User:Fermenter User/Notebook/BROM A11

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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 A11 and F6

Day -3 (Feb 21 Sun):
         2/21/2010 (00:00) Used MD media for first stage cell culturing Autoclaved one 2L flask for culturing. Prepared all necessary media components for fermentation: 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) (Take one third of each above media components, and combine them to make 1 bottles of 1.51L culturing media)

Day -2 (Feb 22 Mon):
          2/22/2010 (00:00)   1 flask of 2L of 500mLBMGY for second stage culturing before fermentation for each wild type.

         2/22/2010 (13:30)    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) Adjutives/Inoculum, 2 x 2-L (tubing connected) 5) Water, 2-L +                              2/22/2010 (14:10)    Assemble bioreactors:                                                                          1. 2 x 3.5-L of BMXY:                                 2. 1 ml of antifoam/ fermenter                               3. 2 x 500 ml of 10x Potassium Phosphate <-- Normally add separate                                6. 2 x 250 ml of 10x Glycerol <-- Normally add separate                                   Autoclave

          2/22/2010 (00:00) Leave the master culturing media containing the following items in the fermenter room: 2) YNB                                                 3) Biotin <-- Normally kept at 4C before addition

Day -1 (Feb 23 Tue): 
         2/23/2010 (10:00)     Cooling bioreactors: 250 rpm 2/23/2010 (10:00)    Purge Air 1 L/min at 400 rpm 2/23/2010 (10:20 - 10:45)    Transfer Adjutives using pumps. Combine: 4. 2 x 500 ml of 10x YNB 5. 2 x 10 ml of 500x Biotin 2/23/2010 (10:21)    Start Fermenter #1 software 2/23/2010 (10:22)    Start Fermenter #2 software 2/23/2010 (10:40)    Set rpm at 900 2/23/2010 (13:00)    Start pH control Acid (500 ml) Base (400 ml): Mut-: 1M Base, Mut+: 4M Base 2/23/2010 (130:00)    Calibrate dO2 probes 2/23/2010 (13:00)    Start dO2 control (O2 valve Max)

Day 0 (Feb 25 Thu):
  2/25/2010 (14:00)     Fermentation 0:00 Flask (WT) OD: 2 Flask (mut) OD: 2 Inoculate 2 fermentors with Sung-Hye ~300 mL of culture in each fermentor           2/25/2010 (14:00)     Fermentation 00:00 Prepared 1.5L 100% Methanol(non-HPLC grade) filter sterilize.

Day 1 (Feb 26 Fri):  MeOH Induction Day 1
         2/26/2010 (15:00)     Fermentation 25:00 F1 Acid (?? ml) Base (?? ml) F2 Acid (?? ml) Base (?? ml) <Adeleke>          2/26/2010 (15:00)     Fermentation 25:00 (F1)=(O.D:??), contamination? (Y/N) Filter changed (F2)=(O.D:??), contamination? (Y/N) <Sung-Hye>         2/26/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 2 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 2/26/2010 (00:00)    MeOH sensor calibration

Day 2 (Feb 27 Sat): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>         2/27/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) F2 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Adeleke>          2/27/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N) (F2)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)

Day 3 (Feb 28 Sun): <font color=#FF00FF> MeOH Induction Day 3
<Sung-Hye>         2/28/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (500 ml) Base (400 ml) MeOH F2 Acid (500 ml) Base (400 ml) MeOH <Adeleke>          2/28/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N) (F2)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)

Day 4 (Mar 1 Mon): <font color=#FF00FF> MeOH Induction Day 4
<Sung-Hye>         3/1/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH F2 Acid (?? ml) Base (?? ml) MeOH <Adeleke>          3/1/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N) (F2)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)

Day 5 (Mar 2 Tue): <font color=#FF00FF> MeOH Induction Day 5
<Sung-Hye>         3/2/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 F1 Acid (?? ml) Base (?? ml) MeOH F2 Acid (?? ml) Base (?? ml) MeOH <Adeleke>          3/2/2010 (00:00)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (F1)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N) (F2)=(O.D:??, Activity: ??/100ul SN), contamination? (Y/N)