# Title: 29072019-SATAY 1st attempt with Byk832-T πŸ™ πŸ˜• ❌

# Date

Tuesday, 29072019 - 31072019

# Objective

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

# 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 πŸ• (14:30) 😊

    • 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 πŸ• (14:30) 😊

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

    # Benoit Data

    Strain Preculture
    OD start OD stop Time
    strain # 1 0.234 4.33 20h
    strain # 2 0.233 4.02 20h
    strain # 3 0.235 3.85 20h
    strain # 4 0.227 4.04 20h
    strain # 5 0.229 3.94 20h
    strain # 6 0.235 4.07 20h

    # Our data

    Strain (2 technical replicates) Preculture
    OD start (29072019-πŸ• -> 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
  3. 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-πŸ•₯ -> 10:30 start T=0 of induction.

    • To evaluate the background:

      • [x] Spread 200ul of this inoculum on SD-ADE
      • [x] Dilute the inoculum 1000x, spread 200ul on SD-URA and SD complete
        • score after 2 days:

        • [x] πŸ˜” 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

      # 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

      # 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
  4. 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.

# Results

# Conclusion