User:Fermenter User/Notebook/MOLD C1

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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


MOLD C1


Day -5 (Jun 18 Fri):

      <Raj>                  6/18/2010 (pm)
                                        Pick a colony from MD plate and grew them in 5-ml MD
SDS-PAGE of small-scale C4 & C6 insulin supernatants (left) and western blot stained with anti-human insulin antibody (right)
SDS-PAGE of small-scale C4 & C6 insulin supernatants (left) and western blot stained with anti-human insulin antibody (right)
Minmal dextrose plate of picZαA-C4(+L) insulin transformed pichia KM71H (MutS)
Minmal dextrose plate of picZαA-C4(+L) insulin transformed pichia KM71H (MutS)

Day -2 (Jun 21 Mon):

      <Meeding>              6/21/2010 (11:15 - 11:45)
      <Raj>                  6/21/2010 (before go home)
                                        Take 50 ul of culture and grow them in fresh 5-ml MD media
      <Raymond>              6/21/2010 (before go home)
                                        Thaw glycerol stock and grow them in fresh 5-ml MD media

Day -1 (Jun 22 Tue): MEDIA PREPARATION DAY

      <Raj and Raymond>  6/22/2010 (before 11am)
                              Give BMXY (yeast extract + peptone for 3.5-L) to Sung-Hye 
                              Prepare:
                              YNB, Phosphate buffer, Glycerol, Biotin, Acid (5N Acetic Acid)
<Sung-Hye> 6/22/2010 (11:00) Assemble bioreactors: 1. Calibrate pH sensors 2. 2 x 3.5-L of BMXY: 3. 2 x 1-ml Antifoam
<Sung-Hye> 6/22/2010 (12:00) Prepare 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) Additives/Inoculum, 2 x 1-L (tubing connected) 5) Water, 2-L

<Sung-Hye> 6/22/2010 (12:53) Autoclave 6/22/2010 (14:20) Autoclave completed!
      <RAj & Raymond>    6/22/2010 (before go home)
                               Spind down cells and resuspend in fresh 50-ml BMGY media
                               Incubator in Fermentation Suite used


Day 0 (June 23 Wed): INOCULATION DAY

      <Raj and Raymond>  6/23/2010 (before 10am)
                             Bring additives to fermenter room:
                                                 1) Potassium Phosphate
                                                 2) YNB (Bromme lab added biotin here and kept at 4C)
                                                 3) Glycerol
                                                 4) Biotin  <-- from 4C 
Transfer Adjutives using pumps. 6/23/2009 (10:15-11:30) 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 6. 2 x 500 ml of 10x Glycerol

6/23/2009 (10:34) Start Fermenter #1 software 6/23/2009 (10:35) Start Fermenter #2 software
<Sung-Hye> 6/23/2009 (11:35) Cooling bioreactors Purge Air 1 L/min at 100 rpm
6/23/2009 (11:35) Set rpm at 900 6/23/2009 (13:30) Calibrate dO2 probes 6/23/2009 (13:50) Strt dO2 control <Everyone> 6/23/2009 (14:00) Fermentation 0:00 Flask OD: Cat K (F1) - 15.2 <-Raymond & Dannis Flask OD: (F2) - 9.2 <--Raj Inoculate 2 fermentors (No contamination) <Sung-Hye> 6/23/2009 (14:05) Start pH control Acid (7.5M 300 ml) Base (2M 200 ml) <Raj & Raymond> 6/23/2010 (pm) make glycerol stock --------------------------------------------------------------------------------------------------------- F2 used wrong acid (swiched with MeOH!) so it will be terminated! New fermentation for F2 will be prepared

Day 1 (June 24 Thu):

      <Raj>              6/24/2010 (14:00)      Fermentation 24:00  
                            F2 Acid (200 ml) Base (150 ml) OD (16.98) Contamination (N) Pressure (N) Change Filter (N)
Pichia, 24 hr, Before Induction (x100 Objective Lens)
Pichia, 24 hr, Before Induction (x100 Objective Lens)

Day 2 (June 25 Fri): MeOH Induction Day 1

      <Raj>              6/25/2010 (14:00)      Fermentation 48:00  
                            F2 Acid (20ml topped up with 466ml)Base(150 ml)OD(17.80)Contamination(N)Pressure(N)Change Filter(N)
<Raj> 6/25/2010 (14:00) Fermentation 48:00 Prepared 1L 100% Methanol(non-HPLC grade) filtered.
<Sung-Hye> 6/25/2010 (14:00) MeOH sensor calibration 6/25/2010 (14:00) Fermentation 48:00 MeOH induction 00:00 Connect MeOH 7. 1000 ml of MeOH: Tasfer MeOH into MeOH feeding bottles.
Pichia, 48hr, Before Induction (x100 Objective Lens)
Pichia, 48hr, Before Induction (x100 Objective Lens)
Calibration curve of MeOH sensor
Calibration curve of MeOH sensor

Day 3 (June 26 Sat): MeOH Induction Day 2

      <Sung-Hye>         6/26/2010 (10:00)      Fermentation 68:00  MeOH induction 20:00  
                            Checked fermenter. Everything look ok. 
                            Seems like MeOH (0.7%) is still decreasing slowly to 0.5%.  MeOH bottle shows no change yet.
                            6559 mV 
      <Raj>              6/26/2010 (14:00)      Fermentation 72:00  MeOH induction 24:00  
                            F2: Acid (220 ml) Base (100 ml) MeOH (960 ml) OD (19.2) Contamination (N) Pressure (N) Change Filter (N)
Pichia, 72, MeOH Induction 24 hr (x40 times objective lens)
Pichia, 72, MeOH Induction 24 hr (x40 times objective lens)

Day 4 (June 27 Sun): MeOH Induction Day 3

      <Raj&Sung-Hye>     6/27/2010 (14:00)      Fermentation 96:00  MeOH induction 48:00  
                            F2: Acid (130 ml) Base (100 ml) MeOH (950 ml) OD (18.0) Contamination (N) Pressure (N) Change Filter (N)
Pichia, 96hr, MeOH Induction 48 hr (x100 times objective lens)
Pichia, 96hr, MeOH Induction 48 hr (x100 times objective lens)

Day 5 (June 28 Mon): MeOH Induction Day 4

      <Raj>              6/28/2010 (14:00)      Fermentation 112:00  MeOH induction 72:00  
                            F2: Acid (50 ml) Base (100 ml) MeOH (930 ml) OD (19.8) Contamination (N) Pressure (Y) Change Filter (Y)
      <sung-Hye>         6/28/2010 (16:00)     
                            Added 150 ml of Acid, Total=150 ml
Pichia, 112hr, MeOH Induction 72 hr (x100 times objective lens)
Pichia, 112hr, MeOH Induction 72 hr (x100 times objective lens)

Day 6 (June 29 Tue): MeOH Induction Day 5

      <Raj>              6/29/2010 (14:00)      Fermentation 136:00  MeOH induction 96:00  
                            F2: Acid (120 ml) Base (120 ml) MeOH (900 ml) OD (20.5) Contamination (N) Pressure (N) Change Filter (N)
Pichia, 136hr, MeOH Induction 96 hr (x100 times objective lens)
Pichia, 136hr, MeOH Induction 96 hr (x100 times objective lens)
SDS-PAGE, CBB Staining
SDS-PAGE, CBB Staining
SDS-PAGE, Silver Staining
SDS-PAGE, Silver Staining

Day 7 (June 30 Wed): MeOH Induction Day 6

      <Raj>              6/30/2010 (14:00)      Fermentation 160:00  MeOH induction 120:00  
                            F2: Acid (50 ml) Base (110 ml) MeOH (880 ml) OD (20.5) Contamination (N) Pressure (N) Change Filter (N)
Pichia, 160hr, MeOH Induction 120 hr (x100 times objective lens)
Pichia, 160hr, MeOH Induction 120 hr (x100 times objective lens)
SDS-PAGE, CBB Staining
SDS-PAGE, CBB Staining
SDS-PAGE, Silver Staining
SDS-PAGE, Silver Staining


                            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
   
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