User:Fermenter User/Notebook/BROM F7

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<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">User:Fermenter User/Notebook/BROM F7</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

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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 F7


Day -2 (May 12 Wed):

      <Adeleke>          5/12/2010 (16:00)  
                                        Used MD media for first stage cell culturing 
                                        Autoclaved one 2L flask for culturing.

Day -1 (May 13 Thu):

      <Adeleke>          5/13/2010 (16:00)   2 flask of 1L of 250mLBMGY for second stage culturing before fermentation for A25 mutant.
<Adeleke> 5/13/2010 (12:30) 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> 5/13/2010 (12:30) 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> 5/13/2010 (13:00) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: <-- Peptone was missing! 2. 2 x 1 ml of antifoam/ fermenter Autoclave

Day 0 (May 14 Fri):

      <Sung-Hye>         5/14/2010 (11:14)      Cooling bioreactors: 300 rpm 
                         5/14/2010 (11:15)      Start Fermenter #1 software 
                         5/14/2010 (11:20)      Start Fermenter #2 software 

5/14/2010 (13:15 - 14:35) Transfer Adjutives using pumps. Combine: 3. 1 x 500 ml of 10x Potassium Phosphate 4. 1 x 500 ml of 10x YNB 5. 2 x 10 ml of 500x Biotin <-- Do not forget to add! 2 x 5 ml of Antibiotics 6. 1 x 250 ml of 10x Glycerol <-- Pepton was mixed with Glycerol
5/14/2010 (14:45) Calibrate dO2 probes 5/14/2010 (15:00) Start dO2 control (O2 valve Max)

<Adeleke&SungHye> 5/14/2010 (15:30) Fermentation 0:00 Flask (A25) OD: 7.0 Inoculate 2 fermenters with Sung-Hye 250 mL of culture in each fermentor
<SungHye> 5/14/2010 (16:30) Start pH control Acid (250-300 ml) Base (400 ml):


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

Day 1 (May 15 Sat):

      <Adeleke>          5/15/2010 (11:30)      Fermentation 00:00  
                          F1 Acid ( ml) Base ( ml) Pressure () OD (12.3) Contamination (No) Activity ( /100ul)
                          F2 Acid ( ml) Base ( ml) Pressure () OD (12.8) Contamination (No) Activity ( /100ul)

Day 2 (May 16 Sun): MeOH Induction Day 1

      <Adeleke>          5/16/2010 (12:00)      Fermentation 00:00  
                          F1 Acid (50 ml) Base (390 ml) Pressure (Yes) OD (13.7) Contamination (No) Activity ( /100ul)
                          F2 Acid (80 ml) Base (390 ml) Pressure (Yes) OD (13.9) Contamination (??) Activity ( /100ul)
      <Sung-Hye>         5/16/2010 (16:00)      Fermentation 47:30 
                                                 Refill Acid +500 ml each
                         5/16/2010 (16:30)      Fermentation 48:00  MeOH induction 00:00 
                                                 Connect MeOH
                             7. 2 x 900 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
5/16/2010 (16:30) MeOH sensor calibration
F1 (6650 mV) F2 (5800 mV)

Day 3 (May 17 Mon): MeOH Induction Day 2

      <Sung-Hye>         5/17/2010 (9:30)       Fermentation 65:00  MeOH induction 17:00   
                          F2 Take out samples 600 ml, Change Filter
      <Adeleke>          5/17/2010 (16:30)      Fermentation 72:00  MeOH induction 24:00   
                          F1 Acid (500 ml) Base (360 ml) Pressure (No) OD (14.5) Contamination (No) Activity ( /100ul) MeOH ( mL)
                          F2 Acid (500 ml) Base (360 ml) Pressure (No) OD (14.7) Contamination (Yes) Activity ( /100ul) MeOH ( mL)

Day 4 (May 18 Tue): MeOH Induction Day 3

      <Sung-Hye>         5/18/2010 (16:00)      Fermentation 98:00  MeOH induction 48:00   
                          F1 Acid (500 ml) Base (300 ml) Pressure () OD () Contamination () Activity ( /100ul) MeOH (455 mL)
                          F2 Acid (490 ml) Base (270 ml) Pressure () OD () Contamination () Activity ( /100ul) MeOH (340 mL)
      <Adeleke>          5/18/2010 (16:00)      Fermentation 98:00  MeOH induction 48:00   
                          F1 Acid (490 ml) Base (300 ml) Pressure (Yes) OD (17.8) Contamination (No) Activity ( /100ul) MeOH (460 mL)
                          F2 Acid (490 ml) Base (280 ml) Pressure (No) OD (20.8) Contamination (???) Activity ( /100ul) MeOH (340 mL)

Day 5 (May 19 Wed): MeOH Induction Day 4

      <Sung-Hye>         5/19/2010 (8:30)       Fermentation 96:00  MeOH induction 64:00   
                          Take out 500 ml of sample from F1, change filter
      <Raymond>          5/19/2010 (12:30)      Fermentation 100:00  MeOH induction 68:00   
                          F1 Acid (200 ml) Base (300 ml) Pressure (yes) OD (51.7) Contamination (No) Activity ( /100ul) MeOH (350 mL)
                             Change filter
                          F2 Acid (490 ml) Base (240 ml) Pressure (no) OD (80.9) Contamination (No) Activity ( /100ul) MeOH (75 mL)
      <Sung-Hye>          Refill Acid (F1) + 300 ml
                          Refill MeOH (F1: 350 + 650 = 1000 ml)
                                      (F2: 75 + 925 = 1000 ml)

Day 6 (May 20 Thu): MeOH Induction Day 5

      <Adeleke>          5/20/2010 (15:00)      Fermentation 128:00  MeOH induction 00:00   
                          F1 Acid (480 ml) Base (275 ml) Pressure (No) OD (59.4) Contamination (No) Activity ( /100ul) MeOH ( mL)
                          F2 Acid (480 ml) Base (205 ml) Pressure (No) OD (94.9) Contamination (??) Activity ( /100ul) MeOH ( mL)


Day 7 (May 21 Fri): MeOH Induction Day 6

      <Adeleke>          5/21/2010 (10:45)      Fermentation 152:00  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)
      <Adeleke&Sung-Hye> 5/21/2010 (18:00)      Fermentation 160:00  MeOH induction 00:00   
                          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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