User:Fermenter User/Notebook/BROM F4

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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 F4


Day -4 (Sep 4 Fri):

     
    Discussion about Glycerol Feeding Methods and Inoculum 
1. Glycerol feeding: Start fermentation with BMGY: 1% final concentration of Glycerol in 5-L media. No more additional feeding will be added. Based on BROM F1 experiment, metabolism switch from Glycerol to MeOH occurred around 22hr fermentation 14 hr MeOH induction We don't have record of OD of that time, but OD measurement at 28:30 hr fermentation was 12-13. We want to keep the same condition, and hope that cells are continue to grow to 20 or more in the presence of MeOH (set to 0.5%). 2. Inoculum volume and density: Same as BROM F1, but inoculate late of Day 0 17:00.
Change #1: Use incubator in Fermenter room <Kevin> 9/4/2009 (12:30) Autoclaved one 500ml 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. 0.5L 10x Glycerol (filtered) 4. 20mL 500x Biotin (filtered, excess amount) (Take half of each above media components, and combine them to make 1 bottles of 1.25L culturing media)
Change #2: MeOH concentration is 100% 5. 6L 100% Methanol(non-HPLC grade)

Day -3 (Sep 7 Mon):


 Holiday

Day -2 (Sep 8 Tue):

      <Kevin>              9/8/2009 (12:30) 
                              Used MD media for first stage cell culturing 
                              2 flasks of 250mL BMGY for second stage culturing before fermentation for each mutant. 
      <Kevin>              9/8/2009 (12:50) 
                              Culture 5uL stocks D5 in 6mL MD media at 28.5C over night.

Day -1 (Sep 9 Wed):

 Change #3: Sung-Hye need to check levels!!: ok 
      <Sung-Hye>           9/9/2009 (11:30)    Prepared bottles:
                                                 1) Acid, 1-L (tubing separate) 
                                                 2) Base, 500-ml (tubing connected)
                                                 3) MeOH, 1-L (tubing connected)
                                                 4) Level, 1-L (tubing connected)
                                                 5) Adjutives, 2-L (tubing connected) 
                                                 6) Inoculum, 500-ml (tubing connected)
                                                 7) Water, 2-L 

<Sung-Hye> 9/9/2009 (12:00) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: 2. 1 ml of antifoam/ fermenter Autoclave 9/9/2009 (17:20) Cooling bioreactors: 400 rpm 9/9/2009 (17:30) Purge Air 1 L/min at 400 rpm


      <Kevin>              9/9/2009 (16:00)     Mutant reached O.D =6    
                                                 Take 0.1 mL of each culture and regrew them in 250mL 
                                                 BMGY for next day fermentation.

<Kevin> 9/9/2009 (17:00) Leave the master culturing media containing the following items in the fermenter room: 1) Potassium Phosphate 2) YNB 3) Biotin 4) Glycerol

Day 0 (Sep 10 Thu):

                           9/10/2009 (10:42)      Start Fermenter #1 software 
                           9/10/2009 (10:44)      Start Fermenter #2 software 
                           9/10/2009 (11:00)      Connect Acid and Base bottles 
                                                  Acid (500 ml) Base (400 ml)<--- Use (4M) for Base
 Change #5: Feeding method: Use Pumps 
       <Sung-Hye>                                 Transfer Adjutives using pumps.  
                           9/10/2009 (11:30-12:30)  Kevin already combine in a sterilized bottle: Combine                       
                              3. 2 x 500 ml of 10x Potassium Phosphate
                              4. 2 x 500 ml of 10x YNB 
                              5. 2 x 10 ml of 500x Biotin
                              6. 2 x 250 ml of 10x Glycerol

9/10/2009 (13:40) Set rpm at 900 9/10/2009 (13:40) Start pH control 9/10/2009 (13:40) Calibrate dO2 probes 9/10/2009 (13:40) Start dO2 control (O2 valve Max) <Sung-Hye> 9/10/2009 (14:00) Fermentation 0:00 Inoculate cells (O.D: 1.2, Volume: 250 ml x 2) Cells were transferred to bottles/tube and inoculated using pumps.

Day 1 (Sep 11 Fri): MeOH Induction Day 1

      <Sung-Hye>           9/11/2009 (09:00)      Fermentation 19:00 
                                                   F1 Acid (490 ml) Base (390 ml)
                                                   F2 Acid (415 ml) Base (385 ml) 
                           9/11/2009 (12:00)      Fermentation 22:00 
                                                 Connect MeOH
                              7. 2 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
9/11/2009 (12:00) MeOH sensor calibration
<Kevin> 9/11/2009 (12:30) Fermentation 22:30 MeOH induction 00:30 (F1)=(O.D:15.3, Activity:N.A.) Contamination? (Y, 1-2 colonies/100 cells) (F2)=(O.D:15.5, Activity:N.A.) Contamination? (Y, 1-2 colonies/100 cells) <SungHye & Kevin> 9/11/2009 (13:30) Fermentation 23:30 MeOH induction 01:30
See slight contamination in both of the fermenter Add Amp (final 50 ug/ml concentration, 5ml each fermenter of 50mg/ml) added on both fermentaiton through septum.

Day 2 (Sep 12 Sat): MeOH Induction Day 2

      <Sung-Hye>           9/12/2009 (11:00)      Fermentation 45:00  MeOH induction 23:00  
                                                   F1 Acid (480 ml) Base (260 ml) MeOH (400 ml)
                                                   F2 Acid (410 ml) Base (250 ml) MeOH (350 ml)
                                                  Decant >1-L media from each fermenters
                                                  Change filters
      <Kevin>              9/12/2009 (16:13)    
                                                  Refill MeOH to 1.2L (addition of 1L)
                                                  Contamination disappeared!!                                   
                                           (F1)=(O.D:90.5, Activity:N.A.) 
                                           (F2)=(O.D:90.1, Activity:N.A.)

Day 3 (Set 13 Sun): MeOH Induction Day 3

      <Sung-Hye>           9/13/2009 (10:00)      Fermentation 68:00  MeOH induction 46:00  
                                                   F1 Acid (380 ml) Base (200 ml) MeOH (650 ml)
                                                   F2 Acid (410 ml) Base (200 ml) MeOH (700 ml)
<Kevin> 9/13/2009 (20:30) Refill MeOH to 1.2L (addition of 600mL) (F1)=(O.D:118.4, Activity:N.A.) (F2)=(O.D:118.9, Activity:N.A.)

Day 4 (Sep 14 Mon): MeOH Induction Day 4

      <Sung-Hye>           9/14/2009 (10:00)      Fermentation 92:00  MeOH induction 70:00 
                                                   F1 Acid (380 ml) Base (180 ml) MeOH (1000 ml)
                                                   F2 Acid (410 ml) Base (180 ml) MeOH (1000 ml) 
<SungHye & Keving> 9/14/2009 (17:00) Fermentation 99:00 MeOH induction 77:00
F1 Acid (380 ml) Base (180 ml) MeOH (900 ml) F2 Acid (420 ml) Base (180 ml) MeOH (900 ml)
(F1)=(O.D:123.2, Activity:N.A.) (F2)=(O.D:123.0, Activity:N.A.) 9/14/2009 (16:40) 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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