User:Sung-Hye Grieco/Notebook/DEMO

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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 F2 (F1) and (F2)

Contents

Day -8 (Nov 10th Mon):

      <Kevin>              11/10/2008 (tim here)

Day -7 (Nov 11th Tue):

      <Kevin>              11/11/2008 (time here)

Day -6 (Nov 12th Wed):

        
    Discussion about BROM F1 (F1) 
 :

Day -5 (Nov 13th Thu):

Day -4 (Nov 14th Fri):

 Change #1: Change head plate configuration to add more open ports 
      <Sung-Hye>           11/14/2008 (12:00)      Change configuration of head plate:
                                                   Add open port (x1) for Fermenter #2
     
    Discussion about Glycerol Feeding Methods and Inoculum 
1. Glycerol feeding: Start fermentation with BMGY: 1% final concentration of Glycerol in 5-L media. No more additional feeding will be added. Based on BROM F1 experiment, metabolism switch from Glycerol to MeOH occurred around 22hr fermentation 14 hr MeOH induction We don't have record of OD of that time, but OD measurement at 28:30 hr fermentation was 12-13. We want to keep the same condition, and hope that cells are continue to grow to 20 or more in the presence of MeOH (set to 0.5%). 2. Inoculum volume and density: Same as BROM F1, but inoculate late of Day 0 17:00.
      <Kevin>              11/14/2008 (time here)  Autoclaved two 100ml flasks and two 500ml flasks for culturing. 
                                        Prepared all necessary media components for fermentation:
                                         1. 1.5L 10x YNB (filtered, excess amount)
                                         2. 1.5L Phosphate buffer, pH 6.0 (autoclaved, excess amount)
                                         3. 1L 10x Glycerol (filtered)
                                         4. 100mL 500x Biotin (filtered, excess amount)
 Change #2: MeOH concentration is 100% 
                                         5. 1.5L 100% Methanol: Re-use from BROM F1 experiments (no need to prepare)

Day -3 (Nov 15th Sat):

      <Kevin>              11/15/2008 (time here) 
                              Used previous MD media (1-week old, < 500 ml) for first stage cell culturing 
                              500mL BMGY for second stage culturing before fermentation for each mutant. 
      <Kevin>              11/15/2008 (time here) 
                              Culture 20uL stocks of Y98C and D5 in 5mL MD media at 30C over night.

Day -2 (Nov 16th Sun):

      <Kevin>              11/16/2008 (time here)  
                            O.D.(both mutants)= 6.0. after 16h
                            Spin down the cells (Y98C and D5) at 2000g for 5 minutes and resuspend them in 10mL BMGY.
                            Transfer 100uL of the resuspended culture of each mutant to 50mL BMGY for over night culturing.

Day -1 (Nov 17th Mon):

 Change #3: Sung-Hye need to check levels!!: ok 
      <Sung-Hye>           11/17/2008 (12:00)    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) Adjutives, 1-L (tubing connected)<-- Make it 2-L next time 
                                                 6) Inoculum, 500-ml (tubing connected)
                                                 7) Water, 2-L 

<Kevin> 11/17/2008 (14:30) Gave all the necessary media components to Sung-Hye.

<Sung-Hye> 11/17/2008 (14:45) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: (forgot to add Antifoam) Autoclave <Sung-Hye> 11/17/2008 (16:45) Add antifoam: 2. 2 ml of antifoam/ fermenter

Change #4: Delay inoculation time: Reduce volume of cell culture (from 1 ml to 0.5 ml) <Kevin> 11/17/2008 (time here) Both mutant cultures (Y98C and D5) reached O.D =6. Take 0.5 mL of each culture and regrew them in 500mL BMGY for next day fermentation.

<Kevin> 11/17/2008 (time here) Leave following items in fermenter room: 1) Potassium Phsphate 2) YNB 3) Biotin 4) Glycerol

Day 0 (Nov 18th Tue):

     <Sung-Hye>            11/18/2008 (9:30)      Cooling bioreactors: 150 rpm 
     <Sung-Hye>            11/18/2008 (9:30-11:10)Transfer nutrients into bioreactors: 
 Change #5: Feeding method: Use Pumps 
     <Sung-Hye>                                   Transfer Adjutives using pumps.  
                           11/18/2008 (9:30-10:30)  1-L sterilized bottle: Combine                       
                              3. 2 x 500 ml of 10x Potassium Phosphate
                              4. 2 x 500 ml of 10x YNB 
                           11/18/2008 (10:33-11:10)  Repeat same procedure with:
                              5. 2 x 10 ml of 500x Biotin
                              6. 2 x 500 ml of 10x Glycerol

11/18/2008 (9:44) Start OD probe software 11/18/2008 (9:47) Start Fermenter #1 software 11/18/2008 (9:49) Start Fermenter #2 software 11/18/2008 (11:10) Connect Acid and Base bottles Acid (250 ml) Base (400 ml) 11/18/2008 (11:15) Set rpm at 900 11/18/2008 (11:15) Purge Air 1 L/min 11/18/2008 (11:25) Start pH control 11/18/2008 (11:50) Calibrate dO2 probes 11/18/2008 (11:50) Start dO2 control (O2 valve Max) 11/18/2008 (12:40) Autozeroing OD sensor


 
   Discussion about Amp: Do not add!! 
  
    1. We don't know whether yeast cells will express Amp resistance gene or not
        (becuase promoter for Amr is from E.coli
    2. We don't know the effect of Amp on yeast growth in general
        Find some reference


 Change #6: Delayed inoculatation time (from 10:00 to 17:00)
      <Kevin>              11/18/2008 (time here) Reduce rpm of incoulum culture

11/18/2008 (16:50-17:20) Fermentation 0:00 Inoculate cells (O.D: < 6.0 Volume: 500 ml) Cells were transferred to bottles/tube and inoculated using pumps.


Day 1 (Nov 19th Wed):

      <Sung-Hye>           11/19/2008 (9:00)      Fermentation 15:00 
                                                   F1 Acid (200 ml) Base (225 ml)
                                                   F2 Acid (180 ml) Base (300 ml) 
                           11/19/2008 (9:00)      Fermentation 15:00 
                                                   Connect MeOH 
                              7. 2 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
11/19/2008 (9:00-9:30) Fermentation 15:00 MeOH Induction 0:00 MeOH sensor calibration
      <Sung-Hye and Kevin> 11/19/2008 (9:35)      Fermentation 15:35  MeOH Induction 0:35
                             Y98C (F1)=(O.D:15, AU:, Activity: ) Acid (200 ml) Base (210 ml) MeOH (405 ml)
                               D5 (F2)=(O.D:14.5, AU:, Activity: ) Acid (150 ml) Base (290 ml) MeOH (440 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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