User:Fermenter User/Notebook/BROM A8

From OpenWetWare

Jump to: navigation, search
Customize your entry pages

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):

      <Raymond>              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):

      <Raymond>           10/26/2009 (10am)   2 flasks of 2L of 500mLBMGY for second stage culturing before fermentation for each wild type.
<Sung-Hye> 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

<Sung-Hye> 10/26/2009 (13:30) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: Autoclave <Raymond> 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):

      <Sung-Hye>          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) <Raymond&SungHye> 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

      <Sung-Hye>          10/28/2009 (09:00)      Fermentation 20:00 
                                                   F1 Acid (N.A.) Base (N.A.)
                                                   F2 Acid (N.A.) Base (N.A.) 
      <Raymond>           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
      <Sung-Hye>          10/28/2009 (13:00)      Fermentation 24:00  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): MeOH Induction Day 2

      <Sung-Hye>           10/29/2009 (09:00)      Fermentation 44:00  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  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  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): MeOH Induction Day 3

      <Sung-Hye>           10/30/2009 (09:00)      Fermentation 68:00  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 MeOH induction 48:00 (F1)=(OD: 17.23) (F2)=(O.D:18.38)

Day 4 (Oct 31 Sat): MeOH Induction Day 4

      <Raymond>            10/31/2009 (04:00)      Fermentation 87:00  MeOH induction 63:00   
                                                    (F1)= Add Biotin 4am
                                                    (F2)= Add Biotin 4am
      <Sung-Hye>           10/31/2009 (16:00)      Fermentation 99:00  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): MeOH Induction Day 5

      <Sung-Hye>           11/1/2009 (10:30)      Fermentation 117:30  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  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): MeOH Induction Day 6

      <Sung-Hye>           11/2/2009 (09:00)      Fermentation 140:00  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  MeOH induction 120:00   
                                                    (F1)=(Activity:0.00) 
                                                    (F2)=(Activity:0.00)
                                                 NO HARVEST

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
   



Recent changes



Personal tools