User:Fermenter User/Notebook/BROM I1

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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 H1

Day -2 (Jun 5 Sun):
          6/19/2011 (13:00) Thaw glycerol stock and grow in 5-ml MD media Incubator in Fermentation Suite used           6/19/2011 Preparing 5-L of Basal Medium (Defined media) PTM1 Salt is from BROM-A16

Day -1 (Jun 20 Mon): MEDIA PREPARATION DAY
           6/20/2010 (??:?? - ??:??) Bring powder to make 5-L basal salts

        6/20/2011 (14:00)    Assemble bioreactors: 1. Calibrate pH sensors 2. 5.0-L of Basal Salts 3. 1-ml Antifoam         6/20/2011 (14:00)  Prepare bottles 1) no Acid need                                           2) Base, 500-ml (tubing connected) x1                                            3) MeOH, 1-L (tubing connected) x1                                            4) Additives/Inoculum, 500-ml (tubing connected) x1                                            5) No Water, 2-L                                             6) Fed-Batch, 500-ml (tubing connected) x1                  6/20/2011 (15:13)  Autoclave

           6/20/2011 (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

Day 0 (Jun 21 Tue): INOCULATION DAY
           6/21/2011 (10:30)  Check contamination of starter culture Flask OD: 32.10

        6/21/2011 (11:00)  Set temperature to 28.5°C         6/21/2011 (11:00)  Purge Air 1L/min at 900 rpm            6/21/2011 (11:10)  Add 21.75 ml of PTM1 Salt/5-L media <-- Prepared at BROM A16 Re-filtered by Jason (4.35 ml PTM1 salt per 1-L media = 21.75 ml in 5-L))      <Jason>            6/21/2011 (11:30)  Adjust pH to ~3.0 (with ~100 ml NH4OH)        <Jason>            6/21/2011 (11:40)  Adjust pH to 5.0 with **2M NaOH** <-- 4M is better!                                             (borrowed from Bromme lab, 500 ml)                                             150 ml used        <Sung-Hye>         6/21/2011 (11:45)  Start pH control                                              F1: No Acid Base, 2M (350 ml)<-- 4M is better!       <Sung-Hye>         6/21/2011 (11:45)  Calibrate dO2 probes                           6/21/2011 (11:45)  Strt dO2 control        <Sung-Hye>         6/21/2011 (11:46)  Start Fermenter #1 software        <Jason&Sung-Hye>   6/21/2011 (12:00)  Fermentation 0:00                                              Inoculate 55 ml/bioreactor Cells were transferred using pumps.

<Sung-Hye>        6/21/2011 (15:00)  Change dO configuration

Day 1 (Jun 22 Wed):
<Jason>          6/22/2011 (13:20)     Fermentation 25:20 F1:  Base(315 ml) OD(4.70) Contamination(N) Pressure(N) Change Filter(N)

Day 2 (Jun 23 Thu): <font color=#FF00FF> Fed-Batch and MeOH Induction Day 1
<Jason>           6/23/2011 (9:00)     Fermentation 45:00 F2: Base(30 ml) OD(31.75) Contamination(n) Pressure(n) Change Filter(n) <Sung-Hye>        6/23/2011 (9:40 - 12:40) Fermentation 44:40 - 47:40 F2: Fed-Batch 400 ml 50% Glycerol + 4.8 ml PTM1 at 90.75ml/hr/5-L (RPM 65 w/ 2RPM pump) <Sung-Hye>        6/23/2011 (11:00) F2: Take out 500-ml media <Sung-Hye>        6/23/2011 (11:07) Manually inject ~5 ml of conc. NH4OH via septum (pH: 4.75 → 5.20) <Jason>           6/23/2011 (11:50) F2: Refill Base (0 ml +500 ml = 500 ml): pH reading: 5.0 <Jason>           6/23/2011 (12:40)     Fermentation 48:00 F1: Base(480 ml) OD(44.30) Contamination(n) Pressure(n) Change Filter(n) <Sung-Hye>        6/23/2011 (13:30)     Fermentation 49:30 <font color=#FF00FF> MeOH induction 00:00 Calibrate MeOH sensor F2: 6231 mV = 0.5% (MeOH 750 ml)<-- Mix with PTM1 Start MeOH induction <Sung-Hye>        6/23/2011 (17:00) F2: Take out 500-ml media <Jason>           6/23/2011 (17:40) F2: Replace Base (2M, 350 ml) → Base (4M, 500 ml)

Day 3 (Jun 24 Fri): <font color=#FF00FF> MeOH Induction Day 2
<Jason>          6/24/2011 (13:30)     Fermentation 73:30 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 3
<Jason>          6/25/2011 (13:30)     Fermentation 97:30 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 4
<Jason>           6/26/2011 (13:30)     Fermentation 121:30 <font color=#FF00FF> 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): <font color=#FF00FF> MeOH Induction Day 5
<Jason>          6/27/2011 (12:00)     Fermentation 144:00 <font color=#FF00FF> 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