User:Fermenter User/Notebook/BROM A14T

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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


BROM A14T



Day -2 (Jun 23 Wed):

      <Raymond>          6/23/2010 (before go home)
                               Thaw glycerol stock and grow them in fresh 5-ml MD media
                               Incubator in Fermentation Suite used
      <Raymond>          6/23/2010 (before go home)
                              Give BMXY (yeast extract + peptone for 3.5-L) to Sung-Hye 
                              Prepare:
                              YNB, Phosphate buffer, Glycerol, Biotin, Acid (5M Acetic Acid)

Day -1 (Jun 24 Thu): MEDIA PREPARATION DAY

      <Sung-Hye>         6/24/2010 (9:00)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 3.5-L of BMXY:  
                              3. 1-ml Antifoam 
<Sung-Hye> 6/24/2010 (9:00) Prepare bottles 1) Acid, 1-L (tubing separate) 2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected) 4) Additives/Inoculum, 2 x 1-L (tubing connected) 5) Water, 2-L

<Sung-Hye> 6/24/2010 (9:35) Autoclave 6/24/2010 (11:00) Autoclave completed! <Raymond> 6/24/2010 (15:20) 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. <Sung-Hye> 6/24/2010 (16:00-17:15) Combine: 3. 500 ml of 10x Potassium Phosphate 4. 500 ml of 10x YNB 5. 10 ml of 500x Biotin 6. 500 ml of 10x Glycerol

6/24/2009 (17:00) Start Fermenter #1 software


      <Raymond>          6/24/2010 (before go home)
                               Spind down cells and resuspend in fresh 50-ml BMGY media
                               Incubator in Fermentation Suite used

Day 0 (June 25 Fri): INOCULATION DAY

      <Sung-Hye>         6/25/2009 (8:40)      Cooling bioreactors 
                                                Purge Air 1 L/min at 500 rpm
<Sung-Hye> 6/25/2009 (9:00) Calibrate dO2 probes 6/25/2009 (9:00) Strt dO2 control <Sung-Hye&Raymond> 6/25/2009 (12:00) Fermentation 0:00 Flask OD: Cat K (F1) - 10.9 <-Raymond & Dannis <Sung-Hye> 6/25/2009 (12:00) Start pH control Acid (300 ml) Base (180 ml) <Dannis> 6/27/2010 (12:00) Fermentation 48:00 Prepared 0.5L 100% Methanol(non-HPLC grade) filtered.


Day 1 (June 26 Sat):

      <Dannis>           6/26/2010 (12:00)      Fermentation 24:00  
                            F1 Acid (40 ml) Base (135 ml) OD (20.5) Contamination (N) Pressure (N) Change Filter (N)

Day 2 (June 27 Sun): MeOH Induction Day 1

      <Dannis>           6/27/2010 (12:00)      Fermentation 48:00  
                            F1 Acid (35 ml, then add 500 mL to make 535 mL) Base (135 ml) OD (22.3)
                            Contamination (N? but I saw one bacteria swimming, thus I assume it's from the syringe.) Pressure (N)$ Change Filter (N)
      <Sung-Hye>         6/27/2010 (12:50)      Fermentation 48:50    
                            Changed syringe
      <Sung-Hye>         6/27/2010 (12:58)      MeOH sensor calibration 
                         6/27/2010 (12:58)      Fermentation 48:58  MeOH induction 00:00 
                            Connect MeOH
                              7. 325 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 

Day 3 (June 28 Mon): MeOH Induction Day 2

      <Dennis>          6/28/2010 (10:30)      Fermentation 72:00  MeOH induction 24:00  
                            F1: Acid (500 ml) Base (135 ml) MeOH (315 ml) OD (28.1) Contamination (N) Pressure (N) Change Filter (N) 
                            Activity assay (161500 units / 5L), (3.23 units/100 ul),(0.756 mg/5-L)

Day 4 (June 29 Tue): MeOH Induction Day 3

      <Dennis>          6/29/2010 (12:00)      Fermentation 96:00  MeOH induction 48:00  
                            F1: Acid (440 ml) Base (135 ml) MeOH (290 ml) OD (22.1) Contamination (N) Pressure (N) Change Filter (N)
                            Activity: (4.77 units/100 ul),(1.12 mg/5-L).  
                            I tried doing a sample where a performed syringe sterilization with the supernatant.  
                            It gave an activity of 2.98, thus I do not believe the syringe makes a difference.

Day 5 (June 30 Wed): MeOH Induction Day 4

      <Dennis>          6/30/2010 (12:00)      Fermentation 112:00  MeOH induction 72:00  
                            F1: Acid (420 ml) Base (135 ml) MeOH (250 ml) OD (21.45) Contamination (N) Pressure (N) Change Filter (N)
                            Activity: (1.05 units/5 ul),(4.914 mg/5-L)
                            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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