User:Fermenter User/Notebook/OVER D1

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Background color codes:  Pastel Green: Major change of setup this time  yellow: Sung-Hye's correction or question to user

Letter color codes: Black: Basic comment Red: Emergency Information (Power shutdown, building access, Manager's absence etc) Blue: Fermentation Setup information Green: Discussion Orange: Fermentation hrs Purple: Induction hrs

OVER D1

Day -4 (Oct 24 Sat):
           10/24/2009 (11am) streak GS115/MMP27 clone 19 onto MD and MM plates, incubate over weekend at 30C

Day -2 (Oct 26 Mon):
     10/26/2009 (14:15) inoculate 5ml BMGY (50ml Falcon tube) with GS115/MMP27 clone 19, incubate 28C 250rpm o/n (important incubate set to 28C but temp fluctuates (never over 30C))

Day -1 (Oct 27 Tue):
             10/27/2009 (9:30)  Autoclave 2x 500ml PBS 1x 700ml BM_Y cool BM_Y to room temp, then add 100ml 10xYNB, 100ml 10x Glycerol 100ml 1M Potassiumphosphate buffer pH 6.0 and 2ml 500x Biotin            10/27/2009 (11:30)    Prepared bottles: 1) Acid, 1-L (tubing separate)                                                  2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected)                                                 4) Adjutives and Inoculum, 1-L (tubing connected) 5) Water, 2-L                          10/27/2009 (13:30)    Assemble bioreactors:                                                                          1. 1 x PBS 1-L of BMXY:                                    Autoclave               10/27/2009 (14:30) inoculate 20 ml BMGY in baffled flask with 100 ul preculture (OD600= 8.2)                                          inoculate o/n 250rpm 28C

Day 0 (Oct 28 Wed):
              10/28/2009 (9:27)   measured OD600= 10.5             10/28/2009 (9:30 - 10:00) Drain PBS bf and transfer media (1-L) using pump. (Verena already combine media in a sterilized bottle:) 2. 1 x 500 ml of 10x Potassium Phosphate 3. 1 x 500 ml of 10x YNB 4. 1 x 10 ml of 500x Biotin 5. 1 x 250 ml of 10x Glycerol 10/28/2009 (9:30) Purge Air 1 L/min at 800 rpm 10/28/2009 (9:50) Start Fermenter #3 software 10/28/2009 (10:00) Add Antifoam 6. 0.5 ml of antifoam/ fermenter 10/28/2009 (10:30) Set temperature Acid (500 ml) Base (500 ml) 10/28/2009 (10:30) Set rpm at 900 10/28/2009 (10:30) Calibrate dO2 probes 10/28/2009 (10:30) Start dO2 control (O2 valve Max)      10/28/2009 (10:40) Fermentation 0:00 Inoculate cells (O.D: 14, Volume: 17 ml) Cells were inoculated via ruber septum using 10-ml syringe needle.             10/28/2009 (10:30) Connect Acid and Base bottles Acid (500 ml) Base (400 ml) 10/28/2009 (10:30) Start pH control

Day 1 (Oct 29 Thu):  MeOH Induction Day 1
              10/29/2009 (10:30) Fermentation 24:00 take sample, measure OD600= 20,2 no contamination seen under the microscope leave growing till afternoon since till budding cells observed. <Verena>              10/29/2009 (14:30) Fermentation 28:00 sample measurement OD600= 27.5, no contamination, cells look healthy, still budding to sample for SDS-PAGE (60ulSN+10ulSDS-SB; cell lysate) <Sung-Hye>            10/29/2009 (15:00) Fermentation 28:30 <font color=#FF00FF> MeOH Induction 00:00 Connect MeOH 7. 1 x 1000 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. MeOH sensor calibration: 0.3% MeOH (3 ml/1-L) reads 9087 mV                               Maximum allowance for sensor: 9000 mV ≈ around 2.5 - 2.8% or so.

Day 2 (Oct 30 Fri): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>            10/30/2009 (9:30) Fermentation 47:00 <font color=#FF00FF> MeOH Induction 18:30 F3 Acid (490 ml) Base (370 ml) MeOH (970 ml) NOTE: Definately, it is Mut+ strain. It took more MeOH than Mut- or Muts. Also, it consumed lot of O2 which is indication of continuous cell growth. MeOH sensor was above measurement limit (>9000 mV) and inactivated during overnight. <Sung-Hye>            10/30/2009 (9:30) Fermentation 47:00 <font color=#FF00FF> MeOH Induction 18:30 Change MeOH setpoint to 8000 mM and reduce MeOH pump speed to half. <Verena>              10/30/2009 (10:30) Fermentation 48:00 <font color=#FF00FF> MeOH Induction 19:30 OD600= 30 no contamination, cells have huge vacuoles, because of starvation o/n and are still budding.

Day 3 (Oct 31 Sat): <font color=#FF00FF> MeOH Induction Day 3
<Verena>              10/31/2009 (14:30) Fermentation 76:00 <font color=#FF00FF> MeOH Induction 47:30 no contamination, budding yeasts and some with big vacuoles OD600= 37,3 <Sung-Hye>            10/31/2009 (16:00) Fermentation 77:30 <font color=#FF00FF> MeOH Induction 49:00 MeOH sensor was stabilized at 8000 mV. Everything looked fine.

Day 4 (Nov 1 Sun): <font color=#FF00FF> MeOH Induction Day 4
<Verena>              11/1/2009 (10:30) Fermentation 96:00 <font color=#FF00FF> MeOH Induction 67:30 OD600= 79.2, no contamination <Sung-Hye>            11/1/2009 (10:30) Fermentation 96:00 <font color=#FF00FF> MeOH Induction 67:30 Acid (490 ml), Base (300 ml), MeOH (800 ml) Pressurized vessel -> Change filter

Day 5 (Nov 2 Mon): <font color=#FF00FF> MeOH Induction Day 5
<Sung-Hye>            11/2/2009 (9:30)  Fermentation 119:00 <font color=#FF00FF> MeOH Induction 90:30 MeOH was in "safty cut-off" mode, meaning MeOH feeding speed was (not) too slow. Resumed MeOH pump --> NEXT time, use other MeOH pump! <Verena>              11/2/2009 (11:00) Fermentation 120:00 <font color=#FF00FF> MeOH Induction 91:30 no contamination, OD600= 84.2

Day 5 (Nov 3 Tue): <font color=#FF00FF> MeOH Induction Day 6
<Verena&SungHye>      11/3/2009 (11:00) Fermentation 148:00 <font color=#FF00FF> MeOH Induction 115:30 Acid (480 ml) Base (250 ml) Harvest OD600= 109,2