User:Fermenter User/Notebook/BROM F2

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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
Pink: MeOH Induction hrs

BROM F2 (F1) and (F2)



Day -6 (Nov 12th Wed):

        
    Discussion about BROM F1 (F1) 
 : The activity of this mutant can not be monitored; therefore, grow D5 (F2) simultaneously with Y98C may allow us to estimate the totally amount of mutant protein in F1

Day -5 (Nov 13th Thu):

Day -4 (Nov 14th Fri):

 Change #1: Change head plate configuration to add more open ports 
      <Sung-Hye>           11/14/2008 (12:00)      Change configuration of head plate:
                                                   Add open port (x1) for Fermenter #2
     
    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.
      <Kevin>              11/14/2008 (16:25)  Autoclaved two 100ml flasks and two 500ml flasks 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. 1L 10x Glycerol (filtered)
                                         4. 100mL 500x Biotin (filtered, excess amount)
 Change #2: MeOH concentration is 100% 
                                         5. 1.5L 100% Methanol: Re-use from BROM F1 experiments (no need to prepare)

Day -3 (Nov 15th Sat):

      <Kevin>              11/15/2008 (23:00) 
                              Used previous MD media (1-week old, < 500 ml) for first stage cell culturing 
                              500mL BMGY for second stage culturing before fermentation for each mutant. 
      <Kevin>              11/15/2008 (23:30) 
                              Culture 20uL stocks of Y98C and D5 in 5mL MD media at 30C over night.

Day -2 (Nov 16th Sun):

      <Kevin>              11/16/2008 (16:00)  
                            O.D.(both mutants)= uncertain  time: ~17h
                            Spin down the cells (Y98C and D5) 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 (Nov 17th Mon):

 Change #3: Sung-Hye need to check levels!!: ok 
      <Sung-Hye>           11/17/2008 (12:00)    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> 11/17/2008 (14:30) Gave all the necessary media components to Sung-Hye.

<Sung-Hye> 11/17/2008 (14:45) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: (forgot to add Antifoam)<-- Do NOT forget! Autoclave <Sung-Hye> 11/17/2008 (16:45) Add antifoam: 2. 2 ml of antifoam/ fermenter

Change #4: Delay inoculation time: Reduce volume of cell culture (from 1 ml to 0.5 ml) <Kevin> 11/17/2008 (21:00) Both mutant cultures (Y98C and D5) reached O.D =6. Take 0.4 mL of each culture and regrew them in 500mL BMGY for next day fermentation.

<Kevin> 11/17/2008 (23:30) Leave following items in fermenter room: 1) Potassium Phsphate 2) YNB 3) Biotin 4) Glycerol

Day 0 (Nov 18th Tue):

     <Sung-Hye>            11/18/2008 (9:30)      Cooling bioreactors: 150 rpm 
     <Sung-Hye>            11/18/2008 (9:30-11:10)Transfer nutrients into bioreactors: 
 Change #5: Feeding method: Use Pumps 
     <Sung-Hye>                                   Transfer Adjutives using pumps.  
                           11/18/2008 (9:30-10:30)  1-L sterilized bottle: Combine                       
                              3. 2 x 500 ml of 10x Potassium Phosphate
                              4. 2 x 500 ml of 10x YNB 
                           11/18/2008 (10:33-11:10)  Repeat same procedure with:
                              5. 2 x 10 ml of 500x Biotin
                              6. 2 x 500 ml of 10x Glycerol

11/18/2008 (9:44) Start OD probe software 11/18/2008 (9:47) Start Fermenter #1 software 11/18/2008 (9:49) Start Fermenter #2 software 11/18/2008 (11:10) Connect Acid and Base bottles Acid (250 ml) Base (400 ml)<--- Use (4M) for Base 11/18/2008 (11:15) Set rpm at 900 11/18/2008 (11:15) Purge Air 1 L/min 11/18/2008 (11:25) Start pH control 11/18/2008 (11:50) Calibrate dO2 probes 11/18/2008 (11:50) Start dO2 control (O2 valve Max) 11/18/2008 (12:40) Autozeroing OD sensor


 
   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


 Change #6: Delayed inoculatation time (from 10:00 to 17:00)
      <Kevin>              11/18/2008 (11:00) Reduce rpm of incoulum culture

11/18/2008 (16:50-17:20) Fermentation 0:00 Inoculate cells (O.D: < 6.0 Volume: 500 ml) Cells were transferred to bottles/tube and inoculated using pumps.

Day 1 (Nov 19th Wed):

      <Sung-Hye>           11/19/2008 (9:00)      Fermentation 16:00 
                                                   F1 Acid (200 ml) Base (225 ml)
                                                   F2 Acid (180 ml) Base (300 ml) 
                           11/19/2008 (9:00)      Fermentation 16:00 
                                                   Connect MeOH 
                              7. 2 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
11/19/2008 (9:00-9:30) Fermentation 16:00 MeOH Induction 0:00 MeOH sensor calibration
<Sung-Hye and Kevin> 11/19/2008 (9:35) Fermentation 16:35 MeOH Induction 0:35 Y98C (F1)=(O.D:10.127, AU: 0.44, Activity:N.A. ) Acid (200 ml) Base (210 ml) MeOH (405 ml) D5 (F2)=(O.D:14.5, AU: 1.319, Activity:N.A. ) Acid (150 ml) Base (290 ml) MeOH (440 ml)
<Sung-Hye and Kevin> 11/19/2008 (10:00) Fermentation 17:00 MeOH Induction 1:00 Start MeOH control 11/19/2008 (10:00) Fermentation 17:00 MeOH Induction 1:00 Set level to 5.5-L: <-- shows as "contact", WHY?
<Kevin> 11/19/2008 (13:10) Fermentation 20:00 MeOH induction 4:00
Y98C (F1)=(O.D:17.24, AU: 0.5, Activity:N.A. ) Acid (N.A.) Base (N.A.) MeOH (N.A.) D5 (F2)=(O.D:15.76, AU: 1.38, Activity: N.A. ) Acid (N.A.) Base (N.A.) MeOH (N.A.)
<Sung-Hye> 11/19/2008 (16:30) Fermentation 23:30 MeOH Induction 7:30 F1 Acid (195 ml) Base (150 ml) MeOH (340 ml) F2 Acid (120 ml) Base (230 ml) MeOH (390 ml)

Day 2 (Nov 20th Thu):

      <Sung-Hye>           11/20/2008 (8:30)       Fermentation 39:30  MeOH induction 23:30 
                           
                           Resume MeOH pump cut-off! <-- Next time use different pump (60 rpm instead of 6 rpm)
                           
      <Sung-Hye>           11/20/2008 (8:30)       Fermentation 39:30  MeOH induction 23:30 
                                                   Remove 300 ml of media
                                                   F1 Acid (180 ml) Base (40 ml) MeOH (150 ml)
                                                   F2 Acid (95 ml) Base (35 ml) MeOH (150 ml) 
11/20/2008 (9:30) Fermentation 40:30 MeOH induction 24:30 Change MeOH pumps (from 6 rpm to 60 rpm) Got MeOH from Bromme lab MeOH level before refill F1 (100 ml) F2 (100 ml) Refill MeOH: add 900 ml each
11/20/2008 (10:00) Fermentation 41:00 MeOH induction 25:00 Acid, Base level before refill F1 Acid (180ml) Base (20 ml) F2 Acid (70 ml)Base (0 ml) Refill Acid: 300 ml Base: Add 300 ml each F1 Acid (480 ml) Base (340 ml) MeOH (1000 ml) F2 Acid (370 ml) Base (335 ml) MeOH (1000 ml)
11/20/2008 (13:00) Fermentation 44:00 MeOH induction 28:00 F1 Acid (480 ml) Base (190 ml) MeOH (850 ml)<-- 50 ml/hr F2 Acid (360 ml) Base (190 ml) MeOH (850 ml)<-- 50 ml/hr
<Kevin and Sung-Hye> 11/20/2008 (13:10) Fermentation 44:20 MeOH induction 28:20
Y98C (F1)=(O.D:24.16, AU: 0.588, Activity: 0.047 /100 ul) Acid (480 ml) Base (180 ml) MeOH (825 ml) D5 (F2)=(O.D:23.62, AU: 1.621, Activity:6.33/100 ul) Acid (350 ml) Base (190 ml) MeOH (845 ml)<-- Release pressure, replace condenser filter
<Sung-Hye> 11/20/2008 (16:00) Fermentation 47:00 MeOH induction 31:00 F1 Acid (470 ml) Base (95 ml) MeOH (700 ml)<-- 50 ml/hr F2 Acid (340 ml) Base (130 ml) MeOH (730 ml)<-- 40 ml/hr
<Sung-Hye> 11/20/2008 (17:00) Fermentation 48:00 MeOH induction 32:00 Refill F1 4M Base +230 ml(300 ml) MeOH +340 ml(1000 ml) Refill F2 4M Base +200 ml(300 ml) MeOH +310 ml(1000 ml)
<Kevin> 11/20/2008 (17:00-18:00) Fermentation 48:00 MeOH induction 32:00 Pump out about 1-L media from each fermenter and save.

Day 3 (Nov 21st Fri):

      <Sung-Hye>           11/20/2008 (8:00)       Fermentation 63:00  MeOH induction 47:00 
                           
                           Based on biocontroller record, O2 tank ran out around 1:00  (Fermentation 57:00)
                           New O2 tank opened 8:00 (Fermentation 63:00)
                           


      <Sung-Hye>           11/21/2008 (8:00)       Fermentation 63:00  MeOH induction 47:00 
                                                   F1 Acid (445 ml) Base (240 ml) MeOH (600 ml)
                                                   F2 Acid (325 ml) Base (220 ml) MeOH (600 ml)
                                          NOTE: Considering there was O2 depeletion between 61:00 - 68:00, 
                                                Amount of MeOH consumption during that time might be small.
<Sung-Hye> 11/21/2008 (11:00) Fermentation 66:00 MeOH induction 50:00 F1 Acid (420 ml) Base (215 ml) MeOH (490 ml)<-- 36.6 ml/hr F2 Acid (310 ml) Base (200 ml) MeOH (490 ml)<-- 36.6 ml/hr
<Kevin> 11/21/2008 (13:00) Fermentation 68:00 MeOH induction 52:00
Y98C (F1)=(O.D:76.1, AU: 0.66, Activity: 0) Acid (420 ml) Base (205 ml) MeOH (390 ml) D5 (F2)=(O.D:73.5, AU: 1.95, Activity: 16.4/100ul) Acid (310 ml) Base (195 ml) MeOH (395 ml)
<Sung-Hye> 11/21/2008 (14:00) Fermentation 69:00 MeOH induction 53:00 F1 Acid (420 ml) Base (200 ml) MeOH (350 ml)<-- 46.6 ml/hr F2 Acid (310 ml) Base (195 ml) MeOH (355 ml)<-- 45.0 ml/hr
11/21/2008 (17:00) Fermentation 72:00 MeOH induction 56:00 F1 Acid (420 ml) Base (190 ml) MeOH (245 ml)<-- 35.0 ml/hr F2 Acid (310 ml) Base (180 ml) MeOH (250 ml)<-- 35.0 ml/hr
11/21/2008 (17:00) Fermentation 72:00 MeOH induction 56:00 Refill F1 MeOH (1000 ml) Refill F2 MeOH (1000 ml)
<Kevin> 11/21/2008 (17:00) Fermentation 72:00 MeOH induction 56:00 Pump out about 1-L media from each fermenter and save.

Day 4 (Nov 22nd Sat):

      <Sung-Hye>           11/21/2008 (10:00)      Fermentation 89:00  MeOH induction 73:00 
                           
                           Based on biocontroller record, O2 tank ran out around 3:00  (Fermentation 82:00)
                           New (my last) O2 tank opened 10:00 (Fermentation 89:00)
                           One full O2 tank only last about 20 hr
                           
      <Sung-Hye>           11/22/2008 (10:00)      Fermentation 89:00  MeOH induction 73:00 
                                                   F1 Acid (410 ml) Base (150 ml) MeOH (630 ml)
                                                   F2 Acid (305 ml) Base (135 ml) MeOH (650 ml) 
<Kevin> 11/22/2008 (13:00) Fermentation 92:00 MeOH induction 76:00
Y98C (F1)=(O.D:106.2, AU: 0.652, Activity: 0.026 /100ul ) Acid (N.A) Base (N.A.) MeOH (N.A.) D5 (F2)=(O.D:113.6, AU: 1.690, Activity:20.3/100ul) Acid (N.A) Base (N.A.) MeOH (N.A.)
<Sung-Hye> 11/22/2008 (15:00) Fermentation 94:00 MeOH induction 78:00 F1 Acid (405 ml) Base (130 ml) MeOH (500 ml)<-- 26.0 ml/hr F2 Acid (300 ml) Base (115 ml) MeOH (530 ml)<-- 24.0 ml/hr
<Sung-Hye and Kevin> 11/22/2008 (15:00) Fermentation 94:00 MeOH induction 78:00 Bring O2 tank from B2 level (Bromme lab) and bring up to fermenter room
11/22/2008 (17:00) Fermentation 96:00 MeOH induction 80:00 Refill F1 MeOH (1000 ml) Refill F2 MeOH (1000 ml)
<Kevin> 11/21/2008 (17:00) Fermentation 96:00 MeOH induction 80:00 Pump out about 1-L media from each fermenter and save.

Day 5 (Nov 23rd Sun):

       No Access to building 7:00 - 19:00
                       Fume hood locked
      


      <Sung-Hye>           11/23/2008 (19:00)      Fermentation 122:00  MeOH induction 106:00 
                                                   F1 Acid (410 ml) Base (80 ml) MeOH (395 ml)<-- 22.4 ml/hr
                                                   F2 Acid (300 ml) Base (60 ml) MeOH (440 ml)<-- 20.7 ml/hr 
<Kevin> 11/23/2008 (19:20) Fermentation 122:20 MeOH induction 106:20
Y98C (F1)=(O.D:117, AU: 0.68, Activity: N.A.) D5 (F2)=(O.D:114, AU: 1.79, Activity: 22.7/100ul)
<Sung-Hye> 11/23/2008 (20:00) Fermentation 123:00 MeOH induction 107:00 Refill F1 MeOH (1000 ml) Refill F2 MeOH (1000 ml)

Day 6 (Nov 24th Mon):

      <Sung-Hye>           11/24/2008 (8:30)      Fermentation 135:30  MeOH induction 119:30 
                                                   F1 Acid (405 ml) Base (60 ml) MeOH (795 ml)<-- 15.1 ml/hr
                                                   F2 Acid (305 ml) Base (50 ml) MeOH (820 ml)<-- 13.3  ml/hr 
<Kevin> 11/24/2008 (9:30) Fermentation 136:30 MeOH induction 120:30
Y98C (F1)=(O.D:117.6, AU: 0.66, Activity: N.A.) Acid (400 ml) Base (60 ml) MeOH (485 ml)<-- 10.0 ml/hr D5 (F2)=(O.D:116.4, AU: 1.791, Activity: 17.2/100ul) Acid (305 ml) Base (50 ml) MeOH (805 ml)<-- 15.5 ml/hr
      <Sung-Hye and Keinv> 11/24/2008 (10:20)      Fermentation 137:20  MeOH induction 121:20 
                               Havest samples and Finish Fermentations

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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