User:Fermenter User/Notebook/BROM A14T

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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 A14T

Day -2 (Jun 23 Wed):
         6/23/2010 (before go home) Thaw glycerol stock and grow them in fresh 5-ml MD media Incubator in Fermentation Suite used          6/23/2010 (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 (Jun 24 Thu): MEDIA PREPARATION DAY
        6/24/2010 (9:00)    Assemble bioreactors: 1. Calibrate pH sensors 2. 3.5-L of BMXY: 3. 1-ml Antifoam         6/24/2010 (9:00)   Prepare bottles 1) Acid, 1-L (tubing separate)                                                  2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected)                                                 4) Additives/Inoculum, 2 x 1-L (tubing connected) 5) Water, 2-L                 6/24/2010 (9:35)    Autoclave                          6/24/2010 (11:00)   Autoclave completed!                 6/24/2010 (15:20)                              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.                  6/24/2010 (16:00-17:15)  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                            6/24/2009 (17:00)     Start Fermenter #1 software

         6/24/2010 (before go home) Spind down cells and resuspend in fresh 50-ml BMGY media Incubator in Fermentation Suite used

Day 0 (June 25 Fri): INOCULATION DAY
        6/25/2009 (8:40)     Cooling bioreactors Purge Air 1 L/min at 500 rpm         6/25/2009 (9:00)     Calibrate dO2 probes 6/25/2009 (9:00)    Strt dO2 control  6/25/2009 (12:00)    Fermentation 0:00 Flask OD: Cat K (F1) - 10.9 <-Raymond & Dannis <Sung-Hye>        6/25/2009 (12:00)     Start pH control Acid (300 ml) Base (180 ml) <Dannis>          6/27/2010 (12:00)     Fermentation 48:00 Prepared 0.5L 100% Methanol(non-HPLC grade) filtered.

Day 1 (June 26 Sat):
<Dannis>          6/26/2010 (12:00)     Fermentation 24:00 F1 Acid (40 ml) Base (135 ml) OD (20.5) Contamination (N) Pressure (N) Change Filter (N)

Day 2 (June 27 Sun): <font color=#FF00FF> MeOH Induction Day 1
<Dannis>          6/27/2010 (12:00)     Fermentation 48:00 F1 Acid (35 ml, then add 500 mL to make 535 mL) Base (135 ml) OD (22.3) Contamination (N? but I saw one bacteria swimming, thus I assume it's from the syringe.) Pressure (N)$ Change Filter (N) <Sung-Hye>        6/27/2010 (12:50)     Fermentation 48:50 Changed syringe <Sung-Hye>        6/27/2010 (12:58)     MeOH sensor calibration 6/27/2010 (12:58)    Fermentation 48:58 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 325 ml of MeOH: Tasfer MeOH into MeOH feeding bottles.

Day 3 (June 28 Mon): <font color=#FF00FF> MeOH Induction Day 2
<Dennis>         6/28/2010 (10:30)     Fermentation 72:00 <font color=#FF00FF> MeOH induction 24:00 F1: Acid (500 ml) Base (135 ml) MeOH (315 ml) OD (28.1) Contamination (N) Pressure (N) Change Filter (N) Activity assay (161500 units / 5L), (3.23 units/100 ul),(0.756 mg/5-L)

Day 4 (June 29 Tue): <font color=#FF00FF> MeOH Induction Day 3
<Dennis>         6/29/2010 (12:00)     Fermentation 96:00 <font color=#FF00FF> MeOH induction 48:00 F1: Acid (440 ml) Base (135 ml) MeOH (290 ml) OD (22.1) Contamination (N) Pressure (N) Change Filter (N) Activity: (4.77 units/100 ul),(1.12 mg/5-L). I tried doing a sample where a performed syringe sterilization with the supernatant. It gave an activity of 2.98, thus I do not believe the syringe makes a difference.

Day 5 (June 30 Wed): <font color=#FF00FF> MeOH Induction Day 4
<Dennis>         6/30/2010 (12:00)     Fermentation 112:00 <font color=#FF00FF> MeOH induction 72:00 F1: Acid (420 ml) Base (135 ml) MeOH (250 ml) OD (21.45) Contamination (N) Pressure (N) Change Filter (N) Activity: (1.05 units/5 ul),(4.914 mg/5-L) Harvest