User:Fermenter User/Notebook/BROM A15

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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 A15



Day -2 (July 11 Sun):

      <Dennis>           7/11/2010 (before go home)
                               Thaw glycerol stock and grow them in fresh 5-ml MD media
                               Incubator in Fermentation Suite used
      <Dennis>           7/11/2010 (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 (July 12 Mon): MEDIA PREPARATION DAY

      <Sung-Hye>         7/12/2010 (14:00)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 3.5-L of BMXY:  
                              3. 1-ml Antifoam 
<Sung-Hye> 7/12/2010 (14:00) Prepare bottles 1) Acid, 1-L (tubing separate) 2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected) 4) Additives/Inoculum, 1 x 500-ml (tubing connected) 5) Water, 2-L

<Sung-Hye> 7/12/2010 (15:00) Autoclave 7/12/2010 (17:00) Autoclave completed!


      <Dennis>           7/12/2010 (before go home)
                               Spind down cells and resuspend in fresh 50-ml BMGY media
                               Incubator in Fermentation Suite used

Day 0 (July 13 Tue): INOCULATION DAY

      <Dennis>           7/13/2010 (10:00)
                             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> 7/13/2010 (10:00-11:22) 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

7/13/2009 (10:05) Start Fermenter #2 software <Sung-Hye> 7/13/2009 (11:40) Cooling bioreactors Purge Air 1 L/min at 500 rpm
<Sung-Hye> 7/13/2009 (11:22) Calibrate dO2 probes 7/13/2009 (11:25) Strt dO2 control <Sung-Hye&Dennis> 7/13/2009 (12:00) Fermentation 0:00 Flask OD: Cat K (F1) - 34 <-Dennis Inoculate 50 ml <Sung-Hye> 7/13/2009 (12:00) Start pH control Acid (500 ml) Base (400 ml)

Day 1 (July 14 Wed):

      <Dennis>           7/14/2010 (12:00)      Fermentation 24:00  
                            F2 Acid(285 ml) Base(375 ml) OD(22.9) Contamination(N) Pressure(N) Change Filter(N)

Day 2 (July 15 Thu): MeOH Induction Day 1

      <Dennis>           7/15/2010 (12:00)      Fermentation 48:00  
                            F2 Acid(150 ml) Base(375 ml) MeOH(--ml) OD(20.6) Contamination(N) Pressure(Y) Change Filter(Y)
      <Sung-Hye>         7/15/2010 (12:58)      MeOH sensor calibration 
                         7/15/2010 (12:58)      Fermentation 48:58  MeOH induction 00:00 
                            Connect MeOH
                              7. 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 

Day 3 (July 16 Fri): MeOH Induction Day 2

      <Dennis>           7/16/2010 (11:00)      Fermentation 72:00  MeOH induction 24:00  
                            F1: Acid(95 ml) Base(375 ml) MeOH(480 ml) OD(20.55) Contamination(N) Pressure(N) Change Filter(N) 
                            Activity assay (0.311 units/100ul), (0.073 mg/5-L)

Day 4 (July 17 Sat): MeOH Induction Day 3

      <Dennis>           7/17/2010 (12:00)      Fermentation 96:00  MeOH induction 48:00  
                            F1: Acid(50 ml; then add acid to 400 mL) Base(375 ml) MeOH(450 ml) OD(22) Contamination(N) Pressure(N) Change Filter(Y/N) 
                            Activity assay (1.256 units/100ul), (0.294 mg/5-L)

Day 5 (July 18 Sun): MeOH Induction Day 4

      <Dennis>           7/18/2010 (12:30)      Fermentation 112:00  MeOH induction 72:00  
                            F1: Acid(400 ml) Base(375 ml) MeOH(425 ml) OD(19.75) Contamination(N) Pressure(N) Change Filter(N) 
                            Activity assay (1.76 units/100ul), (0.412 mg/5-L)

Day 6 (July 19 Mon): MeOH Induction Day 5

      <Dennis>           7/19/2010 (12:00)      Fermentation 136:00  MeOH induction 96:00  
                            F1: Acid(375 ml) Base(375 ml) MeOH(400 ml) OD(19.5) Contamination(N) Pressure(N) Change Filter(N) 
                            Activity assay (2.26 units/100ul), (0.529 mg/5-L)

Day 7 (July 20 Tue): MeOH Induction Day 6

      <Dennis>           7/20/2010 (12:00)      Fermentation 160:00  MeOH induction 120:00  
                            F1: Acid(355 ml) Base(370 ml) MeOH(472 ml) OD(21.7) Contamination(N) Pressure(N) Change Filter(N) 
                            Activity assay (5.19 units/100ul), (1.21 mg/5-L)

Day 8 (July 21 Wed): MeOH Induction Day 7

      <Dennis>           7/21/2010 (12:00)      Fermentation 184:00  MeOH induction 144:00  
                            F1: Acid(350 ml) Base(360 ml) MeOH(337 ml) OD(20.95) Contamination(N) Pressure(N) Change Filter(N) 
                            Activity assay (3.98 units/100ul), (0.93 mg/5-L)

Day 9 (July 22 Thu): MeOH Induction Day 8

      <Dennis>           7/22/2010 (12:00)      Fermentation 208:00  MeOH induction 168:00  
                            F1: Acid(??ml) Base(??ml) MeOH(?? ml) OD(??) Contamination(Y/N) Pressure(Y/N) Change Filter(Y/N) 
                            Activity assay (?? units/100ul), (?? 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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