User:Fermenter User/Notebook/BROM A13T

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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 A13T

Day -2 (Mar 1 Mon):
         3/29/2010 (16:00) Used MD media for first stage cell culturing Autoclaved one 2L flask for culturing.

Day -1 (Mar 2 Tue):
          3/30/2010 (16:00)   1 flask of 2L of 500mLBMGY for second stage culturing before fermentation for wild type.

Day 0 (Mar 3 Wed):
          3/31/2010 (9:00)   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           3/31/2010 (9:30)    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 +                              3/31/2010 (11:00)    Assemble bioreactors:                                                                          1. 2 x 3.5-L of BMXY:                                 2. 2 x 1 ml of antifoam/ fermenter                                  Autoclave                   3/31/2010 (12:45)     Cooling bioreactors: 150 rpm                            3/31/2010 (13:00)     Start Fermenter #1 software                            3/31/2010 (13:02)     Start Fermenter #2 software                             3/31/2010 (13:45 - 00:00)     Transfer Adjutives using pumps.                                 Combine:                                   3. 1 x 500 ml of 10x Potassium Phosphate                                                 4. 1 x 500 ml of 10x YNB                                6. 1 x 250 ml of 10x Glycerol 3/31/2010 (15:00)    Calibrate dO2 probes 3/31/2010 (15:00)    Start dO2 control (O2 valve Max)   3/31/2010 (16:00)     Fermentation 0:00 5. 2 x 10 ml of 500x Biotin <-- Do not forget to add! 2 x 5 ml of Antibiotics Flask (WT) OD: >5.5 Inoculate 2 fermenters with Sung-Hye ~300 mL of culture in each fermentor           3/31/2010 (16:30)     Start pH control Acid (250-300 ml) Base (400 ml):

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

Day 1 (Apr 1 Thr):
         4/1/2010 (9:30)     Fermentation 17:30 F1 Acid (250 ml) Base (300 ml) Pressure (no) OD Contamination  Activity ( /100ul) F2 Acid (200ml) Base (290 ml) Pressure (no) OD Contamination  Activity ( /100ul)

<Raymond>         4/1/2010 (11:00)     Fermentation 20:00 F1 Acid (250 ml) Base (300 ml) Pressure (no) OD (12.15) Contamination (Maybe-1 spec) Activity ( /100ul) F2 Acid (190 ml) Base (290 ml) Pressure (no) OD (19.2) Contamination (Maybe-1 spec) Activity ( /100ul)

<Sung-Hye>        4/1/2010 (17:00)     Fermentation 26:00 F1 Acid (+350 ml = 500 ml) F2 Acid (+400 ml = 500 ml)

Day 2 (Apr 2 Fri): <font color=#FF00FF> MeOH Induction Day 1
<Raymond>         4/2/2010 (11:30)     Fermentation 43:30 F1 Acid (340 ml) Base (295 ml) Pressure (no) OD (15.7) Contamination (no) Activity ( /100ul) F2 Acid (390 ml) Base (295 ml) Pressure (no) OD (15.6) Contamination (no) Activity ( /100ul)

<Sung-Hye>        4/2/2010 (11:30)     Fermentation 43:30 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 1 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 4/2/2010 (11:30)    MeOH sensor calibration

<Adeleke>         4/2/2010 (18:00)     Fermentation 50:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (300 ml) Base (280 ml) Pressure (No) OD (12.56) Contamination (No) Activity ( 0/100ul) F2 Acid (360 ml) Base (280 ml) Pressure (No) OD (12.22) Contamination (No) Activity ( 0/100ul)

Day 3 (Apr 3 Sat): <font color=#FF00FF> MeOH Induction Day 2
<Raymond>         4/3/2010 (12:00)     Fermentation 66:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( 300ml) Base ( 290ml) Pressure ( No ) OD (13.21) Contamination (Maybe) Activity ( 2.29/100ul) MeOH (500mL) F2 Acid (325 ml) Base ( 298ml) Pressure (No) OD (13.1) Contamination (No) Activity ( 1.40/100ul) MeOH (480mL)

<Adeleke>         4/3/2010 (18:00)     Fermentation 74:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (290 ml) Base (280 ml) Pressure (No) OD (13.39) Contamination (?) Activity ( 4.35/100ul) F2 Acid (310 ml) Base (280 ml) Pressure (No) OD (13.16) Contamination (No) Activity ( 3.98/100ul)

Day 4 (Apr 4 Sun): <font color=#FF00FF> MeOH Induction Day 3
<Raymond>         4/4/2010 (09:00)     Fermentation 90:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( 290 ml) Base ( 299ml) Pressure (Yes) OD (13.31) Contamination (No) Activity (1.12 /100ul) MeOH (475mL) F2 Acid ( 300ml) Base (300ml) Pressure (No) OD (13.09) Contamination (No) Activity ( 1.04/100ul) MeOH (450mL)

<Adeleke>         4/4/2010 (18:00)     Fermentation 98:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( ml) Base ( ml) Pressure OD  Contamination  Activity ( /100ul) F2 Acid ( ml) Base ( ml) Pressure OD  Contamination  Activity ( /100ul)

Day 5 (Apr 5 Mon): <font color=#FF00FF> MeOH Induction Day 4
<Raymond>         4/5/2010 (13:45)     Fermentation 104:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( 250ml) Base ( 290ml) Pressure (Yes) OD (13.21) Contamination (No) Activity (2.21 /100ul) MeOH (450mL) F2 Acid (290 ml) Base ( 299ml) Pressure (No) OD (13.1) Contamination (No) Activity (3.23 /100ul) MeOH (450mL)

<Adeleke>         4/5/2010 (18:00)     Fermentation 112:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (220 ml) Base (270 ml) Pressure (No) OD (13.34) Contamination (No) Activity (7.084 /100ul) MeOH (435mL) F2 Acid (270 ml) Base (290 ml) Pressure (No) OD (12.72) Contamination (No) Activity (4.652 /100ul) MeOH (400mL)

Day 6 (Apr 6 Tue): <font color=#FF00FF> MeOH Induction Day 5
<Raymond>         4/5/2010 (11:15)     Fermentation 128:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( 250ml) Base ( 250ml) Pressure (No) OD (13.22) Contamination (No) Activity (8 /100ul) MeOH (400mL) F2 Acid (290 ml) Base (290 ml) Pressure (No) OD (13.10) Contamination (No) Activity (6 /100ul) MeOH (400mL)

<Adeleke>         4/5/2010 (18:00)     Fermentation 136:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (215 ml) Base (260 ml) Pressure (No) OD (13.99) Contamination (No) Activity (8.251 /100ul) MeOH (mL) F2 Acid (260 ml) Base (275 ml) Pressure (Yes) OD (12.96) Contamination (No) Activity (6.960 /100ul) MeOH (mL)

Day 7 (Apr 7 Wed): <font color=#FF00FF> MeOH Induction Day 6
<Raymond>         4/6/2010 (10:45)     Fermentation 152:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid ( 225ml) Base (250 ml) Pressure (No) OD (13.28) Contamination (No) Activity (6.15 /100ul) MeOH (350mL) F2 Acid ( 280ml) Base ( 280ml) Pressure (No) OD (13.18) Contamination (No) Activity (4.15 /100ul) MeOH (350mL)

<Adeleke>         4/6/2010 (18:00)     Fermentation 160:00 <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)