User:Fermenter User/Notebook/BROM A19

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BROM A19

Day -2 (Jan 31 Mon):
          1/31/2010 Thaw glycerol stock and grow in 5-ml MD media (made fresh 2 wks ago) Incubator in Fermentation Suite used           1/31/2010 (before go home) Preparing 5-L of Basal Medium (Defined media) PTM1 Salt is from BROM-A16

Day -1 (Feb 1 Tue): MEDIA PREPARATION DAY
        2/1/2011 (10:30)    Assemble bioreactors: 1. Calibrate pH sensors 2. 5.0-L of Basal Salts 3. 1-ml Antifoam         2/1/2011 (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                  2/1/2011 (11:30)  Autoclave                2/1/2011 (12:20)  Autoclave completed!                2/1/2011 (12:30)  Set temperature to 28.5°C                 2/1/2011 (13:30)  Purge Air 1L/min at 900 rpm                 2/1/2011 (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)         2/1/2011 (13:30)  Adjust pH to ~2.0 with ~100 ml NH4OH Reduce rpm to 400 overnight

          11/15/2010 (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
        2/2/2011 (9:30)   Increase rpm to 900         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): <font color=#FF00FF> 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 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 2
<Dennis>          2/5/2011 (14:00)     Fermentation 96:00 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 3
<Dennis>          2/6/2011 (14:00)     Fermentation 120:00 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 4
<Dennis>          2/7/2011 (14:00)     Fermentation 144:00 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 5
<Dennis>          2/8/2011 (14:00)     Fermentation 168:00 <font color=#FF00FF> 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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