User:Fermenter User/Notebook/BROM F7

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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

BROM F7

Day -2 (May 12 Wed):
         5/12/2010 (16:00) Used MD media for first stage cell culturing Autoclaved one 2L flask for culturing.

Day -1 (May 13 Thu):
         5/13/2010 (16:00)   2 flask of 1L of 250mLBMGY for second stage culturing before fermentation for A25 mutant.          5/13/2010 (12:30)   Prepared all necessary media components for fermentation: <-- Prepare following media separate, do not combine! 1. 2 x 1.0L 10x YNB (filtered) 2. 2 x 1.0L Phosphate buffer, pH 6.0 (filtered) 3. 2 x 1.0L 10x Glycerol (filtered) 4. 2 x 20mL 500x Biotin (filtered) <-- keep at 4C before addition          5/13/2010 (12:30)    Prepared bottles: 1) Acid, 2 x 1-L (tubing separate)                                                  2) Base, 2 x 500-ml (tubing connected) 3) MeOH, 2 x 1-L (tubing connected)                                                 4) Adjutives/Inoculum, 2 x 2-L (tubing connected) 5) Water, 2-L +                 5/13/2010 (13:00)    Assemble bioreactors:                                                                          1. 2 x 3.5-L of BMXY:  <-- Peptone was missing!                                2. 2 x 1 ml of antifoam/ fermenter                                  Autoclave

Day 0 (May 14 Fri):
        5/14/2010 (11:14)     Cooling bioreactors: 300 rpm 5/14/2010 (11:15)    Start Fermenter #1 software 5/14/2010 (11:20)    Start Fermenter #2 software 5/14/2010 (13:15 - 14:35)    Transfer Adjutives using pumps. Combine: 3. 1 x 500 ml of 10x Potassium Phosphate 4. 1 x 500 ml of 10x YNB 5. 2 x 10 ml of 500x Biotin <-- Do not forget to add! 2 x 5 ml of Antibiotics 6. 1 x 250 ml of 10x Glycerol <-- Pepton was mixed with Glycerol 5/14/2010 (14:45)    Calibrate dO2 probes 5/14/2010 (15:00)    Start dO2 control (O2 valve Max)  5/14/2010 (15:30)     Fermentation 0:00 Flask (A25) OD: 7.0 Inoculate 2 fermenters with Sung-Hye 250 mL of culture in each fermentor          5/14/2010 (16:30)     Start pH control Acid (250-300 ml) Base (400 ml):

Prepared 1.0L 100% Methanol(non-HPLC grade) filter sterilize.

Day 1 (May 15 Sat):
         5/15/2010 (11:30)     Fermentation 00:00 F1 Acid ( ml) Base ( ml) Pressure OD (12.3) Contamination (No) Activity ( /100ul) F2 Acid ( ml) Base ( ml) Pressure OD (12.8) Contamination (No) Activity ( /100ul)

Day 2 (May 16 Sun): <font color=#FF00FF> MeOH Induction Day 1
<Adeleke>         5/16/2010 (12:00)     Fermentation 00:00 F1 Acid (50 ml) Base (390 ml) Pressure (Yes) OD (13.7) Contamination (No) Activity ( /100ul) F2 Acid (80 ml) Base (390 ml) Pressure (Yes) OD (13.9) Contamination (??) Activity ( /100ul)

<Sung-Hye>        5/16/2010 (16:00)     Fermentation 47:30 Refill Acid +500 ml each 5/16/2010 (16:30)    Fermentation 48:00 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 2 x 900 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 5/16/2010 (16:30)    MeOH sensor calibration F1 (6650 mV) F2 (5800 mV)

Day 3 (May 17 Mon): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>        5/17/2010 (9:30)      Fermentation 65:00 <font color=#FF00FF> MeOH induction 17:00 F2 Take out samples 600 ml, Change Filter <Adeleke>         5/17/2010 (16:30)     Fermentation 72:00 <font color=#FF00FF> MeOH induction 24:00 F1 Acid (500 ml) Base (360 ml) Pressure (No) OD (14.5) Contamination (No) Activity ( /100ul) MeOH ( mL) F2 Acid (500 ml) Base (360 ml) Pressure (No) OD (14.7) Contamination (Yes) Activity ( /100ul) MeOH ( mL)

Day 4 (May 18 Tue): <font color=#FF00FF> MeOH Induction Day 3
<Sung-Hye>        5/18/2010 (16:00)     Fermentation 98:00 <font color=#FF00FF> MeOH induction 48:00 F1 Acid (500 ml) Base (300 ml) Pressure OD  Contamination  Activity ( /100ul) MeOH (455 mL) F2 Acid (490 ml) Base (270 ml) Pressure OD  Contamination  Activity ( /100ul) MeOH (340 mL)

<Adeleke>         5/18/2010 (16:00)     Fermentation 98:00 <font color=#FF00FF> MeOH induction 48:00 F1 Acid (490 ml) Base (300 ml) Pressure (Yes) OD (17.8) Contamination (No) Activity ( /100ul) MeOH (460 mL) F2 Acid (490 ml) Base (280 ml) Pressure (No) OD (20.8) Contamination (???) Activity ( /100ul) MeOH (340 mL)

Day 5 (May 19 Wed): <font color=#FF00FF> MeOH Induction Day 4
<Sung-Hye>        5/19/2010 (8:30)      Fermentation 96:00 <font color=#FF00FF> MeOH induction 64:00 Take out 500 ml of sample from F1, change filter

<Raymond>         5/19/2010 (12:30)     Fermentation 100:00 <font color=#FF00FF> MeOH induction 68:00 F1 Acid (200 ml) Base (300 ml) Pressure (yes) OD (51.7) Contamination (No) Activity ( /100ul) MeOH (350 mL) Change filter F2 Acid (490 ml) Base (240 ml) Pressure (no) OD (80.9) Contamination (No) Activity ( /100ul) MeOH (75 mL)

<Sung-Hye>         Refill Acid (F1) + 300 ml                           Refill MeOH (F1: 350 + 650 = 1000 ml) (F2: 75 + 925 = 1000 ml)

Day 6 (May 20 Thu): <font color=#FF00FF> MeOH Induction Day 5
<Adeleke>         5/20/2010 (15:00)     Fermentation 128:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (480 ml) Base (275 ml) Pressure (No) OD (59.4) Contamination (No) Activity ( /100ul) MeOH ( mL) F2 Acid (480 ml) Base (205 ml) Pressure (No) OD (94.9) Contamination (??) Activity ( /100ul) MeOH ( mL)

Day 7 (May 21 Fri): <font color=#FF00FF> MeOH Induction Day 6
<Adeleke>         5/21/2010 (10:45)     Fermentation 152:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( ml) Base ( ml) Pressure OD  Contamination  Activity ( /100ul) MeOH ( mL) F2 Acid ( ml) Base ( ml) Pressure OD  Contamination  Activity ( /100ul) MeOH ( mL)

<Adeleke&Sung-Hye> 5/21/2010 (18:00)    Fermentation 160:00 <font color=#FF00FF> MeOH induction 00:00 Harvest