User:Fermenter User/Notebook/BROM A6

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<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">User:Fermenter User/Notebook/BROM A6</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

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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
Pink: MeOH Induction hrs

BROM A6 (F2)

Day -1 (Dec 8th Mon):

 Change #3: Sung-Hye need to check levels!!:  
 
<Adeleke>              12/8/2008 (8:45)  Autoclaved 100ml flasks for culturing. 
                                        Prepared all necessary media components for fermentation:
                                         1. 1L 10x YNB (sterile filtered)
                                         2. 1L Phosphate buffer, pH 6.0 (autoclaved)
                                         3. 1L 10x Glycerol (Autoclaved)
                                         4. 30mL 500x Biotin (sterile filtered)
                                         5. 1L 100% Methanol  

<Adeleke> 12/8/2008 (11:45) Grow 0.5 ml stock culture in 50 ml of BMGY at 30C for 12-24h until OD reaches 6. 50 ml of the cells will be used for fermentation.

<Sung-Hye> 12/8/2008 (13:00) Prepared bottles: 1) Acid, 1-L (tubing separate) 2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected) 4) Level, 1-L (tubing connected) 5) Additives, 2-L (tubing connected) 6) Inoculum, 500-ml (tubing connected) 7) Water, 2-L

<Adeleke> 12/8/2008 (15:00) Gave 3.5-L BMGY to Sung-Hye.

<Sung-Hye> 12/8/2008 (15:20) Assemble bioreactors: 1. 1 x 3.5-L of BMXY: 2. 2 ml of antifoam/ fermenter Autoclave <Adeleke> 12/8/2008 (20:00) Left additives in fume food

Day 0 (Dec 9th Tue):

<Sung-Hye>             12/9/2008 (9:33)      Cooling bioreactors: 150 rpm 
                       12/9/2008 (9:45 - 11:30)      In 2-L sterilized bottle: Combine                       
                               3. 1 x 500 ml of 10x Potassium Phosphate
                               4. 1 x 500 ml of 10x YNB 
                               5. 1 x 10 ml of 500x Biotin
                               6. 1 x 500 ml of 10x Glycerol
                               Picture was taken during transfer of media 

12/9/2008 (9:47) Start OD probe software 12/9/2008 (9:50) Start Fermenter #2 software 12/9/2008 (11:17) Connect Acid and Base bottles Acid (490 ml) Base (400 ml) 12/9/2008 (11:17) Set rpm at 900 12/9/2008 (11:17) Purge Air 1 L/min 12/9/2008 (11:17) Start pH control 12/9/2008 (13:05) Calibrate dO2 probes 12/9/2008 (13:05) Start dO2 control (O2 valve Max) 12/9/2008 (13:05) Autozeroing OD sensor

<Sung-Hye and Adeleke> 12/9/2008 (14:00) Fermentation 0:00 Inoculate cells (O.D: 5.2 Volume: 50 ml) Cells were transferred to bottles/tube and inoculated using pumps. <Adeleke> 12/9/2008 (18:24) Fermentation 4:24 OD check (O.D: 0.68 Volume: 1 ml )

Day 1 (Dec 10th Wed): MeOH Induction Day 0

<Sung-Hye>             12/10/2008 (9:00)     Fermentation 19:00 
                                                        Acid (285 ml) Base (195 ml)
                      Memo: dO2 is >SP. Cells are fully grown and reay for MeOH induction.

<Adeleke> 12/10/2008 (11:50) Fermentation 21:50 (O.D:12.6 ) Contamination? (No) Acid (285 ml) Base (195 ml)

<Adeleke> 12/10/2008 (16:45) Fermentation 26:45 (OD :13.4 ) Contamination? (No)

<Sung-Hye and Adeleke> 12/10/2008 (17:00) Fermentation 27:00 MeOH Induction 00:00 Connect MeOH 7. 1 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. MeOH sensor calibration Set level to 5.5-L
<Adeleke> 12/10/2008 (18:30) Fermentation 28:30 MeOH induction 01:30 (OD :13.3 ) Contamination? (No) Activity (0.43)what is unit?


Day 2 (Dec 11th Thu): MeOH Induction Day 1

<Sung-Hye>             12/11/2008 (9:00)      Fermentation 43:00  MeOH induction 16:00 
                                             Acid (285 ml) Base (195 ml)
                                             Picture of bioreator was taken
<Adeleke> 12/11/2008 (18:46) Fermentation 52:46 MeOH induction 25:46 (O.D:14.89 , Activity: 0.57) Contamination? (No) MeOH (510 ml)

Day 3 (Dec 12th Fri): MeOH Induction Day 2

<Sung-Hye>             12/12/2008 (9:00)      Fermentation 67:00  MeOH induction 40:00 
                                              Acid (260 ml) Base (195 ml) MeOH (510 ml)
<Adeleke>              12/12/2008 (17:30)      Fermentation 75:30  MeOH induction 48:30  
                       (O.D: 14:37, Activity: 0.74) Contamination? (No) MeOH (510 ml)

Day 4 (Dec 13th Sat): MeOH Induction Day 3

<Adeleke>              12/13/2008 (time here)      Fermentation 00:00  MeOH induction 00:00  
                       (O.D: , Activity: ) Contamination? (Y/N) MeOH ( ml) 


Day 5 (Dec 14th Sun): MeOH Induction Day 4

<Adeleke>              12/14/2008 (time here)      Fermentation 00:00  MeOH induction 00:00  
                       (O.D: , Activity: ) Contamination? (Y/N) MeOH ( ml) 


Day 6 (Dec 15th Mon): MeOH Induction Day 5

<Adeleke>              12/15/2008 (time here)      Fermentation 00:00  MeOH induction 00:00  
                       (O.D: , Activity: ) Contamination? (Y/N) MeOH ( ml) 


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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