User:Fermenter User/Notebook/MOLD B1

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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

MOLD B1

Day -2 (June 17 Tue):
             6/19/2010 (12:30) Used BMGY media for first stage cell culturing Prepared all necessary media components for fermentation: 1. 0.5 L 10x YNB (filtered, excess amount) 2. 0.5 L Phosphate buffer, pH 6.0 (autoclaved, excess amount) 3. 0.5 L 10x Glycerol (filtered) 4. 10mL 500x Biotin (filtered, excess amount)

Day -1 (June 16 Wed):
              6/19/2010 (10:30)     Leave the master culturing media containing the following items in the fermenter room: 1) Potassium Phosphate                                                 2) YNB 3) Glycerol                      6/19/2010 (10:30)    Prepare 3-5L BMXY (1.4-L /fermenter x 2 + extra)                  6/19/2010 (11:30)    Assemble bioreactors:                                 1. Calibrate pH sensors                                                                        2. 2 x 1.4-L of BMXY:                                 3. 2 x 1-ml Antifoam                  6/19/2010 (12:00)    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) Additives/Inoculum, 2 x 1-L (tubing connected)                                                  5) Water, 2-L          6/19/2010 (13:30)    Autoclave Media leaked! (sample port was missing!) Sample ports tubing was connected and clamped!               6/19/2010 (15:30)    Made 2 x 1.4-L BMXY media again          6/19/2010 (16:00) 1. 2 x 1.4-L of BMXY 2. 2 x 1-ml Antifoam 6/19/2010 (16:09)   Autoclave again 6/19/2010 (17:45)   Autoclave completed!

Day 0 (Jun 17 Thu):
         6/17/2010 (11:00)     Cooling bioreactors Purge Air 1 L/min at 100 rpm Transfer Adjutives using pumps. 6/17/2010 (10:30-11:30) Combine: 3. 2 x 200 ml of 10x Potassium Phosphate 4. 2 x 200 ml of 10x YNB 5. 2 x 4 ml of 500x Biotin <--- from 4C 6. 2 x 200 ml of 10x Glycerol 6/17/2010 (13:30)    Set rpm at 250  <-- little bit low 6/17/2010 (13:30)    Calibrate dO2 probes 6/17/2010 (13:30)    Fermenter #2: Start dO2 control (O2 valve Max) 6/17/2010 (13:45)    Start pH control Acid (250 ml) Base (100 ml)       6/17/2010 (14:00)     Fermentation 0:00 Flask OD: 1.5? Inoculate 2 fermentors (F#1 5-ml, F#2 3.5-ml) 6/17/2010 (15:16)    Start Fermenter #1 software 6/17/2010 (15:18)    Start Fermenter #2 software

Day 1 (Jun 18 Fri):
6/18/2010 (14:00)    Fermentation 24:00 Check contamination with Raymond. Both F1 and F2 were containing mainly bacteria, but rarely Pichia cells. Starter cultures were also containing heavily contaminated culture. Teminate fermentation.


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