User:Fermenter User/Notebook/OVER D1

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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


OVER D1


Day -4 (Oct 24 Sat):

<Verena>            10/24/2009 (11am) streak GS115/MMP27 clone 19 onto MD and MM plates, 
                                      incubate over weekend at 30C

Day -3 (Oct 25 Sun):


 

Day -2 (Oct 26 Mon):

<Verena>      10/26/2009 (14:15) inoculate 5ml BMGY (50ml Falcon tube) with GS115/MMP27 clone 19, 
                                 incubate 28C 250rpm o/n 
                                 (important incubate set to 28C but temp fluctuates (never over 30C))

Day -1 (Oct 27 Tue):

<Verena>              10/27/2009 (9:30)  Autoclave 2x 500ml PBS
                                                   1x 700ml BM_Y
                                         cool BM_Y to room temp, then add 100ml 10xYNB, 100ml 10x Glycerol
                                         100ml 1M Potassiumphosphate buffer pH 6.0 and 2ml 500x Biotin 
<Sung-Hye> 10/27/2009 (11:30) Prepared bottles: 1) Acid, 1-L (tubing separate) 2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected) 4) Adjutives and Inoculum, 1-L (tubing connected) 5) Water, 2-L

<Sung-Hye> 10/27/2009 (13:30) Assemble bioreactors: 1. 1 x PBS 1-L of BMXY: Autoclave
<verena> 10/27/2009 (14:30) inoculate 20 ml BMGY in baffled flask with 100 ul preculture (OD600= 8.2) inoculate o/n 250rpm 28C


Day 0 (Oct 28 Wed):

<Verena>               10/28/2009 (9:27)   measured OD600= 10.5
<Sung-Hye>             10/28/2009 (9:30 - 10:00) Drain PBS bf and transfer media (1-L) using pump.
                           (Verena already combine media in a sterilized bottle:)                       
                              2. 1 x 500 ml of 10x Potassium Phosphate
                              3. 1 x 500 ml of 10x YNB 
                              4. 1 x 10 ml of 500x Biotin
                              5. 1 x 250 ml of 10x Glycerol

10/28/2009 (9:30) Purge Air 1 L/min at 800 rpm 10/28/2009 (9:50) Start Fermenter #3 software 10/28/2009 (10:00) Add Antifoam 6. 0.5 ml of antifoam/ fermenter 10/28/2009 (10:30) Set temperature Acid (500 ml) Base (500 ml) 10/28/2009 (10:30) Set rpm at 900 10/28/2009 (10:30) Calibrate dO2 probes 10/28/2009 (10:30) Start dO2 control (O2 valve Max) <Verena&Sung-Hye> 10/28/2009 (10:40) Fermentation 0:00 Inoculate cells (O.D: 14, Volume: 17 ml) Cells were inoculated via ruber septum using 10-ml syringe needle. <Sung-Hye> 10/28/2009 (10:30) Connect Acid and Base bottles Acid (500 ml) Base (400 ml) 10/28/2009 (10:30) Start pH control

Day 1 (Oct 29 Thu): MeOH Induction Day 1

<Verena>               10/29/2009 (10:30)  Fermentation 24:00 
                                          take sample, measure OD600= 20,2
                                          no contamination seen under the microscope
                                          leave growing till afternoon since till budding cells observed.
<Verena>               10/29/2009 (14:30)  Fermentation 28:00 
                                          sample measurement OD600= 27.5,
                                          no contamination, cells look healthy, still budding
                                          to sample for SDS-PAGE (60ulSN+10ulSDS-SB; cell lysate)
<Sung-Hye>             10/29/2009 (15:00)  Fermentation 28:30  MeOH Induction 00:00 
                               Connect MeOH
                               7. 1 x 1000 ml of MeOH: Tasfer MeOH into MeOH feeding bottles.
                               MeOH sensor calibration: 0.3% MeOH (3 ml/1-L) reads 9087 mV
                               Maximum allowance for sensor: 9000 mV ≈ around 2.5 - 2.8% or so.

Day 2 (Oct 30 Fri): MeOH Induction Day 2

<Sung-Hye>             10/30/2009 (9:30)  Fermentation 47:00  MeOH Induction 18:30 
                                           F3 Acid (490 ml) Base (370 ml) MeOH (970 ml)
                        NOTE: Definately, it is Mut+ strain. It took more MeOH than Mut- or Muts. 
Also, it consumed lot of O2 which is indication of continuous cell growth. MeOH sensor was above measurement limit (>9000 mV) and inactivated during overnight.


<Sung-Hye> 10/30/2009 (9:30) Fermentation 47:00 MeOH Induction 18:30 Change MeOH setpoint to 8000 mM and reduce MeOH pump speed to half.
<Verena> 10/30/2009 (10:30) Fermentation 48:00 MeOH Induction 19:30 OD600= 30 no contamination, cells have huge vacuoles, because of starvation o/n and are still budding.

Day 3 (Oct 31 Sat): MeOH Induction Day 3

<Verena>               10/31/2009 (14:30)  Fermentation 76:00  MeOH Induction 47:30    
                                          no contamination, budding yeasts and some with big vacuoles
                                          OD600= 37,3
<Sung-Hye>             10/31/2009 (16:00)  Fermentation 77:30  MeOH Induction 49:00  
                                          MeOH sensor was stabilized at 8000 mV.
                                          Everything looked fine.

Day 4 (Nov 1 Sun): MeOH Induction Day 4

<Verena>               11/1/2009 (10:30)  Fermentation 96:00  MeOH Induction 67:30    
                                           OD600= 79.2, no contamination
<Sung-Hye>             11/1/2009 (10:30)  Fermentation 96:00  MeOH Induction 67:30  
                                           Acid (490 ml), Base (300 ml), MeOH (800 ml)
                                           Pressurized vessel -> Change filter

Day 5 (Nov 2 Mon): MeOH Induction Day 5

<Sung-Hye>             11/2/2009 (9:30)   Fermentation 119:00  MeOH Induction 90:30   
                                          MeOH was in "safty cut-off" mode, meaning MeOH feeding speed was (not) too slow.
                                          Resumed MeOH pump --> NEXT time, use other MeOH pump!
<Verena> 11/2/2009 (11:00) Fermentation 120:00 MeOH Induction 91:30 no contamination, OD600= 84.2

Day 5 (Nov 3 Tue): MeOH Induction Day 6

<Verena&SungHye>       11/3/2009 (11:00)  Fermentation 148:00  MeOH Induction 115:30  
                                         Acid (480 ml) Base (250 ml)
                                         Harvest
                                         OD600= 109,2

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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