User:Fermenter User/Notebook/BROM J1

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 J1

Day -4 (Jun 8 Fri):

Day -3 (Jul 9 Sat):

Day -2 (Jul 10 Sun):

      <Vidhu>           7/10/2011 (13:00)
                              Thaw glycerol stock and grow in 5-ml MD media and 
                              inoculated from MD plate as well and o/n incubation at 30deg , 200rpm

Day -1 (Jul 11, Mon): MEDIA PREPARATION DAY

      <Vidhu>            7/11/2010 (14:30)
                               Bring powder to make 5-L basal salts

      <Sung-Hye>         7/11/2011 (15:00)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 5.0-L of Basal Salts: Phosphoric Acid was 10-ml short.
                              3. 1-ml Antifoam 

      <Sung-Hye>         7/11/2011 (15:00)  Prepare bottles
                                           1) no Acid need
                                           2) Base, 500-ml (tubing connected) x1
                                           3) MeOH, 1-L (tubing connected) x1 <-- No need
                                           4) Additives/Inoculum, 500-ml (tubing connected) x1
                                           5) No Water, 2-L 
                                           6) Fed-Batch, 500-ml (tubing connected) x1

 
      <Sung-Hye>         7/11/2011 (15:16)  Autoclave

      <Vidhu>            7/11/2011 (15:00)
                               1.Transfer cells (5ml MD media) to 50ml BMGY media.
                                 Incubator in Fermentation Suite used
                               2.Prepare sterilized Methanol by filtration
                               3.Prepare sterilized NaOH (4M, 500-ml)

Day 0 (Jul 12 Tue): INOCULATION DAY

      <Vidhu>            7/12/2011 (10:30)  Bring the following items to Fermentation room.
                                            :NaOH (4M, 500 ml), MeOH (1000 ml), and 50% Glycerol (500ml)
                         7/12/2011 (11:00)  Check contamination of starter culture.
                                            Flask OD: 32.10
                         7/12/2011 (11:50)  Bring inoculum to Fermentation room.
      <Vidhu&Sung-Hye>   7/12/2011 (11:30)  Set temperature to 28.5°C 
      <Vidhu&Sung-Hye>   7/12/2011 (11:30)  Purge Air 1L/min at 900 rpm 
      <Vidhu&Sung-Hye>   7/12/2011 (11:30)  Adjust pH to ~4.75 (with ~90 ml NH4OH)
      <Vidhu&Sung-Hye>   7/12/2011 (11:40)  Adjust pH to 5.0 with **4M NaOH**
                                            (borrowed from Bromme lab, 500 ml)
                                            50 ml used
      <Vidhu&Sung-Hye>   7/12/2011 (11:45)  Start pH control 
                                            F1: No Acid Base, 4M (450 ml)
      <Vidhu&Sung-Hye>   7/12/2011 (12:00)  Calibrate dO2 probes 
                         7/12/2011 (12:00)  Strt dO2 control 
      <Vidhu&Sung-Hye>   7/12/2011 (12:04)  Start Fermenter #1 software 
      <Vidhu&Sung-Hye>   7/12/2011 (12:15)  Fermentation 0:00 
                                            Inoculate 55 ml/bioreactor
                                            Cells were transferred using pumps.
      <Sung-Hye>         7/12/2011 (12:20)  Add 21.75 ml of PTM1 Salt/5-L media <-- Prepared at BROM A16
                                            (4.35 ml PTM1 salt per 1-L media = 21.75 ml in 5-L))

Day 1 (Jul 13 Wed):

      <vidhu>            7/13/2011 (12:15)      Fermentation 24:00  
                            F1:   Base(350 ml) OD(20) Contamination(N) Pressure(N) Change Filter(N)

cells look good...

Day 2 (Jul 14 Thu): Fed-Batch and MeOH Induction Day 1

      <Vidhu>            7/14/2011 (9:00)      Fermentation 45:00 
                            F2: Base(30 ml) OD(31.75) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         7/14/2011 (9:00 - 12:00)  Fermentation 45:00 - 48:00 
                            F2: Fed-Batch
                            400 ml 50% Glycerol + 4.8 ml PTM1 at 90.75ml/hr/5-L (RPM 75 w/ 2RPM pump)
      <Sung-Hye>         7/14/2011 (11:00)
                            F2: Take out 500-ml media
      <Vidhu>            7/14/2011 (12:40)      Fermentation 48:00  
                            F1: Base(480 ml) OD(44.30) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         7/14/2011 (13:30)      Fermentation 49:30  MeOH induction 00:00 
                            Calibrate MeOH sensor  
                            F2: 6231 mV = 0.5% (MeOH 750 ml)<-- Mix with PTM1
                            Start MeOH induction

Day 3 (Jun 24 Fri): MeOH Induction Day 2

      <Jason>           6/24/2011 (13:30)      Fermentation 73:30  MeOH induction 24:00  
                            F2: Base(390 ml) MeOH( 480 ml) OD(58.70) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (3.12 units/100 ul), (0.73 mg/5-L)
      <Sung-Hye>        6/24/2011 (14:45)
                            F2: Take out 500-ml media
      <Jason>           6/24/2011 (16:30)
                            F2: Refill MeOH (450 ml +500 ml = 950 ml)

Day 4 (Jun 25 Sat): MeOH Induction Day 3

      <Jason>           6/25/2011 (13:30)      Fermentation 97:30  MeOH induction 48:00  
                            F2: Base(380 ml) MeOH(650 ml) OD(65.05) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (18.72 units/100ul), (4.38 mg/5-L)

Day 5 (Jun 26 Sun): MeOH Induction Day 4

      <Jason>            6/26/2011 (13:30)      Fermentation 121:30  MeOH induction 72:00 
                            F2: Base(370 ml) MeOH(350 ml) OD(68.10) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (15.08 units/100ul), (3.529 mg/5-L)
                            F2: Refill MeOH (350 ml +650 ml = 1000 ml)

Day 6 (Jun 27 Mon): MeOH Induction Day 5

      <Jason>           6/27/2011 (12:00)      Fermentation 144:00  MeOH induction 94:30 

                            F1: Base(370 ml) MeOH(800 ml) OD(70.05) Contamination(N) Pressure(Yes) Change Filter(Yes)
                                Activity assay (18.87 units/100ul), (4.41 mg/5-L)
      <Sung-Hye>         HARVEST

Suggestion for next trials:

1.  Add PTM1 salt into MeOH (12 ml/L of MeOH)
2.  Even add Glycerol together with MeOH
3.  Extend fed-batch hours (from 3 hrs to overnight) before start MeOH induction
4.  Measure samples twice a day

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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