User:Fermenter User/Notebook/BROM A8

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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 A8

Day -2 (Oct 25 Sun):
             10/25/2009 (12:30) Used MD media for first stage cell culturing Autoclaved two 2L flask for culturing. Prepared all necessary media components for fermentation: 1. 1.0L 10x YNB (filtered, excess amount) 2. 1.0L Phosphate buffer, pH 6.0 (autoclaved, excess amount) 3. 1.0L 10x Glycerol (filtered) 4. 20mL 500x Biotin (filtered, excess amount) (Take half of each above media components, and combine them to make 1 bottles of 1.51L culturing media)

Day -1 (Oct 26 Mon):
          10/26/2009 (10am)   2 flasks of 2L of 500mLBMGY for second stage culturing before fermentation for each wild type.          10/26/2009 (13:00)    Prepared bottles: 1) Acid, 2 x 1-L (tubing separate)                                                  2) Base, 2 x 500-ml (tubing connected) 3) MeOH, 2 x 1-L (tubing connected)                                                 4) Adjutives/Inoculum, 2 x 1-L (tubing connected) 5) Water, 2-L                              10/26/2009 (13:30)    Assemble bioreactors:                                                                          1. 2 x 3.5-L of BMXY:                                    Autoclave                  10/26/2009 (13:30) Leave the master culturing media containing the following items in the fermenter room:                                                  1) Potassium Phosphate 2) YNB                                                 3) Biotin 4) Glycerol

Day 0 (Oct 27 Tue):
         10/27/2009 (11:00)     Cooling bioreactors: 400 rpm 10/27/2009 (11:00)   Assemble bioreactors (continued): 2. 1 ml of antifoam/ fermenter 10/27/2009 (11:30)    Purge Air 1 L/min at 400 rpm 10/27/2009 (12:22)    Start Fermenter #1 software 10/27/2009 (12:23)    Start Fermenter #2 software Transfer Adjutives using pumps. 10/27/2009 (11:30-12:30) Combine: 3. 2 x 500 ml of 10x Potassium Phosphate 4. 2 x 500 ml of 10x YNB 5. 2 x 10 ml of 500x Biotin <--- Missed!!! 6. 2 x 250 ml of 10x Glycerol 10/27/2009 (13:40)    Set rpm at 900 10/27/2009 (13:40)    Calibrate dO2 probes 10/27/2009 (13:40)    Start dO2 control (O2 valve Max)   10/27/2009 (13:00)     Fermentation 0:00 Flask 1 OD: 7.23, FLask 2 OD: 9.14 Inoculate 2 fermentors with Sung-Hye 10/27/2009 (13:10)    Start pH control Acid (500 ml) Base (400 ml)

Day 1 (Oct 28 Wed):  MeOH Induction Day 1
         10/28/2009 (09:00)     Fermentation 20:00 F1 Acid (N.A.) Base (N.A.) F2 Acid (N.A.) Base (N.A.)           10/28/2009 (13:00)     Fermentation 24:00 Prepared 1.5L 100% Methanol(non-HPLC grade) filter sterilize. Fermentor 1 OD: 17.83, Fermentor 2 OD: 18.33 Microscope: Fermentor 1 has very little contamination less than 1% on the slides. Fermentor 2 has no contamination on slides. Begin induction with Methanol with Sung-Hye          10/28/2009 (13:00)     Fermentation 24:00 <font color=#FF00FF> MeOH induction 00:00 Connect MeOH 7. 2 x 700 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 10/28/2009 (13:00)    MeOH sensor calibration

Day 2 (Oct 29 Thu): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>          10/29/2009 (09:00)     Fermentation 44:00 <font color=#FF00FF> MeOH induction 20:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) F2 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Raymond>           10/29/2009 (13:00)     Fermentation 48:00 <font color=#FF00FF> MeOH induction 24:00 (F1)=(O.D:18.5 ) same amount of contamination, one needs to look for it. Possible sources of contamination due to syringe or coverslip etc.                                                    (F2)=(O.D:19.43) <Raymond&SungHye>   10/29/2009 (13:30)     Fermentation 48:30 <font color=#FF00FF> MeOH induction 23:00 (F1)=Manuial injection of 15 ml (total 0.8%), MeOH [0.5%], change filter (F2)=Manuial injection of 15 ml (total 0.8%), MeOH [0.5%], change filter

Day 3 (Oct 30 Fri): <font color=#FF00FF> MeOH Induction Day 3
<Sung-Hye>          10/30/2009 (09:00)     Fermentation 68:00 <font color=#FF00FF> MeOH induction 40:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) F2 Acid (?? ml) Base (?? ml) MeOH (?? ml) NOTE: Cells are not taking MeOH. MeOH concentration did not change at 0.8%. Possible reason: Colony chosed for this fermentation might not the best ones. My recommendation: Terminate this fermentation and go back to colony screening. <Raymond>           10/30/2009 (13:00)     Fermentation 72:00 <font color=#FF00FF> MeOH induction 48:00 (F1)=(OD: 17.23) (F2)=(O.D:18.38)

Day 4 (Oct 31 Sat): <font color=#FF00FF> MeOH Induction Day 4
<Raymond>           10/31/2009 (04:00)     Fermentation 87:00 <font color=#FF00FF> MeOH induction 63:00 (F1)= Add Biotin 4am (F2)= Add Biotin 4am <Sung-Hye>          10/31/2009 (16:00)     Fermentation 99:00 <font color=#FF00FF> MeOH induction 75:00 F1: Acid tubing was damaged. Remove Acid (Output#3) from pH control. Noticed MeOH leve was going down on both F1 and F2

Day 5 (Nov 1 Sun): <font color=#FF00FF> MeOH Induction Day 5
<Sung-Hye>          11/1/2009 (10:30)     Fermentation 117:30 <font color=#FF00FF> MeOH induction 93:30 F1 Acid (430 ml) Base (380 ml) MeOH (680 ml) F2 Acid (360 ml) Base (450 ml) MeOH (700 ml) MeOH level gone down since biotin addition, but DO level is >100% which is indication of unhealthy cells. <Raymond>           11/1/2009 (13:00)     Fermentation 120:00 <font color=#FF00FF> MeOH induction 96:00 (F1)=(O.D:17.23, Activity:0.18) (F2)=(O.D:18.96, Activity:0.00)

Day 6 (Nov 2 Mon): <font color=#FF00FF> MeOH Induction Day 6
<Sung-Hye>          11/2/2009 (09:00)     Fermentation 140:00 <font color=#FF00FF> MeOH induction 116:00 F1 Acid (?? ml) Base (?? ml) MeOH (?? ml) F2 Acid (?? ml) Base (?? ml) MeOH (?? ml) <Raymond>           11/2/2009 (13:00)     Fermentation 144:00 <font color=#FF00FF> MeOH induction 120:00 (F1)=(Activity:0.00) (F2)=(Activity:0.00)

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