User:Fermenter User/Notebook/MACG E3

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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 E3 (F1/F2)

Day -1 (Feb 2 Mon)
 pH sensor: monitor or control pH   dO2 sensor: monitor or control dO2 Condenser: Gas outlet OD probe: monitor cell growth Triple: Addition of media, Acid or Base if necessary MeOH sensor: Monitor EtOH (byproduct)  MeOH sensors also detects ethanol! :See reference #6] Volume of F2: keep as same 1-L Media (F2): 1-L Additional blade (higher than normal):no need Harvest time will be quicker when activity is Max.            2/2/2009 (13:20)  Pour media 2/2/2009 (13:20) pH calibration 2/2/2009 (13:40) Autoclave bioreactor 3 empty bottles/fermenter Carbon source, Inoculum, Feeding bottle 2/2/2009 (15:30) Antifoam added :added later! 2/2/2009 (15:35) Start Temp Control 2/2/2009 (15:45) Add Carbon sources using pumps 2/2/2009 (15:46) Start OD software 2/2/2009 (15:50) Start F1 software 2/2/2009 (15:52) Start F2 software

Day 0 (Feb 3 Tue)
Sung-Hye Class 9:30-11:00

           2/3/2009 (8:15)   Calibrate dO2 2/3/2009 (8:15)  Set dO2   2/3/2009 (9:00)  Overnight culture KLABDM3 OD: 60.8, no contamination 2/3/2009 (10:00) Fermentation 0:00 Inoculation 30 ml each                2/3/2009 (17:00)  Fermentation 7:00 F1: Sampling: OD (??) Contamination? (N) F2: Sampling: OD (??) Contamination? (N)

Day 1 (Feb 4 Wed)
               2/4/2009 (8:00)  Fermentation 22:00 F1: OD (62.4) Contamination? (N)budding well, no vacuoles. F2: OD (49.1) Contamination? (N)budding well, no vacuoles, clumpy. 5 am - 9:15 am : O2 tanks ran out            2/4/2009 (9:15) Fermentation 23:15 Open O2 tank                2/4/2009 (10:00)  Fermentation 24:00 F1: OD (65.3) Contamination? (N) F2: OD (35.0) Contamination? (N) Cells turning pink!!- not sure if 8am reading was anomalously high?                2/4/2009 (13:00)  Fermentation 27:00 F1: OD (62.8) Contamination? (N) F2: OD (49.5) Contamination? (N) <Sung-Hye and Isis>  2/4/2009 (15:00) Fermentation 29:00 F1: Connect Pump to 500 ml of Feeding media: Induction 0:00 F2: Connect Pump to 500 ml of Feeding media (Wait till tomorrow) Watson pump: 10 rpm ==> Same as MACG E1 (F1) <Isis>               2/4/2009 (16:00)  Fermentation 30:00  Induction 1:00 F1: OD (86.9) Contamination? (N)no vacuoles, filter change due to pressure buildup. F2: OD (55.6) Contamination? (N)no vacuoles <Sung-Hye>           2/4/2009 (16:30)  Fermentation 30:30  Induction 1:30 F1: 1uM Pepstatin added to F1 to limit activity of aspartyl proteases (Paper 8) <Isis>               2/4/2009 (17:00)  Fermentation 31:00 F1: OD (112.9) Contamination? (N) Induction 2:00 F2: OD (59.1) Contamination? (N)

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Day 2 (Feb 5 Thu)
Sung-Hye Class 9:30 - 11:00 <Isis>               2/5/2009 (8:00) Fermentation 46:00 F1: OD (131.4) Contamination? (N) 1-5 vacuoles present. Pellet cream color with pink patches Induction 17:00 F2: OD (52.8) Contamination? (N) Cell pellet visibly pink now. One large vesicle in approximately 1/2 cells, Takes much much of the periplasmic space. <Sung-Hye and Isis>  2/5/2009 (9:00) Fermentation 47:00 1uM Pepstatin added to F2 to limit activity of aspartyl proteases (Paper 8) F2: Connect Pumps to 300 ml of Feeding media: Induction 0:00 Watson pump: 8 rpm ==> Same as MACG E1    <Isis>                2/5/2009 (11:00) Fermentation 49:00 F1: OD (139.4) Contamination? (N) Induction 20:00 Pellet is cream colored again, not much pink remains. F2: OD (60.5) Contamination? (N) Induction 2:00 Active budding, vacoules still present. Filter change due to pressure buildup. <Isis>               2/5/2009 (13:00) Fermentation 51:00 F1: OD (??) Contamination? (Y/N) Induction 22:00 F2: OD (??) Contamination? (Y/N) Induction 4:00 <Isis>               2/5/2009 (15:00)  Fermentation 53:00 F1: OD (128.3) Contamination? (N) Induction 24:00 Harvest <Isis>               2/5/2009 (16:00)  Fermentation 54:00 F2: OD (57.0) Contamination? (N) Induction 7:00 <Isis>               2/5/2009 (19:00)  Fermentation 57:00 F2: OD (66.0) Contamination? (N) Induction 10:00

Day 3 (Feb 6 Fri)
<Isis>               2/6/2009 (6:00) Fermentation 68:00 F2: OD (62.2) Contamination? (N) lots of budding. Induction 21:00 <Isis>               2/6/2009 (7:00) Fermentation 69:00 F2: OD (64.2) Contamination? (N) Induction 22:00 2/6/2009 (7:30) Fermentation 69:00 Picture of bioreactor (F2) was taken Induction 22:30 <Isis>               2/6/2009 (8:00) Fermentation 70:00 F2: OD (68.7) Contamination? (N) Induction 23:00 <Isis>               2/6/2009 (9:00) Fermentation 71:00 F2: Harvest Induction 23:00