User:Fermenter User/Notebook/BROM J1

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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 J1

Day -2 (Jul 10 Sun):
          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
           7/11/2010 (14:30) Bring powder to make 5-L basal salts

        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         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                  7/11/2011 (15:16)  Autoclave

           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
           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.   7/12/2011 (11:30)  Set temperature to 28.5°C   7/12/2011 (11:30)  Purge Air 1L/min at 900 rpm   7/12/2011 (11:30)  Adjust pH to ~4.75 (with ~90 ml NH4OH)   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):
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): <font color=#FF00FF> 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>        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>        7/14/2011 (11:00) F2: Take out 500-ml media <Sung-Hye>        7/14/2011 (11:07) Manually inject ~5 ml of conc. NH4OH via septum (pH: 4.75 → 5.20) <Vidhu>           7/14/2011 (11:50) F2: Refill Base (0 ml +500 ml = 500 ml): pH reading: 5.0 <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 <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>      7/14/2011 (17:00) F2: Take out 500-ml media <Vidhu>           7/14/2011 (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