User:Fermenter User/Notebook/BROM F3

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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 F3


Day -4 (Mar 27 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> 3/27/2009 (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. 1.0L 10x Glycerol (filtered) 4. 300mL 500x Biotin (filtered, excess amount) Change #2: MeOH concentration is 100% 5. 6L 100% Methanol(non-HPLC grade)

Day -3 (Mar 28 Sat):

      <Kevin>              3/28/2009 (15:30) 
                              Used previous MD media (3-week old, < 500 ml) for first stage cell culturing 
                              500mL BMGY for second stage culturing before fermentation for each mutant. 
      <Kevin>              3/28/2009 (15:30) 
                              Culture 5uL stocks D5 in 10mL MD media at 28.5C over night.

Day -2 (Mar 29 Sun):

      <Kevin>              3/29/2009 (10:00)  
                            O.D= uncertain  time: ~17h
                            Spin down the cells at 2000g for 5 minutes and resuspend them in 10mL BMGY.
                            Transfer 100uL of the resuspended culture of each mutant to 50mL BMGY for over night culturing.

Day -1 (Mar 30 Mon):

 Change #3: Sung-Hye need to check levels!!: ok 
      <Sung-Hye>           3/30/2009 (13:00)    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, 1-L (tubing connected)<-- Make it 2-L next time 
                                                 6) Inoculum, 500-ml (tubing connected)
                                                 7) Water, 2-L 

<Kevin> 3/30/2009 (13:00) Gave all the necessary media components to Sung-Hye.

<Sung-Hye> 3/30/2009 (15:10) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: 2. 1 ml of antifoam/ fermenter

Autoclave 3/30/2009 (17:00) Cooling bioreactors: 400 rpm 3/30/2009 (17:00) Purge Air 1 L/min at 400 rpm

Change #4: Delay inoculation time: Reduce volume of cell culture (from 1 ml to 0.5 ml) <Kevin> 3/30/2009 (16:00) D5 reached O.D =6 Take 0.4 mL of each culture and regrew them in 500mL BMGY for next day fermentation.

<Kevin> 3/30/2009 (17:00) Leave following items in fermenter room: 1) Potassium Phosphate 2) YNB 3) Biotin 4) Glycerol

Day 0 (Mar 31 Tue):

                           3/31/2009 (10:18)      Start Fermenter #1 software 
                           3/31/2009 (10:20)      Start Fermenter #2 software 
                           3/31/2009 (10:50)      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.  
                           3/31/2009 (11:11-00:00)  2-L 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 500 ml of 10x Glycerol <-- only used 375 ml/fermenter

3/31/2009 (12:40) Set rpm at 900 3/31/2009 (12:40) Start pH control 3/31/2009 (12:40) Calibrate dO2 probes 3/31/2009 (12:40) Start dO2 control (O2 valve Max)


 
   Discussion about Amp: Do not add!! 
  
    1. We don't know whether yeast cells will express Amp resistance gene or not
        (becuase promoter for Amr is from E.coli
    2. We don't know the effect of Amp on yeast growth in general
        Find some reference


</font>
      <Kevin>              3/31/2009 (10:00) Fermentation 0:00  
                                                  Inoculate cells  (O.D: 1.5, Volume: 250 ml)  
                                                  Cells were transferred to bottles/tube and inoculated using pumps.

Day 1 (Apr 1 Wed):

      <Sung-Hye>           4/1/2009 (07:00)      Fermentation 18:00 
                                                   F1 Acid (480 ml) Base (390 ml)
                                                   F2 Acid (420 ml) Base (380 ml) 
      <Sung-Hye and Kevin> 4/1/2009 (11:00)      Fermentation 22:00  
                               D5 (F1)=(O.D:9.82, Activity:N.A.) 
                               D5 (F2)=(O.D:10.02, Activity:N.A.)                                           
                           4/1/2009 (11:00)      Fermentation 22:00 
                                                 Connect MeOH
                              7. 2 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
4/1/2009 (11:10-11:37) MeOH sensor calibration
4/1/2009 (12:00) Fermentation 23:00 MeOH Induction 00:00 <Sung-Hye> 4/1/2009 (15:00) Fermentation 26:00 MeOH Induction 03:00 Change Temp <Sung-Hye> 4/1/2009 (16:00) Fermentation 27:00 MeOH Induction 04:00 D5 (F1)= Acid (480 ml) Base (380 ml) MeOH (760 ml) D5 (F2)= Acid (400 ml) Base (360 ml) MeOH (850 ml)

Day 2 (Apr 2 Thu):

      <Sung-Hye>           4/2/2009 (07:00)       Fermentation 42:00  MeOH induction 19:00 
                           MeOH pump:ok
                           O2 tank:ok
      <Sung-Hye>           4/2/2009 (07:00)       Fermentation 42:00  MeOH induction 19:00 
                                                   F1 Acid (480 ml) Base (300 ml) MeOH (300 ml)
                                                   F2 Acid (400 ml) Base (270 ml) MeOH (320 ml) 
4/2/2009 (08:00) Fermentation 43:00 MeOH induction 20:00 Decant 1-L of media/fermenter F1 high pressure F1 and F2 both gas outlet filters replaced

<Kevin> 4/2/2009 (00:00) Fermentation 00:00 MeOH induction 00:00 D5 (F1)=(O.D:24.16, Activity: 4.73/100 ul) D5 (F2)=(O.D:23.62, Activity: 5.78/100 ul) <Sung-Hye> 4/2/2009 (12:00) Fermentation 47:00 MeOH induction 32:00 D5 (F1) MeOH=50 ml Refill 950 ml Total 1000 ml D5 (F2) MeOH=50 ml Refill 950 ml Total 1000 ml

Day 3 (Apr 3 Fri):

      <Sung-Hye>           4/3/2009 (07:00)       Fermentation 66:00  MeOH induction 43:00 
                           MeOH pump:ok
                           O2 tank:ok
      <Sung-Hye>           4/3/2009 (07:00)       Fermentation 66:00  MeOH induction 43:00 
                                                   F1 Acid (480 ml) Base (195 ml) MeOH (60 ml)
                                                   F2 Acid (400 ml) Base (170 ml) MeOH (140 ml) 
4/3/2009 (07:00) Fermentation 66:00 MeOH induction 43:00 Decant 1-L of media/fermenter F1 and F2 both gas outlet filters replaced

<Sung-Hye> 4/3/2009 (9:30) Fermentation 68:30 MeOH induction 45:30 D5 (F1) MeOH=<50 ml Refill to 500 ml D5 (F2) MeOH=<100 ml Refill to 500 ml
<Kevin> 4/3/2009 (00:00) Fermentation 00:00 MeOH induction 11:00
D5 (F1)=(O.D:89.9, Activity: 14.0/100ul) D5 (F2)=(O.D:90.8, Activity: 16.6/100ul)
<Sung-Hye> 4/3/2009 (15:30) Fermentation 74:30 MeOH induction 51:30 D5 (F1) MeOH=<50 ml Refill to 500 ml D5 (F2) MeOH=<100 ml Refill to 500 ml

Day 4 (Apr 4 Sat):

      <Sung-Hye>           4/4/2009 (10:30)       Fermentation 93:30  MeOH induction 70:30 
                           MeOH pump:ok
                           O2 tank:ok
      <Sung-Hye>           4/4/2009 (10:30)       Fermentation 93:30  MeOH induction 70:30 
                                                   F1 Acid (480 ml) Base (180 ml) MeOH (550 ml)
                                                   F2 Acid (400 ml) Base (130 ml) MeOH (550 ml) 
<Sung-Hye> 4/4/2009 (10:30) Fermentation 93:30 MeOH induction 70:30 D5 (F1) MeOH Refill to 1000 ml D5 (F2) MeOH Refill to 1000 ml
4/4/2009 (10:30) Fermentation 93:30 MeOH induction 70:30 Decant 1-L of media/fermenter F1 and F2 both gas outlet filters replaced

<Kevin> 4/4/2009 (11:00) Fermentation 00:00 MeOH induction 00:00
D5 (F1)=(O.D:91.9, Activity: 27.2/100ul) D5 (F2)=(O.D:99.4, Activity: 28.3/100ul)

Day 5 (Apr 5 Sun):

      <Kevin>              4/5/2009 (14:00)      Fermentation 00:00  MeOH induction 00:00 
D5 (F1)=(O.D:94.3, Activity: 17.5/100ul) D5 (F2)=(O.D:94.3, Activity: 25.6/100ul)
<Keving and Sung-Hye>4/5/2009 (16:00) Fermentation 123:00 MeOH induction 100:00
F1 Acid (480 ml) Base (150 ml) MeOH (550 ml) F2 Acid (400 ml) Base (110 ml) MeOH (550 ml)
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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