User:Fermenter User/Notebook/MACG E8T

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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

MACG E8T

Day -1 (Apr 13 Mon)
 pH sensor: monitor or control pH   dO2 sensor: monitor or control dO2 Condenser: Gas outlet OD probe: monitor cell growth ==> Not used! Replate it with septum Triple: Addition of media, Acid or Base if necessary MeOH sensor: Monitor EtOH (byproduct)  MeOH sensors also detects ethanol! :See reference #6]            4/13/2009 (11:00)  Pour media 4/13/2009 (11:00) pH calibration 4/13/2009 (11:00) Add antifoam 0.5 ml/fermenter 4/13/2009 (11:30) Autoclave bioreactor ==> 3 bioreactors 3 empty bottles/fermenter (no need this time!) Carbon source, Inoculum, Feeding bottle 4/13/2009 (14:00) Start Temp Control

Day 0 (Apr 14 Tue)
4/14/2009 (8:47) Start F1 software 4/14/2009 (8:48) Start F2 software 4/14/2009 (8:49) Start F3 software 4/14/2009 (8:45) Connect Base (400 ml) 4/14/2009 (9:20) Add Carbon source using pump at 28.5°C 4/14/2009 (9:25) Calibrate dO2 4/14/2009 (9:25) Set dO2 4/14/2009 (9:25) Set pH        4/14/2009 (9:30) Fermentation 00:00 Inoculation 30 ml each using a pump. Sample in YP Dex stored for SDSPaGE                no readings taken today

Day 1 (Apr 15 Wed)
           4/15/2009 (7:00) Fermentation 21:30 F1: Base (380 ml) F2: Base (395 ml) F3: Base (370 ml)                4/15/2009 (10:30)  Fermentation 25:00 F1: OD (71.0) Contamination? (N)white, stationary, large ovoid cells with large vesicle. F2: OD (65.0) Contamination? (N)white, stationary, large ovoid cells with large vesicle. F3: OD (58.4) Contamination? (N)white, stationary, large ovoid cells with large vesicle. High pressure released and new filter used.   4/15/2009 (13:30)  Fermentation 28:00  Induction 00:00 Pepstatin added (13:30) Stop dO2 control F1: Watson pump: 20 rpm (2rpm pump was used, 10rpm = 12.8 ml/h) OD (67.5)Connect Pump to 250 ml of Feeding media (400g/l Gal)Cell viability: 99.5% F2: Watson pump: 20 rpm (2rpm pump was used, 10rpm = 12.8 ml/h) OD (64.3.0)Connect Pump to 250 ml of Feeding media (1/10th YP; 400g/l Gal)Cell viability: 99.5% F3: Watson pump: 1 rpm (32rpm pump was used, 10rpm = 209.3 ml/h) OD (54.9)Connect Pump to 250 ml of Feeding media (1xYP; 400g/l Gal)Cell viability: 99.3%

<Isis>               4/15/2009 (15:30)  Fermentation 30.50 Induction 02.50 F1: OD (86.9) Contamination? (N)white, large ovoid cells with large vesicle, budding. F2: OD (85.5) Contamination? (N)white, large ovoid cells with large vesicle, budding. F3: OD (56.4) Contamination? (N)white, large ovoid cells with large vesicle, budding.F2 AND F3 smell like IAA, F1 smells nasty. High pressure released and new filter used. <Isis>               4/15/2009 (23:30)  Fermentation 38.5 Induction 10.0 F1: OD (106.0) Contamination? (N)white, budding. F2: OD (107.8) Contamination? (N)white, budding. F3: OD (81.8) Contamination? (N)white, budding. all smell like IAA

Day 2 (Apr 16 Thu)
<Sung-Hye>           4/16/2009 (7:00) Fermentation 46.5  Induction 18.0 F1: Base (360 ml) F2: Base (380 ml) F3: Base (370 ml) <Isis>               4/16/2009 (7:30)  Fermentation 46.5  Induction 18.0 F1: OD (119.6) Contamination? (N) white, cells, some fat large buds still, lots of small vesicles in cells, 99.99% viability F2: OD (107.0) Contamination? (N) white, cells, some fat large buds still, lots of small vesicles (approx 5/cell), >99.99% viability F3: OD (105.5) Contamination? (N) white, cells, still budding, small and large buds seen, some small vesicles in cells, >99.99% viability <Isis>               4/16/2009 (10:00)  Fermentation 48:50  Induction 20.5 F1: OD (122.1) Contamination? (N) <Isis>                   F2: OD (124.8) Contamination? (N) F3: OD (115.2) Contamination? (N) all still budding a little, and lots of vacuoles. <Isis>               4/16/2009 (11:30)  Fermentation 50:00  Induction 22.0 F1: OD (125.2) Contamination? (N) F2: OD (126.6) Contamination? (N) F3: OD (113.0) Contamination? (N) lots of vacuoles. <Isis>               4/16/2009 (18:00)  Fermentation 55.00  Induction 28.5 F1: OD (120.6) Contamination? (N) 3 dead cells F2: OD (122.2) Contamination? (N) 5 dead cells F3: OD (113.8) Contamination? (N) 2 dead cells <Isis>               4/16/2009 (19:30)  Fermentation 54:50  Induction 30.0 no ODs taken- 50ml samples only.

Day 3 (Apr 17 Fri)
<Sung-Hye>           4/17/2009 (07:00) Fermentation 70.5  Induction 32.0 F1: Base (330 ml) F2: Base (360 ml) F3: Base (300 ml) <Isis>               4/17/2009 (00:00) Fermentation ?? Induction ?? F1: OD (??) Contamination? (N) F2: OD (??) Contamination? (N) F3: OD (??) Contamination? (N) <Sung-Hye>           4/17/2009 (00:00) Fermentation ?? Induction ?? Terminate Fermentation