User:Fermenter User/Notebook/BROM A13T

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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 A13T


Day -2 (Mar 1 Mon):

      <Adeleke>          3/29/2010 (16:00)  
                                        Used MD media for first stage cell culturing 
                                        Autoclaved one 2L flask for culturing.

Day -1 (Mar 2 Tue):

      <Adeleke>           3/30/2010 (16:00)   1 flask of 2L of 500mLBMGY for second stage culturing before fermentation for wild type.

Day 0 (Mar 3 Wed):

      <Adeleke>           3/31/2010 (9:00)   Prepared all necessary media components for fermentation:
<-- Prepare following media separate, do not combine! 1. 2 x 1.0L 10x YNB (filtered) 2. 2 x 1.0L Phosphate buffer, pH 6.0 (filtered) 3. 2 x 1.0L 10x Glycerol (filtered) 4. 2 x 20mL 500x Biotin (filtered) <-- keep at 4C before addition

<Sung-Hye> 3/31/2010 (9:30) 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 +

<Sung-Hye> 3/31/2010 (11:00) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: 2. 2 x 1 ml of antifoam/ fermenter Autoclave

<Sung-Hye> 3/31/2010 (12:45) Cooling bioreactors: 150 rpm 3/31/2010 (13:00) Start Fermenter #1 software 3/31/2010 (13:02) Start Fermenter #2 software

3/31/2010 (13:45 - 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
3/31/2010 (15:00) Calibrate dO2 probes 3/31/2010 (15:00) Start dO2 control (O2 valve Max)

<Adeleke&SungHye> 3/31/2010 (16:00) Fermentation 0:00 5. 2 x 10 ml of 500x Biotin <-- Do not forget to add! 2 x 5 ml of Antibiotics Flask (WT) OD: >5.5 Inoculate 2 fermenters with Sung-Hye ~300 mL of culture in each fermentor
<SungHye> 3/31/2010 (16:30) Start pH control Acid (250-300 ml) Base (400 ml):



                                         Prepared 1.0L 100% Methanol(non-HPLC grade) filter sterilize.

Day 1 (Apr 1 Thr):

      <Sung-Hye>          4/1/2010 (9:30)      Fermentation 17:30  
                          F1 Acid (250 ml) Base (300 ml) Pressure (no) OD () Contamination () Activity ( /100ul)
                          F2 Acid (200ml) Base (290 ml) Pressure (no) OD () Contamination () Activity ( /100ul)
      <Raymond>          4/1/2010 (11:00)      Fermentation 20:00  
                          F1 Acid (250 ml) Base (300 ml) Pressure (no) OD (12.15) Contamination (Maybe-1 spec) Activity ( /100ul)
                          F2 Acid (190 ml) Base (290 ml) Pressure (no) OD (19.2) Contamination (Maybe-1 spec) Activity ( /100ul)
      <Sung-Hye>         4/1/2010 (17:00)      Fermentation 26:00  
                          F1 Acid (+350 ml = 500 ml) 
                          F2 Acid (+400 ml = 500 ml)

Day 2 (Apr 2 Fri): MeOH Induction Day 1

      <Raymond>          4/2/2010 (11:30)      Fermentation 43:30  
                          F1 Acid (340 ml) Base (295 ml) Pressure (no) OD (15.7) Contamination (no) Activity ( /100ul)
                          F2 Acid (390 ml) Base (295 ml) Pressure (no) OD (15.6) Contamination (no) Activity ( /100ul)
      <Sung-Hye>         4/2/2010 (11:30)      Fermentation 43:30  MeOH induction 00:00 
                                                 Connect MeOH
                             7. 1 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
4/2/2010 (11:30) MeOH sensor calibration
      <Adeleke>          4/2/2010 (18:00)      Fermentation 50:00  MeOH induction 00:00   
                          F1 Acid (300 ml) Base (280 ml) Pressure (No) OD (12.56) Contamination (No) Activity ( 0/100ul)
                          F2 Acid (360 ml) Base (280 ml) Pressure (No) OD (12.22) Contamination (No) Activity ( 0/100ul)

Day 3 (Apr 3 Sat): MeOH Induction Day 2

      <Raymond>          4/3/2010 (12:00)      Fermentation 66:00  MeOH induction 00:00   
                          F1 Acid ( 300ml) Base ( 290ml) Pressure ( No ) OD (13.21) Contamination (Maybe) Activity ( 2.29/100ul) MeOH (500mL)
                          F2 Acid (325 ml) Base ( 298ml) Pressure (No) OD (13.1) Contamination (No) Activity ( 1.40/100ul) MeOH (480mL)
      <Adeleke>          4/3/2010 (18:00)      Fermentation 74:00  MeOH induction 00:00   
                          F1 Acid (290 ml) Base (280 ml) Pressure (No) OD (13.39) Contamination (?) Activity ( 4.35/100ul)
                          F2 Acid (310 ml) Base (280 ml) Pressure (No) OD (13.16) Contamination (No) Activity ( 3.98/100ul)

Day 4 (Apr 4 Sun): MeOH Induction Day 3

      <Raymond>          4/4/2010 (09:00)      Fermentation 90:00  MeOH induction 00:00   
                          F1 Acid ( 290 ml) Base ( 299ml) Pressure (Yes) OD (13.31) Contamination (No) Activity (1.12 /100ul) MeOH (475mL)
                          F2 Acid ( 300ml) Base (300ml) Pressure (No) OD (13.09) Contamination (No) Activity ( 1.04/100ul) MeOH (450mL)
      <Adeleke>          4/4/2010 (18:00)      Fermentation 98:00  MeOH induction 00:00   
                          F1 Acid ( ml) Base ( ml) Pressure () OD () Contamination () Activity ( /100ul)
                          F2 Acid ( ml) Base ( ml) Pressure () OD () Contamination () Activity ( /100ul)

Day 5 (Apr 5 Mon): MeOH Induction Day 4

      <Raymond>          4/5/2010 (13:45)      Fermentation 104:00  MeOH induction 00:00   
                          F1 Acid ( 250ml) Base ( 290ml) Pressure (Yes) OD (13.21) Contamination (No) Activity (2.21 /100ul) MeOH (450mL)
                          F2 Acid (290 ml) Base ( 299ml) Pressure (No) OD (13.1) Contamination (No) Activity (3.23 /100ul) MeOH (450mL)
      <Adeleke>          4/5/2010 (18:00)      Fermentation 112:00  MeOH induction 00:00   
                          F1 Acid (220 ml) Base (270 ml) Pressure (No) OD (13.34) Contamination (No) Activity (7.084 /100ul) MeOH (435mL)
                          F2 Acid (270 ml) Base (290 ml) Pressure (No) OD (12.72) Contamination (No) Activity (4.652 /100ul) MeOH (400mL)

Day 6 (Apr 6 Tue): MeOH Induction Day 5

      <Raymond>          4/5/2010 (11:15)      Fermentation 128:00  MeOH induction 00:00   
                          F1 Acid ( 250ml) Base ( 250ml) Pressure (No) OD (13.22) Contamination (No) Activity (8 /100ul) MeOH (400mL)
                          F2 Acid (290 ml) Base (290 ml) Pressure (No) OD (13.10) Contamination (No) Activity (6 /100ul) MeOH (400mL)
      <Adeleke>          4/5/2010 (18:00)      Fermentation 136:00  MeOH induction 00:00   
                          F1 Acid (215 ml) Base (260 ml) Pressure (No)  OD (13.99) Contamination (No) Activity (8.251 /100ul) MeOH (mL)
                          F2 Acid (260 ml) Base (275 ml) Pressure (Yes) OD (12.96) Contamination (No) Activity (6.960 /100ul) MeOH (mL)

Day 7 (Apr 7 Wed): MeOH Induction Day 6

      <Raymond>          4/6/2010 (10:45)      Fermentation 152:00  MeOH induction 00:00   
                          F1 Acid ( 225ml) Base (250 ml) Pressure (No) OD (13.28) Contamination (No) Activity (6.15 /100ul) MeOH (350mL)
                          F2 Acid ( 280ml) Base ( 280ml) Pressure (No) OD (13.18) Contamination (No) Activity (4.15 /100ul) MeOH (350mL)
      <Adeleke>          4/6/2010 (18:00)      Fermentation 160:00  MeOH induction 00:00   
                          F1 Acid ( ml) Base ( ml) Pressure () OD () Contamination () Activity ( /100ul) MeOH (mL)
                          F2 Acid ( ml) Base ( ml) Pressure () OD () Contamination () Activity ( /100ul) MeOH (mL)

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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