User:Fermenter User/Notebook/BROM A12T

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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 A12T

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

Day -1 (Mar 2 Tue):
          3/2/2010 (00:00)   1 flask of 2L of 500mLBMGY for second stage culturing before fermentation for each wild type.           3/2/2010 (00:00)   Prepared all necessary media components for fermentation: <-- Prepare following media separate, do not combine! 1. 1.0L 10x YNB (filtered, excess amount) 2. 1.0L Phosphate buffer, pH 6.0 (autoclaved, excess amount) 3. 1.0L 10x Glycerol (filtered) 4. 20mL 500x Biotin (filtered, excess amount) <-- keep at 4C before addition

         3/2/2010 (14:00)    Prepared bottles: 1) Acid, 1 x 1-L (tubing separate)                                                  2) Base, 1 x 500-ml (tubing connected) 3) MeOH, 1 x 1-L (tubing connected)                                                 4) Adjutives/Inoculum, 1 x 2-L (tubing connected) 5) Water, 2-L +                              3/2/2010 (14:20)    Assemble bioreactors:                                                                          1. 2 x 3.5-L of BMXY:                                 2. 1 ml of antifoam/ fermenter                                  Autoclave

          3/2/2010 (00:00) Leave the master culturing media containing the following items in the fermenter room: 2) YNB                                                 3) Phosphate buffer 4) Glycerol                                                 <-- keep Biotin at 4C before addition

Day 0 (Mar 3 Wed):
         3/3/2010 (10:20)     Cooling bioreactors: 250 rpm 3/3/2010 (10:20)    Purge O2 <0.1 L/min at 250 rpm 3/3/2010 (10:27)    Start Fermenter #1 software 3/3/2010 (11:45 - 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 3/3/2010 (16:30)    Set rpm at remote 300-1200 3/3/2010 (16:30)    Start pH control Acid (400 ml) Base (300 ml): 3/3/2010 (12:00)    Calibrate dO2 probes 3/3/2010 (12:00)    Start dO2 control (O2 valve Max)   3/3/2010 (16:30)     Fermentation 0:00 5. 1 x 10 ml of 500x Biotin <-- Do not forget to add! Flask (WT) OD: >6.0 <-- We have to wait until OD reaches at least 6 or higher! Inoculate 1 fermenter with Sung-Hye ~100 mL of culture in each fermentor           3/3/2010 (00:00)     Fermentation 00:00 Prepared 1.0L 100% Methanol(non-HPLC grade) filter sterilize.

Day 1 (Mar 4 Thr):
         3/4/2010 (16:00)     Fermentation 24:00 F1 Acid (400 ml) Base (240 ml)           3/4/2010 (16:00)     Fermentation 24:00 (F1)=(O.D:7.5), contamination? (No)

Day 2 (Mar 5 Fri): <font color=#FF00FF> MeOH Induction Day 1
<Sung-Hye>         3/5/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Adeleke>          3/5/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??), contamination? (Y/N) <Sung-Hye>         3/4/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 1 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 3/4/2010 (00:00)    MeOH sensor calibration

Day 3 (Mar 6 Sat): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>         3/6/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Adeleke>          3/6/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)

Day 4 (Mar 7 Sun): <font color=#FF00FF> MeOH Induction Day 3
<Sung-Hye>         3/7/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Adeleke>          3/7/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)

Day 5 (Mar 8 Mon): <font color=#FF00FF> MeOH Induction Day 4
<Sung-Hye>         3/8/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Adeleke>          3/8/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)