User:Fermenter User/Notebook/BROM A15

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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 A15

Day -2 (July 11 Sun):
          7/11/2010 (before go home) Thaw glycerol stock and grow them in fresh 5-ml MD media Incubator in Fermentation Suite used           7/11/2010 (before go home) Give BMXY (yeast extract + peptone for 3.5-L) to Sung-Hye Prepare: YNB, Phosphate buffer, Glycerol, Biotin, Acid (5M Acetic Acid)

Day -1 (July 12 Mon): MEDIA PREPARATION DAY
        7/12/2010 (14:00)    Assemble bioreactors: 1. Calibrate pH sensors 2. 3.5-L of BMXY: 3. 1-ml Antifoam         7/12/2010 (14:00)   Prepare bottles 1) Acid, 1-L (tubing separate)                                                  2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected)                                                 4) Additives/Inoculum, 1 x 500-ml (tubing connected) 5) Water, 2-L                 7/12/2010 (15:00)   Autoclave                          7/12/2010 (17:00)   Autoclave completed!

          7/12/2010 (before go home) Spind down cells and resuspend in fresh 50-ml BMGY media Incubator in Fermentation Suite used

Day 0 (July 13 Tue): INOCULATION DAY
          7/13/2010 (10:00) 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.         7/13/2010 (10:00-11:22)  Combine: 3. 500 ml of 10x Potassium Phosphate 4. 500 ml of 10x YNB 5. 10 ml of 500x Biotin 6. 500 ml of 10x Glycerol 7/13/2009 (10:05)    Start Fermenter #2 software         7/13/2009 (11:40)     Cooling bioreactors Purge Air 1 L/min at 500 rpm         7/13/2009 (11:22)     Calibrate dO2 probes 7/13/2009 (11:25)    Strt dO2 control  7/13/2009 (12:00)     Fermentation 0:00 Flask OD: Cat K (F1) - 34 <-Dennis Inoculate 50 ml      <Sung-Hye>         7/13/2009 (12:00)     Start pH control Acid (500 ml) Base (400 ml)

Day 1 (July 14 Wed):
<Dennis>          7/14/2010 (12:00)     Fermentation 24:00 F2 Acid(285 ml) Base(375 ml) OD(22.9) Contamination(N) Pressure(N) Change Filter(N)

Day 2 (July 15 Thu): <font color=#FF00FF> MeOH Induction Day 1
<Dennis>          7/15/2010 (12:00)     Fermentation 48:00 F2 Acid(150 ml) Base(375 ml) MeOH(--ml) OD(20.6) Contamination(N) Pressure(Y) Change Filter(Y) <Sung-Hye>        7/15/2010 (12:58)     MeOH sensor calibration 7/15/2010 (12:58)    Fermentation 48:58 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles.

Day 3 (July 16 Fri): <font color=#FF00FF> MeOH Induction Day 2
<Dennis>          7/16/2010 (11:00)     Fermentation 72:00 <font color=#FF00FF> MeOH induction 24:00 F1: Acid(95 ml) Base(375 ml) MeOH(480 ml) OD(20.55) Contamination(N) Pressure(N) Change Filter(N) Activity assay (0.311 units/100ul), (0.073 mg/5-L)

Day 4 (July 17 Sat): <font color=#FF00FF> MeOH Induction Day 3
<Dennis>          7/17/2010 (12:00)     Fermentation 96:00 <font color=#FF00FF> MeOH induction 48:00 F1: Acid(50 ml; then add acid to 400 mL) Base(375 ml) MeOH(450 ml) OD(22) Contamination(N) Pressure(N) Change Filter(Y/N) Activity assay (1.256 units/100ul), (0.294 mg/5-L)

Day 5 (July 18 Sun): <font color=#FF00FF> MeOH Induction Day 4
<Dennis>          7/18/2010 (12:30)     Fermentation 112:00 <font color=#FF00FF> MeOH induction 72:00 F1: Acid(400 ml) Base(375 ml) MeOH(425 ml) OD(19.75) Contamination(N) Pressure(N) Change Filter(N) Activity assay (1.76 units/100ul), (0.412 mg/5-L)

Day 6 (July 19 Mon): <font color=#FF00FF> MeOH Induction Day 5
<Dennis>          7/19/2010 (12:00)     Fermentation 136:00 <font color=#FF00FF> MeOH induction 96:00 F1: Acid(375 ml) Base(375 ml) MeOH(400 ml) OD(19.5) Contamination(N) Pressure(N) Change Filter(N) Activity assay (2.26 units/100ul), (0.529 mg/5-L)

Day 7 (July 20 Tue): <font color=#FF00FF> MeOH Induction Day 6
<Dennis>          7/20/2010 (12:00)     Fermentation 160:00 <font color=#FF00FF> MeOH induction 120:00 F1: Acid(355 ml) Base(370 ml) MeOH(472 ml) OD(21.7) Contamination(N) Pressure(N) Change Filter(N) Activity assay (5.19 units/100ul), (1.21 mg/5-L)

Day 8 (July 21 Wed): <font color=#FF00FF> MeOH Induction Day 7
<Dennis>          7/21/2010 (12:00)     Fermentation 184:00 <font color=#FF00FF> MeOH induction 144:00 F1: Acid(350 ml) Base(360 ml) MeOH(337 ml) OD(20.95) Contamination(N) Pressure(N) Change Filter(N) Activity assay (3.98 units/100ul), (0.93 mg/5-L)

Day 9 (July 22 Thu): <font color=#FF00FF> MeOH Induction Day 8
<Dennis>          7/22/2010 (12:00)     Fermentation 208:00 <font color=#FF00FF> MeOH induction 168:00 F1: Acid(??ml) Base(??ml) MeOH(?? ml) OD(??) Contamination(Y/N) Pressure(Y/N) Change Filter(Y/N) Activity assay (?? units/100ul), (?? mg/5-L) Harvest