User:Fermenter User/Notebook/MOLD C1

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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/MOLD C1</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
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)
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)

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)
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)

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)

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)

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)
SDS-PAGE, CBB 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)
SDS-PAGE, CBB 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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