User:Fermenter User/Notebook/BROM J1
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(Difference between revisions)
m (→Day 0 (Jun 21 Tue): INOCULATION DAY) |
(→Day -1 (Jul 11, Mon): MEDIA PREPARATION DAY) |
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<Sung-Hye> 7/11/2011 (14:00) <font color=blue>Assemble bioreactors: </font> | <Sung-Hye> 7/11/2011 (14:00) <font color=blue>Assemble bioreactors: </font> | ||
1. Calibrate pH sensors | 1. Calibrate pH sensors | ||
| - | 2. 5.0-L of Basal Salts | + | 2. 5.0-L of Basal Salts: Phosphoric Acid was 10-ml short. |
3. 1-ml Antifoam <br> | 3. 1-ml Antifoam <br> | ||
<Sung-Hye> 7/11/2011 (14:00) Prepare bottles | <Sung-Hye> 7/11/2011 (14:00) Prepare bottles | ||
| Line 57: | Line 57: | ||
<b>Incubator in Fermentation Suite used</b> | <b>Incubator in Fermentation Suite used</b> | ||
2.Prepare sterilized Methanol by filtration | 2.Prepare sterilized Methanol by filtration | ||
| - | 3.Prepare sterilized NaOH | + | 3.Prepare sterilized NaOH (4M, 500-ml) |
== Day 0 (Jun 21 Tue): <font color=red> INOCULATION DAY </font>== | == Day 0 (Jun 21 Tue): <font color=red> INOCULATION DAY </font>== | ||
Revision as of 18:24, 11 July 2011
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Background color codes: Letter color codes:
Day -4 (Jun 3 Fri):Day -3 (Jun 4 Sat):Day -2 (Jul 10 Sun): <Vidhu> 7/10/2011 (13:00)
Thaw glycerol stock and grow in 5-ml MD media
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 (14:00) Assemble bioreactors:
1. Calibrate pH sensors
2. 5.0-L of Basal Salts: Phosphoric Acid was 10-ml short.
3. 1-ml Antifoam
<Jason> 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 (4M, 500-ml)
Day 0 (Jun 21 Tue): INOCULATION DAY <Jason> 6/21/2011 (10:30) Check contamination of starter culture<-- Before 11:00AM
Flask OD: 32.10
Bring NaOH and MeOH to Fermentation room.<-- Before 10:30AM
Bring inoculum to Fermentation room.<-- 11:50AM
<Sung-Hye> 6/21/2011 (11:00) Set temperature to 28.5°C
<Sung-Hye> 6/21/2011 (11:00) Purge Air 1L/min at 900 rpm
<Jason> 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)
Prepare 400 ml 50% Glycerol (autoclaved) for Fed-Batch next day.
Day 2 (Jun 23 Thu): 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 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): 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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