78. Title: 29072019-SATAY 1st attempt with Byk832-T :pray: :confused: :x:#

78.1. Date#

Tuesday, 29072019 - 31072019

78.2. Objective#

To learn about the whole technique and detect the main issues and bottlenecks when used it for the target strain.

78.3. Method#

  1. Streak the chosen Byk832 transformed cells that display full growth in -URA and medium growth in -ade, in -ura.

  2. Pre-culture step - 29072019 :clock230: (14:30) :blush:

    • Scrape cells from SD-URA, inoculate 20-30mL SD-URA 0.2% glucose +2% Raffinose at OD600=0.16-0.24 (OD=0.06 works as well)

      it is more than one cell, just take with a pipette tip many cells and put them in a eppi with 1ml MiliQ

      Measure the OD of a 100x diluted stock (10ul of diluted cells + 990ml MiliQ)

       | Strain  | OD (100x diluted) |
 |---|---|
 |Byk832T_1   | 0.15 |
 |Byk832T_2   | 0.24 |
 |Byk832T_3   | 0.20 |

      Dilution to OD=0,2 to a final volume of 20mL

      Enzo did three flasks more , as technical replicates.

    • Incubate at 30C, 140-160 rpm, to OD=4-6 (around 20h)

      Started at :clock230: (14:30) :blush:

    • It is recommended to start with 3-4 precultures and later chose which to reseed in SD-ADE 2% Glucose.

78.3.1. Benoit Data#

Strain

OD start

OD STOP

Time

strain # 3

0.235

3.85

20h

strain # 5

0.229

3.94

20h

strain # 2

0.233

4.02

20h

strain # 4

0.227

4.04

20h

strain # 6

0.235

4.07

20h

strain # 1

0.234

4.33

20h

78.3.2. Our data#

Strain (2 technical replicates)

Preculture

OD start (29072019-:clock230: -> 14:30)

OD stop (~2X higher
than Benoit data)

Time

Byk832T_1(Leila)

0.19

~ 12.6

19h

Byk832T_2 (Leila)

0.14

~11.2

19h

Byk832T_3(Leila)

0.17

~ 10.8

19h

Byk832T_1(Enzo)

0.19

~ 8

19h

Byk832T_2(Enzo)

0.14

~ 10

19h

Byk832T_3(Enzo)

0.17

~ 9.7

19h

  1. Induction

    • Inoculate 200ml SD-URA 2% Galactose at OD600=0.2 from the saturated preculture.

      We choose base on the OD values Byk832T_1(Enzo),Byk832T_2(Enzo), Byk832T_3(Enzo), and Byk832T_3(Leila) forthe induction in 150mL of media

    • Incubate at 30C , 140 to 160 rpm for 50-52 hours

      30072019-:clock1030: -> 10:30 start T=0 of induction.

    • To evaluate the background:

      • Spread 200ul of this inoculum on SD-ADE

      • Dilute the inoculum 1000x, spread 200ul on SD-URA and SD complete

        • score after 2 days:

        • :pensive: Expect ~200-400 ADE+ clones/mL culture (~ 40-80 clones for 200ul), i.e. ~0.01%-0.02% of the total number of cells. These are cells that repaired the ADE2 gene by homologous recombination WITHOUT transposition

          The plates next morning had easily more than 200 colonies , which means 5 times more than indicated for Benoit.

    • Reproduce the following plots, with the chosen technical replicates for induction: 

      - Step 1: At the time of induction we plate 200ul from the induction culture at OD=0.2

    We could not plate at T=0 from the induction culture, because we did not have enough plates.

       - Step 2: Next day morning  repeat Step 1 (17 hours)

    31072019 - We aborted this attempt because of a high background

       - Step 3: 3 hours later  repeat Step 1  (20 hours)
   - Step 4: Next morning  repeat Step 1 (42 hours)
   - Step 5: Same day afternoon , repeat Step 1 (51 hours)
   - Step 6-8 : Every morning Step 1, for 5 days more.

    • The OD should have reached 4-5 after 20h of induction.

      Measure OD , next day after induction.

    • In order to know how well the transposition is going before reseeding:

      • Spread 200ul directly on SD-ADE

      • Spread 200ul of a 40000X dilution on SD and on SD-URA

      • Score under a binocular after 30h (i.e. closer to the time of reseed)

      • Expect around 0.05-0.1% of the cells to have become ADE+ . Incubate the plate for one more day for a final count.

      • Pursue with the culture that gives the highest number of clones, starting from a minimum of background.

      • 500-1000X more ADE+ clones at T=51 hours than at T=0 of inductions is in the ballpark

78.3.3. Benoit Data#

Strain

Induction

OD start

OD stop

Time

# ADE+ cells/mL
at START

# ADE+ cells/mL
at STOP

 
| strain # 1 | 0.189  | 6.96  |  51h |4.05E+02 | 1.46E+05|

| strain # 2 | 0.198 | 7.17 | 51h | 2.60E+02 |1.15E+05 | | strain # 3 | 0.194 | 7.13 | 51h |2.85E+02 |2.24E+05 | | strain # 4 | 0.196 | 7.11 | 51h |1.43E+02 |3.72E+05 | | strain # 5 | 0.172 | 7.13 | 51h |2.25E+02 |2.58E+05 | | strain # 6 | 0.199 | 7.27 | 51h |2.55E+02 | 1.82E+05|

78.3.4. Our data#

| Strain | Induction | | | |
|—|—|—|—|—|—| | | OD start | OD stop | Time | # ADE+ cells/mL
at START | # ADE+ cells/mL
at STOP | | Byk832T_3 (Leila)| 0.098 | | 51h | | | | Byk832T_1 (Enzo) | 0.484 | | 51h | | | | Byk832T_2 (Enzo) | 0.510 | | 51h | | | | Byk832T_3 (Enzo)| 0.393 | | 51h | | |

  1. Reseed

  • Determine the number of ADE+ clones after 50-52 hours of induction. If you have around 2.35E5 ADE+ cells per mL is GREAT.

  • Inoculate 14E6 cells (14 million of cells) in SD-ADE 2% glucose at OD=0.2 in 2-4L

  • Incubate at 30C, 140 rpm, harvest at OD~2 (80 hours). Note that the final OD will not exceed the value of 2.

  • Fo reasons we ignore, the presence of ade- cells in the mix inhibits the growth of ade+ cells. This is why the culture will not grow above OD=2. This is also why you should not reinoculate at an OD above 0.2

  • To estimate the growth of ADE+ cells: dilute the culture 2000X, spread 200 ul on SD-ADE – dilute 40000X, spread 200 ul on SD and SD-URA – Expect the number of ADE+ cells to have been multiplied by ~1000X.

Note1: If you don’t mind increasing the volume, you can reseed at a lower OD, and thus have less growth inhibition. But in any case you want to inoculate enough cells so as to keep a good complexity in your library.

  • For example, a library containing 1.8E5 ADE+ cells/ml (OD=7.27, 0.15% ADE+ cells) after induction and reseeded at OD0.2 in 2L (1E7 independent transpositions) will reach an OD of 2 and a fold increase of 1250

– the same library reseeded at OD0.1 in 2L (5E6 independent transpositions) will reach OD3 for a fold increase of 3700

  1. Harvest cells and proceed to DNA extraction as in published protocol.

Because of the growth inhibition, only ~70% of the harvested cells are ADE+, meaning that during the sequencing reaction, 30% of the reads will map to the untransposed transposon in ADE2.

78.4. Results#

78.5. Conclusion#