Difference between revisions of "User:Fermenter User/Notebook/BROM J1"

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(Day 6 (Jun 27 Mon): MeOH Induction Day 5)
m (Day 4 (Jul 16 Sat): MeOH Induction Day 3)
Line 116: Line 116:
 
== Day 4 (Jul 16 Sat): <font color=#FF00FF> MeOH Induction Day 3 </font>==  
 
== Day 4 (Jul 16 Sat): <font color=#FF00FF> MeOH Induction Day 3 </font>==  
 
       <Vidhu>          6/25/2011 (13:30)    <font color=orange> Fermentation 97:30 </font><font color=#FF00FF> MeOH induction 48:00 </font>  
 
       <Vidhu>          6/25/2011 (13:30)    <font color=orange> Fermentation 97:30 </font><font color=#FF00FF> MeOH induction 48:00 </font>  
refilled base to 500ml.                             
+
                                              refilled base to 500ml.                             
F2: Base(500 ml) MeOH(700 ml) OD(61.85) Contamination(N) Pressure(N) Change Filter(N)
+
                                              F2: Base(500 ml) MeOH(700 ml) OD(61.85) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (No activity?????)/100ul),
+
                                              Activity assay (No activity?????)/100ul),
  
checked later the incubator temperature was set to 28deg...may be that's the reason for still inactivated form of protein since pepsin would not have activated it...but no sample left to retry...next day try the activity assay again.
+
                        checked later the incubator temperature was set to 28deg...
 +
                        may be that's the reason for still inactivated form of protein since pepsin would not have activated it...
 +
                        but no sample left to retry...next day try the activity assay again.
  
 
== Day 5 (Jul 17th, sat: <font color=#FF00FF> MeOH Induction Day 4 </font> ==   
 
== Day 5 (Jul 17th, sat: <font color=#FF00FF> MeOH Induction Day 4 </font> ==   

Revision as of 12:14, 21 July 2011

<!-- 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/BROM J1</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>

Customize your entry pages <html><img src="/images/a/aa/Help.png" border="0" /></html>

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 J1

Day -4 (Jun 8 Fri):

Day -3 (Jul 9 Sat):

Day -2 (Jul 10 Sun):

      <Vidhu>           7/10/2011 (13:00)
                              Thaw glycerol stock and grow in 5-ml MD media and 
                              inoculated from MD plate as well and o/n incubation at 30deg , 200rpm

Day -1 (Jul 11, Mon): MEDIA PREPARATION DAY

      <Vidhu>            7/11/2010 (14:30)
                               Bring powder to make 5-L basal salts
      <Sung-Hye>         7/11/2011 (15:00)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 5.0-L of Basal Salts: Phosphoric Acid was 10-ml short.
                              3. 1-ml Antifoam 
<Sung-Hye> 7/11/2011 (15:00) Prepare bottles 1) no Acid need 2) Base, 500-ml (tubing connected) x1 3) MeOH, 1-L (tubing connected) x1 <-- No need 4) Additives/Inoculum, 500-ml (tubing connected) x1 5) No Water, 2-L 6) Fed-Batch, 500-ml (tubing connected) x1

<Sung-Hye> 7/11/2011 (15:16) Autoclave
      <Vidhu>            7/11/2011 (15:00)
                               1.Transfer cells (5ml MD media) to 50ml BMGY media.
                                 Incubator in Fermentation Suite used
                               2.Prepare sterilized Methanol by filtration
                               3.Prepare sterilized NaOH (4M, 500-ml)

Day 0 (Jul 12 Tue): INOCULATION DAY

      <Vidhu>            7/12/2011 (10:30)  Bring the following items to Fermentation room.
                                            :NaOH (4M, 500 ml), MeOH (1000 ml), and 50% Glycerol (500ml)
                         7/12/2011 (11:00)  Check contamination of starter culture.
                                            Flask OD: 32.10
                         7/12/2011 (11:50)  Bring inoculum to Fermentation room.
      <Vidhu&Sung-Hye>   7/12/2011 (11:30)  Set temperature to 28.5°C 
      <Vidhu&Sung-Hye>   7/12/2011 (11:30)  Purge Air 1L/min at 900 rpm 
      <Vidhu&Sung-Hye>   7/12/2011 (11:30)  Adjust pH to ~4.75 (with ~90 ml NH4OH)
      <Vidhu&Sung-Hye>   7/12/2011 (11:40)  Adjust pH to 5.0 with **4M NaOH**
                                            (borrowed from Bromme lab, 500 ml)
                                            50 ml used
      <Vidhu&Sung-Hye>   7/12/2011 (11:45)  Start pH control 
                                            F1: No Acid Base, 4M (450 ml)
      <Vidhu&Sung-Hye>   7/12/2011 (12:00)  Calibrate dO2 probes 
                         7/12/2011 (12:00)  Strt dO2 control 
      <Vidhu&Sung-Hye>   7/12/2011 (12:04)  Start Fermenter #1 software 
      <Vidhu&Sung-Hye>   7/12/2011 (12:15)  Fermentation 0:00 
                                            Inoculate 55 ml/bioreactor
                                            Cells were transferred using pumps.
      <Sung-Hye>         7/12/2011 (12:20)  Add 21.75 ml of PTM1 Salt/5-L media <-- Prepared at BROM A16
                                            (4.35 ml PTM1 salt per 1-L media = 21.75 ml in 5-L))

Day 1 (Jul 13 Wed):

      <vidhu>            7/13/2011 (12:15)      Fermentation 24:00  
                            F1:   Base(350 ml) OD(20) Contamination(N) Pressure(N) Change Filter(N)

cells look good...

Day 2 (Jul 14 Thu): Fed-Batch and MeOH Induction Day 1

      <Vidhu>            7/14/2011 (9:00)      Fermentation 45:00 
                            F2: Base(300ml) OD(31.75) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         7/14/2011 (9:00 - 12:00)  Fermentation 45:00 - 48:00 
                            F2: Fed-Batch
                            400 ml 50% Glycerol + 4.8 ml PTM1 at 90.75ml/hr/5-L (RPM 95 w/ 2RPM pump)
      <Sung-Hye>         7/14/2011 (12:10)
                            F2: Take out 500-ml media
      <Vidhu>            7/14/2011 (12:40)      Fermentation 48:00  
                            F1: Base(280 ml) OD(35.5) Contamination(N) Pressure(N) Change Filter(N)
      <Sung-Hye>         7/14/2011 (12:40)      Fermentation 48:00  MeOH induction 00:00 
                            Calibrate MeOH sensor  
                            F2: 6970 mV = 0.5% (MeOH 1000 ml)<-- Mix with 12 ml PTM1 
                            Start MeOH induction

Day 3 (Jul 15, Fri): MeOH Induction Day 2

      <Vidhu>           7/15/2011 (12:58)      Fermentation 73:30  MeOH induction 24:00  
                            F2: Base(160 ml) MeOH( 880 ml) OD(55.80) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (5.5 units/100 ul), (1.28mg/5-L)
      <Sung-Hye>        7/15/2011 (14:15)
                            F2: Take out 500-ml media
      <Vidhu>           7/15/2011 Prepared more base 400ml 4M NaOH

Day 4 (Jul 16 Sat): MeOH Induction Day 3

      <Vidhu>           6/25/2011 (13:30)      Fermentation 97:30  MeOH induction 48:00  
                                              refilled base to 500ml.                             
                                              F2: Base(500 ml) MeOH(700 ml) OD(61.85) Contamination(N) Pressure(N) Change Filter(N)
                                              Activity assay (No activity?????)/100ul),
                        checked later the incubator temperature was set to 28deg...
                        may be that's the reason for still inactivated form of protein since pepsin would not have activated it...
                        but no sample left to retry...next day try the activity assay again.

Day 5 (Jul 17th, sat: MeOH Induction Day 4

      <Vidhu>            6/26/2011 (13:30)      Fermentation 121:30  MeOH induction 72:00 
                            F2: Base(500 ml) MeOH(600 ml) OD(64.20) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (12.08 units/100ul), (2.8 mg/5-L)

Day 6 (Jul 18 Mon): MeOH Induction Day 5

      <Vidhu>          7/18/2011 (12:00)      Fermentation 144:00  MeOH induction 94:30 

                            F1: Base(500 ml) MeOH(520 ml) OD(58.9) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (14 units/100ul), (3.26mg/5-L)
      <Sung-Hye>         HARVEST

Suggestion for next trials:

1.  Add PTM1 salt into MeOH (12 ml/L of MeOH)
2.  Even add Glycerol together with MeOH
3.  Extend fed-batch hours (from 3 hrs to overnight) before start MeOH induction
4.  Measure samples twice a day

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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