User:Fermenter User/Notebook/MOLD C1

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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 C1

Day -5 (Jun 18 Fri):
                 6/18/2010 (pm) Pick a colony from MD plate and grew them in 5-ml MD

Day -2 (Jun 21 Mon):
             6/21/2010 (11:15 - 11:45)                  6/21/2010 (before go home) Take 50 ul of culture and grow them in fresh 5-ml MD media              6/21/2010 (before go home) Thaw glycerol stock and grow them in fresh 5-ml MD media

Day -1 (Jun 22 Tue): MEDIA PREPARATION DAY
 6/22/2010 (before 11am) Give BMXY (yeast extract + peptone for 3.5-L) to Sung-Hye Prepare: YNB, Phosphate buffer, Glycerol, Biotin, Acid (5N Acetic Acid)         6/22/2010 (11:00)    Assemble bioreactors: 1. Calibrate pH sensors 2. 2 x 3.5-L of BMXY: 3. 2 x 1-ml Antifoam         6/22/2010 (12:00)   Prepare 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                 6/22/2010 (12:53)    Autoclave                          6/22/2010 (14:20)    Autoclave completed!

   6/22/2010 (before go home) Spind down cells and resuspend in fresh 50-ml BMGY media Incubator in Fermentation Suite used

Day 0 (June 23 Wed): INOCULATION DAY
 6/23/2010 (before 10am) Bring additives to fermenter room: 1) Potassium Phosphate                                                 2) YNB (Bromme lab added biotin here and kept at 4C) 3) Glycerol                                                 4) Biotin <-- from 4C Transfer Adjutives using pumps. 6/23/2009 (10:15-11:30) Combine: 3. 2 x 500 ml of 10x Potassium Phosphate 4. 2 x 500 ml of 10x YNB 5. 2 x 10 ml of 500x Biotin 6. 2 x 500 ml of 10x Glycerol 6/23/2009 (10:34)    Start Fermenter #1 software 6/23/2009 (10:35)    Start Fermenter #2 software         6/23/2009 (11:35)     Cooling bioreactors Purge Air 1 L/min at 100 rpm 6/23/2009 (11:35)    Set rpm at 900 6/23/2009 (13:30)    Calibrate dO2 probes 6/23/2009 (13:50)    Strt dO2 control <Everyone>        6/23/2009 (14:00)     Fermentation 0:00 Flask OD: Cat K (F1) - 15.2 <-Raymond & Dannis Flask OD: (F2) - 9.2 <--Raj Inoculate 2 fermentors (No contamination) <Sung-Hye>        6/23/2009 (14:05)     Start pH control Acid (7.5M 300 ml) Base (2M 200 ml) <Raj & Raymond>   6/23/2010 (pm)  make glycerol stock -                         F2 used wrong acid (swiched with MeOH!) so it will be terminated! New fermentation for F2 will be prepared

Day 1 (June 24 Thu):
<Raj>             6/24/2010 (14:00)     Fermentation 24:00 F2 Acid (200 ml) Base (150 ml) OD (16.98) Contamination (N) Pressure (N) Change Filter (N)

Day 2 (June 25 Fri): <font color=#FF00FF> MeOH Induction Day 1
<Raj>             6/25/2010 (14:00)     Fermentation 48:00 F2 Acid (20ml topped up with 466ml)Base(150 ml)OD(17.80)Contamination(N)Pressure(N)Change Filter(N) <Raj>             6/25/2010 (14:00)     Fermentation 48:00 Prepared 1L 100% Methanol(non-HPLC grade) filtered. <Sung-Hye>        6/25/2010 (14:00)     MeOH sensor calibration 6/25/2010 (14:00)    Fermentation 48:00 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 1000 ml of MeOH: Tasfer MeOH into MeOH feeding bottles.

Day 3 (June 26 Sat): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>        6/26/2010 (10:00)     Fermentation 68:00 <font color=#FF00FF> MeOH induction 20:00 Checked fermenter. Everything look ok. Seems like MeOH (0.7%) is still decreasing slowly to 0.5%. MeOH bottle shows no change yet. 6559 mV       <Raj>              6/26/2010 (14:00)     Fermentation 72:00 <font color=#FF00FF> MeOH induction 24:00 F2: Acid (220 ml) Base (100 ml) MeOH (960 ml) OD (19.2) Contamination (N) Pressure (N) Change Filter (N)

Day 4 (June 27 Sun): <font color=#FF00FF> MeOH Induction Day 3
<Raj&Sung-Hye>    6/27/2010 (14:00)     Fermentation 96:00 <font color=#FF00FF> MeOH induction 48:00 F2: Acid (130 ml) Base (100 ml) MeOH (950 ml) OD (18.0) Contamination (N) Pressure (N) Change Filter (N)

Day 5 (June 28 Mon): <font color=#FF00FF> MeOH Induction Day 4
<Raj>             6/28/2010 (14:00)     Fermentation 112:00 <font color=#FF00FF> MeOH induction 72:00 F2: Acid (50 ml) Base (100 ml) MeOH (930 ml) OD (19.8) Contamination (N) Pressure (Y) Change Filter (Y) <sung-Hye>        6/28/2010 (16:00) Added 150 ml of Acid, Total=150 ml

Day 6 (June 29 Tue): <font color=#FF00FF> MeOH Induction Day 5
<Raj>             6/29/2010 (14:00)     Fermentation 136:00 <font color=#FF00FF> MeOH induction 96:00 F2: Acid (120 ml) Base (120 ml) MeOH (900 ml) OD (20.5) Contamination (N) Pressure (N) Change Filter (N)

Day 7 (June 30 Wed): <font color=#FF00FF> MeOH Induction Day 6
<Raj>             6/30/2010 (14:00)     Fermentation 160:00 <font color=#FF00FF> MeOH induction 120:00 F2: Acid (50 ml) Base (110 ml) MeOH (880 ml) OD (20.5) Contamination (N) Pressure (N) Change Filter (N)

Harvest