User:Fermenter User/Notebook/BROM A9

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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 A9


Day -2 (Nov 2 Mon):

      <Raymond>              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):

      <Raymond>           10/26/2009 (10am)   1 flask of 2L of 500mLBMGY for second stage culturing before fermentation for each wild type.
<Sung-Hye> 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 +

<Sung-Hye> 11/3/2009 (12:22) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: 2. 1 ml of antifoam/ fermenter Autoclave <Raymond> 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
<Sung-Hye> 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):

      <Raymond&SungHye>   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
<Raymond> 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

      <Sung-Hye>           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 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): MeOH Induction Day 2

      <Sung-Hye>           11/6/2009 (09:00)      Fermentation 46:00  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  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): MeOH Induction Day 3

      <Raymond>            10/31/2009 (04:00)      Fermentation 87:00  MeOH induction 63:00   
                                                    (F1)= Add Biotin 
                                                    (F2)= Add Biotin
      <Sung-Hye>           10/31/2009 (16:00)      Fermentation 99:00  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): MeOH Induction Day 4

      <Sung-Hye>           11/1/2009 (10:30)      Fermentation 117:30  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  MeOH induction 96:00   
                                                    (F1)=(O.D:??, Activity:??) 
                                                    (F2)=(O.D:??, Activity:??)


  testtest

Day 5 (Nov 9 Mon): MeOH Induction Day 5

      <Sung-Hye>           11/2/2009 (09:00)      Fermentation 140:00  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  MeOH induction 120:00   
                                                    (F1)=(O.D:??, Activity:??) 
                                                    (F2)=(O.D:??, Activity:??)
                                                 HARVEST

References

     
    References regarding Feeding: 
                                    Media:DAnjou_(2000)_Biotech._Bioeng._(72).pdf
                                    Media:Loewen_(1997)_Appl._Microbiol._Biotechnol._48_p480.pdf 
    Effect of Ethanol or Acetate on Protein Expression in Pichia pastoris:
                                    Media:Meagher_(2001)_J._Bioscience._Bioeng._92_p337.pdf
   



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