% gal (I also added 6XAde to this media) 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)
- Plate 200ul of the inoculum in SD-ADE+ 2% dextrose (expect 40-80 colonies per 200uL)
- Dilute 1000x and spread 200ul in SD-URA
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
Plating in SD-ADE to check induction after T=22h and SD-URA 1000x dilution.
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

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.
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

Reseed at 13:30
17082020
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
OD measurement for T=92h after reseeding at 12:00
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.
Reseeding
- Library to reseed
- Rule of thumb for the number of cells under certain OD600
OD

approx Number of cells in 1mL

0.1

3,000,000

0.18

6,000,000

0.25

7,500,000

0.27

7,500,000

1

30,000,000

Strain	OD at T=0 (reseed)	Volume	Library
ylic133_2	0.25	3L	7,500,000 * 3 * \(10^3\)= \(22.5 *10^9\)
ylic136_1	0.27	3L	7,500,000 * 3 * \(10^3\)= \(22.5 *10^9\)
ylic136_2	0.18	3L	6,000,000* 3 * \(10^3\)= \(18 *10^9\)

End of reseeding
Harvest of the cell culture.
- 15ml of solid pellet, frozen in -80C. The pellet is pink (?)
{width=50%}

Strain	OD START	OD STOP	ADE+/mL-start induct(backg)	ADE+/mL-start Reseed	ADE+/ml-stop Reseed	URA+/ml-stop Reseed	% of ade+/ura+	Total # of cells Harvest	Time- Reseeding
ylic133_2	0.25	8.3	150	5000	3295000	19200000	16.9	\(7.47 *10^{11}\)	90h
ylic136_1	0.27	10.2	400	10000	5350000	18000000	27.8	\(9.18*10^{11}\)	90h
ylic136_2	0.18	9.5	400	7500	3070000	11600000	30.2	\(8.55*10^{11}\)	90h

The number of ADE+ cells reseeded minus the number of background ADE+ cells is the complexity of the library.
To determine the complexity of the library, you should know:
- #ADE+ per ml at the start of the reseed,
- Volume of reseed.
- If you harvest all the culture:

For strain 1:

OD8 ~8E+7 cells/ml -> 7.47E+11/ 8E+7~10L

The number of URA+ cells per ml at the end of the regrowth is 19200000 -> 7.47E+11/ 19.2E+5 ~ 38L
I reseed in a volume of 3L :

If so, the complexity of the library is 5E+3 * 3*10E+3= 1.5E+7
Complexity = \(ADE+|_{T=0(reseed)}\) * Volume of reseed

The number of cycles that each ADE+ cells has done during the regrowth of the libraries as follow:
- N=ln(#ADE+ per ml STOP reseed/#ADE+ per ml START reseed)/ln(2)
- N_ WT=9.34
- N_dnrp1_1=9.06
- N_dnrp1_2=8.67

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 :)

First round of SATAY :) with WT and dnrp1 strains.
Next time I will:
- Also plate the cells in CSM in order to capture the whole population.
- Also plate the cells during the induction in -URA mainly to know the number of cells before the reseeding and to quantify better the ADe+/ADE- ratio across induction.