User:Fermenter User/Notebook/MOLD C2

From OpenWetWare
Jump to: navigation, search

<!-- 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 C2</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 Help.png

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 C2


Day -3 (Jul 20 Tue):

      <Raj>              7/21/2010 (pm)
                        Pick a colonies of SAP A6 & SAP B6+L from MD plates and 
                        grew them overnight in 15 ml MD medium in 50 ml falcon tubes
SDS-PAGE of SAP A6 & B6+L small-scale supernatants (left) and western blot stained with anti-human SAP antibody (right)
Minimal dextrose plates of PicZαA-SAP(A6) and PicZαA-SAP(B6+L) courtesy of DHL Express !

Day -2 (Jul 21 Wed):

      <Raj>              7/21/2010
                         Let the A6 & B6+L SAP MD cultures continue growing at 30 °C 0/n at 225 rpm

Day -1 (Jul 22 Thu): MEDIA PREPARATION DAY

      <Raj>              7/22/2010 (before 11am)
                              Give 2 x 5-L Basal Medium to Sung-Hye 
<Sung-Hye> 7/22/2010 (11:00) Assemble bioreactors: 1. Calibrate pH sensors 2. 2 x 5.0-L of Basal Medium: 3. 2 x 1-ml Antifoam
<Sung-Hye> 7/22/2010 (12:00) Prepare bottles 1) Base, 2 x 500-ml (tubing connected) 2) MeOH, 2 x 1-L (tubing connected) 3) Inoculum, 2 x 1-L (tubing connected) 4) Water, 2-L

<Sung-Hye> 7/22/2010 (12:00) Autoclave 7/22/2010 (14:20) Autoclave completed!
      <Raj>              7/22/2010 (before go home)
                               Innoculate 2 x 25 ml fresh MD  with 10 μL of SAP A6 o/n
                               Innoculate 2 x 25 ml fresh MD  with 10 μL of SAP B6+L o/n 
                               Incubator in Fermentation Suite used
PTM1 Trace Salts
PTM1 Trace Salts sterile filter

Day 0 (Jul 23 Fri): INOCULATION DAY

      <Sung-Hye>         7/23/2009 (10:10)      Start Fermenter #1 software 
                         7/23/2009 (10:20)      Start Fermenter #2 software 
<Sung-Hye> 7/23/2009 (11:35) Cooling bioreactors Purge Air 1 L/min at 100 rpm
7/23/2009 (13:30) Calibrate dO2 probes 7/23/2009 (13:50) Strt dO2 control <Raj & SungHye> 7/23/2009 (11:00) Fermentation 0:00 hr Flask OD600: SAP A6 = 0.5, SAP B6+L = 0.8 (diluted to 0.5) Inoculate fermentor #1 with 50 ml SAP A6 (No contamination) Inoculate fermentor #2 with 50 ml SAP B6+L (No contamination) <Sung-Hye> 7/23/2009 (14:05) Start pH control Original pH of media after autoclave was around 1.5 Add about 150 ml each of conc. NH4OH to bring pH to 5.0 Base (2M 500 ml)
SAP A6 before innoculation
SAP B6+L before innoculation
Fermenter set-up : #2-SAP B6+L (left) & #1-SAP A6 (right)
#2-SAP B6+L (left)

Day 1 (Jul 24 Sat):

      <Raj>              7/24/2010 (11:00)      Fermentation 24:00 hr  
                            #1 A6  : Base (425 ml) OD (2.48) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0049 g/ml
                            #2 B6+L: Base (375 ml) OD (5.27) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0048 g/ml
                           


SAP_A6 24 hr post-innoculation
SAP_B6+L 24 hr post-innoculation

Day 2 (Jul 25 Sun):

      <Raj>              7/25/2010 (11:00)      Fermentation 48:00 hr  
                         #1 A6  : Base (50 [↑ 600] ml) OD (38.00) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0531 g/ml
                         #2 B6+L: Base (50 [↑ 500] ml) OD (38.60) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0478 g/ml
#2 SAP B6+L after 48 hr growth
SAP A6 48 hr post-innoculation
SAP B6+L 48 hr post-innoculation

Day 3 (Jul 26 Mon): Fed-Batch Day 1

      <Raj>              7/26/2010 (9:00)      Fermentation 70:00 hr  
                            #1 A6  : Base (600 ml) OD (37.90) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0650 g/ml
                            #2 B6+L: Base (350 ml) OD (37.20) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0521 g/ml 
      <Sung-Hye>         7/26/2010 (9:30) - 7/27/2010 (5:30)
                            Feed Glycerol + PTM1 trace salts (370 ml)
                            Feeding flow rate: 18 ml/hr
                            Use 2 rpm Watson pump and set rpm to 13 
                            Feeding hour: 20 hr


SAP A6 72 hr post-innoculation
SAP B6+L 72 hr post-innoculation

File:Watsonpump.tif

Day 4 (Jul 27 Tue): MeOH Induction Day 1

      <Raj>              7/27/2010 (09:00)      Fermentation 96:00 hr
                           #1 A6  : Base (330 ml) OD (61.80) Contamination (N) Pressure (N) Change Filter (N) Methanol (500 ml) DCW 0.1359 g/ml
                           #2 B6+L: Base (200 ml) OD (58.55) Contamination (N) Pressure (N) Change Filter (N) Methanol (500 ml) DCW 0.1003 g/ml
      <Raj>              7/27/2010 (11:00)      Fermentation 96:00 hr
                            Prepared 1L 100% Methanol(non-HPLC grade) filtered.
<Sung-Hye> 7/27/2010 (11:00) MeOH sensor calibration Set Point: 6400 mV for both fermenters. 7/27/2010 (11:00) Fermentation 96:00 hr MeOH induction 00:00 Connect MeOH + PTM1 trace 7. 500 ml of MeOH per fermenter. 8. 6-ml of PTM1 trace salt per fermenter
SAP A6 96 hr post-innoculation (0 hr methanol induction)
SAP B6+L 96 hr post-innoculation (0 hr methanol induction)
Marmie collecting an A6 SAP sample !!
Question <Sung-Hye>: Who's Marmie?
Answer <Raj>: Professor of marmalade studies at London University

Day 5 (Jul 28 Wed): MeOH Induction Day 2

       <Sung-Hye>         7/28/2010 (10:00)
                            Harvest 1-L from #1
       <Raj>              7/28/2010 (11:00)      Fermentation 120:00  MeOH induction 24:00  
                           #1 A6  : Base (200 ml ↑ 500 ml) OD (60.55) Contamination (N) Pressure (Y) Change Filter (Y) Methanol (325 ml) DCW 0.1247 g/ml
                           #2 B6+L: Base ( 90 ml ↑ 500 ml) OD (55.95) Contamination (N) Pressure (N) Change Filter (N) Methanol (400 ml) DCW 0.1033 g/ml
                          7/28/2010 (15:00)
                           #1 A6  : Methanol (275 ml ↑ 750 ml )
                           #2 B6+L: Methanol (400 ml ↑ 750 ml )
       <Sung-Hye>         7/28/2010 (17:00)
                            Harvest 0.5-L from #2


SAP A6 120 hr post-innoculation (24 hr post-induction)
SAP B6+L 120 hr post-innoculation (24 hr post-induction)
SAP A6 120 hr post-innoculation (24 hr post-induction) 175 ml methanol consumed, 1 litre culture removed
SAP B6+L post-innoculation (24 hr post-induction) 100 ml methanol consumed
Fermentation Parameters : SAP A6 120 hr post-innoculation (24 hr post-induction)
Fermentation Parameters : SAP B6+L 120 hr post-innoculation (24 hr post-induction)

Day 6 (Jul 29 Thu): MeOH Induction Day 3

       <Sung-Hye>         7/29/2010 (09:00)
                            Harvest 0.5-L from #2
       <Raj>              7/29/2010 (11:00)      Fermentation 144:00  MeOH induction 48:00  
                           #1 A6  : Base (400 ml) OD (72.50) Contamination (N) Pressure (N) Change Filter (N) Methanol (500 ml) DCW 0.1495 g/ml
                           #2 B6+L: Base (450 ml) OD (59.80) Contamination (N) Pressure (N) Change Filter (N) Methanol (650 ml) DCW 0.0976 g/ml
       <Marmie>           7/29/2010 (12:00)
                            Harvest 0.5-L from #1
       <Raj>              7/29/2010 (16:30)
                           #1 A6  : Methanol (425 ml ↑ 1000 ml)
                           #2 B6+L: Methanol (650 ml ↑ 1000 ml)
                   
SAP A6 144 hr post-innoculation (48 hr post-induction)
SAP B6+L 144 hr post-innoculation (48 hr post-induction)
SAP B6+L 143 hr post-innoculation (23 hr post-induction) 9 am 0.5 L culture removed
SAP A6 145 hr post-innoculation (25 hr post-induction) 11 am 0.5 L culture removed

Day 7 (Jul 30 Fri): Harvest

      <Raj&Sung-Hye>     7/30/2010 (11:00)      Fermentation 168:00  MeOH induction 72:00  
                            
       <Raj>          #1 A6  :   Base (400 ml) OD (84.65) Contamination (N) Pressure (N) Change Filter (N) Methanol (900 ml) DCW 0.2039 g/ml
                      #2 B6+L:   Base (450 ml) OD (77.60) Contamination (N) Pressure (N) Change Filter (N) Methanol (750 ml) DCW 0.1231 g/ml
                            
                          
                      Harvest
SAP A6 167 hr post-innoculation (71 hr post-induction), 1 litre culture removed
Fermentation Parameters : SAP A6 168 hr post-innoculation (72 hr post-induction)
Fermentation Parameters : SAP B6+L 168 hr post-innoculation (72 hr post-induction)
SAP A6 168 hr post-innoculation (72 hr post-induction)
SAP B6+L 168 hr post-innoculation (72 hr post-induction)
Harvesting of SAP A6 culture ...
... into 1 L centrifuge pots
Post-centrifuge SAP A6 culture (1 hr @ 6 krpm)
Pooled clarified SAP A6 supernatant

Day 8 (Aug 1 Sat): Precipitation

Cell pastes frozen (A6: 1.184 kg, B6+L: 0.772 kg)
0.2 μm filtration of A6 supernatant to remove residual yeast cells
Clarified SAP A6 filtered supernatant made to 90% AmmSO4 saturation (610 g/L @ 4 °C)
Overnight AmmSO4 precipitations of SAP A6 & B6+L filtered supernatants @ 4 °C
Transfer SAP A6 AmmSO4 precipitate to 1 L centrifuge pots
Transfer SAP A6 AmmSO4 precipitate to 1 L centrifuge pots
SAP A6 AmmSO4 precipitate
SAP B6+L AmmSO4 precipitate
A6 & B6+L SAP pooled AmmSO4 pellets
SAP A6 near UV scan
SAP B6+L near UV scan
OD600 and Dry Cell Weight against fermentation day


SDS PAGE of concentrated supernatants (coomassie blue)
10 μg secreted protein (colloidal blue)

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
   
  1. How to make wiki table and insert pictures (meta.wikimedia.org)



Recently Edited Notebook Pages