User:Fermenter User/Notebook/BROM A20

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BROM A20


Day -2 (May 15 Sun):

      <Jason>           5/15/2011 (PM)
                               Thaw glycerol stock and grow in 5-ml MD media (made fresh 2 wks ago)
                               Incubator in Fermentation Suite used
      <Jason>           5/15/2011 (before go home)
                              Preparing 5-L of Basal Medium (Defined media)
                              PTM1 Salt is from BROM-A16 <-- Refill

Day -1 (May 16 Mon): MEDIA PREPARATION DAY

      <Sung-Hye>         5/16/2011 (10:30)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 5.0-L of Basal Salts
                              3. 1-ml Antifoam 
<Sung-Hye> 5/16/2011 (10:30) Prepare bottles 1) no Acid need 2) Base, 500-ml (tubing connected) x2 3) MeOH, 1-L (tubing connected) x2 4) Additives/Inoculum, 1 x 500-ml (tubing connected) x2 5) Water, 2-L (Note) Add Fed-Batch, 1 x 500-ml (tubing connected) x2

<Sung-Hye> 5/16/2011 (11:30) Autoclave <Sung-Hye> 5/16/2011 (12:20) Autoclave completed! <Sung-Hye> 5/16/2011 (12:30) Set temperature to 28.5°C <Sung-Hye> 5/16/2011 (13:30) Purge Air 1L/min at 900 rpm <Sung-Hye> 5/16/2011 (13:30) Add 21.75 ml of PTM1 Salt/5-L media <-- Prepared at BROM A16 (4.35 ml PTM1 salt per 1-L media) <Sung-Hye> 5/16/2011 (13:30) Adjust pH to ~2.0 with ~100 ml NH4OH Reduce rpm to 400 overnight


      <Jason>            5/16/2010 (11:50 - 12:00)
                               Transfer cells to fresh 55-ml BMGY media per fermenter.
                               Incubator in Fermentation Suite used

Day 0 (Feb 2 Wed): INOCULATION DAY

      <Sung-Hye>         2/2/2011 (9:30)   Increase rpm to 900 
      <Sung-Hye>         2/2/2011 (10:30)  Adjust pH to 5.0 with additional **NaOH** 
                                           (borrowed from Bromme lab)
      <Sung-Hye>         2/2/2011 (10:30)  Start pH control 
                                           F1: No Acid Base, 2M (500 ml)
                                           F2: No Acid Base, 2M (500 ml)
      <Sung-Hye>         2/2/2011 (10:31)  Start Fermenter #1 (TEST) software 
                         2/2/2011 (10:33)  Start Fermenter #2 (NORM) software  
      <Sung-Hye>         2/2/2011 (10:45)  Calibrate dO2 probes 
                         2/2/2011 (10:45)  Strt dO2 control 
      <Dennis>           2/2/2011 (13:30)  Check contamination of starter culture 
                                            Flask TEST OD: 15.35
                                            Flask NORM OD: 16.15
      <Dennis&Sung-Hye>  2/2/2011 (14:00)  Fermentation 0:00 
                                            Inoculate 50 ml/bioreactor

Day 1 (Feb 3 Thu):

      <Sung-Hye>         2/2/2011 (9:30)    Fermentation 19:00 
                            F1(TEST): Base (500 ml -500 ml = 0 ml)
                            F2(NORM): Base (500 ml -500 ml = 0 ml)
      <Dennis>           2/2/2011 (14:30)      Fermentation 24:00  
                            F1 (Test):   Base(500 ml) OD(5.50) Contamination(N) Pressure(N) Change Filter(N)
                            F2 (Normal): Base(500 ml) OD(9.65) Contamination(N) Pressure(N) Change Filter(N)
      <Sung-Hye>         2/2/2011 (Afternoon)  Prepare Fed-batch bottles
                                            6) Fed-batch (50% Glycerol), 1 x 500-ml (tubing connected) x2

Day 2 (Fev 4 Fri): Fed-Batch and MeOH Induction Day 1

      <Dennis>           2/4/2011 (10:00)      Fermentation 44:00  
                            F1 (Test): Base(300 ml) OD(27.4) Contamination(n) Pressure(n) Change Filter(n)
                            F2 (Norm): Base(300 ml) OD(26.0) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         2/4/2011 (11:30 - 15:30)  Fermentation 45:30 - 49:30 
                            F1 (TEST) and F2 (Norm): Fed-Batch
                            400 ml 50% Glycerol + 4.8 ml PTM1 w/ 90.75ml/hr/5-L (RPM 65 w/ 2RPM pump)
      <Sung-Hye>         2/4/2011 (13:30)
                            F1 (TEST): Take out 500-ml media
                            F2 (Norm): Take out 300-ml media
      <Dennis>           2/4/2011 (15:00)      Fermentation 49:30  
                            F1 (Test): Base(200 ml) OD(44.65) Contamination(n) Pressure(n) Change Filter(n)
                            F2 (Norm): Base(200 ml) OD(41.00) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         2/4/2011 (15:30)      Fermentation 52:00  MeOH induction 00:00 
                            Calibrate MeOH sensor  
                              F1 (TEST): 7399 mV = 0.5% (MeOH 450 ml)
                              F2 (Norm): 6380 mV = 0.5% (MeOH 475 ml) 
                            Start MeOH induction
      <Sung-Hye>         2/4/2011 (18:00)
                            F1 (TEST): Take out 300-ml media
                            F2 (Norm): Take out 800-ml media

Day 3 (Feb 5 Sat): MeOH Induction Day 2

      <Dennis>           2/5/2011 (14:00)      Fermentation 96:00  MeOH induction 24:00  
                            F1(Test): Base(100 ml) MeOH( ml) OD(38.05) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (15.24 units/10ul), (35.66 mg/5-L)
                            F2(Norm): Base(100 ml) MeOH( ml) OD(41.10) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (12.85 units/20ul), (15.03 mg/5-L)
      <Sung-Hye>         2/5/2011 (17:00) 
                            F1 (TEST) Refill Base (100 ml + 250 ml = 350 ml)
                            F2 (Norm) Refill Base (130 ml + 250 ml = 380 ml)
                            ** NaOH ** is from Bromme lab

Day 4 (Feb 6 Sun): MeOH Induction Day 3

      <Dennis>           2/6/2011 (14:00)      Fermentation 120:00  MeOH induction 48:00  
                            F1(Test): Base(340 ml) MeOH(300 ml) OD(56.0) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (14.34 units/5ul), (67.11 mg/5-L)
                            F2(Norm): Base(350 ml) MeOH(285 ml) OD(52.9) Contamination(N) Pressure(Y) Change Filter(Y)
                                Activity assay (18.57 units/5ul), (86.91 mg/5-L)

Day 5 (Feb 7 Mon): MeOH Induction Day 4

      <Dennis>           2/7/2011 (14:00)      Fermentation 144:00  MeOH induction 72:00 
                            F1(Test): Base(325 ml) MeOH(200 ml) OD(53.65) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (26.52 units/5ul), (124.14 mg/5-L)                                 
                            F2(Norm): Base(340 ml) MeOH(175 ml) OD(52.9) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (23.57 units/5ul), (110.31 mg/5-L)

Day 6 (Feb 8 Tue): MeOH Induction Day 5

      <Dennis>           2/8/2011 (14:00)      Fermentation 168:00  MeOH induction 96:00 

                            F1(Test): Base(310 ml) MeOH(100 ml) OD(53.9) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (34.96 units/5ul), (163.61 mg/5-L)
                            F2(Norm): Base(340 ml) MeOH(95 ml) OD(55.7) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (23.94 units/5ul), (112.04 mg/5-L)
      <Sung-Hye>         HARVEST




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