User:Fermenter User/Notebook/BROM A9

{| width="800"
 * style="background-color: #cdde95;" align="center"|
 * style="background-color: #cdde95;" align="center"|




 * align="center" style="background-color: #e5edc8;" |


 * colspan="2" style="background-color: #F2F2F2;" align="right"|Customize your entry pages 
 * colspan="2"|
 * colspan="2"|
 * colspan="2"|

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 A9

Day -2 (Nov 2 Mon):
             11/2/2009 (pm) Used MD media for first stage cell culturing Autoclaved one 2L flask for culturing. Prepared all necessary media components for fermentation: 1. 1.5L 10x YNB (filtered, excess amount) 2. 1.5L Phosphate buffer, pH 6.0 (autoclaved, excess amount) 3. 1.5L 10x Glycerol (filtered) 4. 30mL 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 -1 (Nov 3 Tue):
          10/26/2009 (10am)   1 flask of 2L of 500mLBMGY for second stage culturing before fermentation for each wild type.          11/3/2009 (9:30)    Prepared bottles: 1) Acid, 3 x 1-L (tubing separate)                                                  2) Base, 3 x 500-ml (tubing connected) 3) MeOH, 3 x 1-L (tubing connected)                                                 4) Adjutives/Inoculum, 3 x 2-L (tubing connected) 5) Water, 2-L +                              11/3/2009 (12:22)    Assemble bioreactors:                                                                          1. 2 x 3.5-L of BMXY:                                 2. 1 ml of antifoam/ fermenter                                  Autoclave                  10/26/2009 (13:30) Leave the master culturing media containing the following items in the fermenter room:                                                  1) Potassium Phosphate 2) YNB                                                 3) Biotin 4) Glycerol                 11/3/2009 (15:25)     Cooling bioreactors: 400 rpm                            11/3/2009 (15:25)     Purge Air 1 L/min at 400 rpm                             11/3/2009 (15:43)     Start Fermenter #1 software                            11/3/2009 (15:44)     Start Fermenter #2 software                            11/3/2009 (15:45)     Start Fermenter #2 software                                                  Transfer Adjutives using pumps.                             11/3/2009 (15:30-16:30)  Combine:                                                      3. 3 x 500 ml of 10x Potassium Phosphate                               4. 3 x 500 ml of 10x YNB                                5. 3 x 10 ml of 500x Biotin  (<--- Mixed with YNB!)                                6. 3 x 250 ml of 10x Glycerol                             11/3/2009 (16:00)     Start pH control Acid (500 ml) Base (400 ml) 11/3/2009 (16:40)    Set rpm at 900 11/3/2009 (16:40)    Calibrate dO2 probes 11/3/2009 (16:40)    Start dO2 control (O2 valve Max)

Day 0 (Nov 4 Wed): 
  11/4/2009 (11:00)     Fermentation 0:00 Flask 1 OD: 18.2 Inoculate 3 fermentors with Sung-Hye ~180mL of culture in each fermentor           11/4/2009 (13:00)     Fermentation 24:00 Prepared 1.5L 100% Methanol(non-HPLC grade) filter sterilize.

Day 1 (Nov 5 Thu):  MeOH Induction Day 1
          11/5/2009 (09:00)     Fermentation 22:00 F1 Acid (470 ml) Base (340 ml) F2 Acid (160 ml) Base (400+160 ml) --> Output connected wrong. F3 Acid (480 ml) Base (290 ml), change filter <Raymond>           11/5/2009 (10:00)     Fermentation 23:00 (F1)=(O.D:18.97), no contamination (F2)=(O.D:19.73), no contamination (F3)=(O.D:17.21), no contamination <Sung-Hye>          11/5/2009 (11:00)     Fermentation 24:00 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 3 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 11/5/2009 (11:00)    MeOH sensor calibration

Day 2 (Nov 6 Fri): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>          11/6/2009 (09:00)     Fermentation 46:00 <font color=#FF00FF> MeOH induction 22:00 F1 Acid (460 ml) Base (340 ml) MeOH (500-480 =20 ml) F2 Acid (150 ml) Base (160 ml) MeOH (470-450 =20 ml) F3 Acid (440 ml) Base (290 ml) MeOH (480-460 =20 ml) <Raymond>           11/6/2009 (13:00)     Fermentation 50:00 <font color=#FF00FF> MeOH induction 26:00 (F1)=(O.D:18.67, Activity: 0.00/100ul SN), no contamination (F2)=(O.D:18.98, Activity: 0.12/100ul SN), no contamination (F3)=(O.D:17.49, Activity: 0.00/100ul SN), no contamination

Day 3 (Nov 7 Sat): <font color=#FF00FF> MeOH Induction Day 3
<Raymond>           10/31/2009 (04:00)     Fermentation 87:00 <font color=#FF00FF> MeOH induction 63:00 (F1)= Add Biotin (F2)= Add Biotin <Sung-Hye>          10/31/2009 (16:00)     Fermentation 99:00 <font color=#FF00FF> MeOH induction 75:00 F1: Acid tubing was damaged. Remove Acid (Output#3) from pH control. Noticed MeOH leve was going down on both F1 and F2

Day 4 (Nov 8 Sun): <font color=#FF00FF> MeOH Induction Day 4
<Sung-Hye>          11/1/2009 (10:30)     Fermentation 117:30 <font color=#FF00FF> MeOH induction 93:30 F1 Acid (430 ml) Base (380 ml) MeOH (680 ml) F2 Acid (360 ml) Base (450 ml) MeOH (700 ml) MeOH level gone down since biotin addition, but DO level is >100% which is indication of unhealthy cells. <Raymond>           11/1/2009 (13:00)     Fermentation 120:00 <font color=#FF00FF> MeOH induction 96:00 (F1)=(O.D:??, Activity:??) (F2)=(O.D:??, Activity:??)

testtest

Day 5 (Nov 9 Mon): <font color=#FF00FF> MeOH Induction Day 5
<Sung-Hye>          11/2/2009 (09:00)     Fermentation 140:00 <font color=#FF00FF> MeOH induction 116:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) F2 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Raymond>           11/2/2009 (13:00)     Fermentation 144:00 <font color=#FF00FF> MeOH induction 120:00 (F1)=(O.D:??, Activity:??) (F2)=(O.D:??, Activity:??)

HARVEST