User:Fermenter User/Notebook/Pichia Workshop 20100809

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WORK A1

Teaching:


Attending:


Day -2 (Aug 9 Mon):

      <Dennis>           8/9/2010 (during workshop)
                               Thaw glycerol stock and grow 5-ul in fresh 5-ml MD media
                               Incubator in Fermentation Suite used
      <Dennis>           8/9/2010 (before go home)
                              Give BMXY (yeast extract + peptone for 3.5-L x2) to Sung-Hye 
                              Prepare:
                              YNB, Phosphate buffer, Glycerol, Biotin, Acid (5M Acetic Acid)
<Sung-Hye> 8/9/2010 (before go home) Preparing 5-L of Basal Medium (Defined media)
Growing starter culture in Incubator/Shaker at 30°C
Growing starter culture in Incubator/Shaker at 30°C
Complex Media for 5-L x 2
Complex Media for 5-L x 2
Defined Media for 5-L
Defined Media for 5-L

Day -1 (Aug 10 Tue): MEDIA PREPARATION DAY

      <Workshop>         8/10/2010 (10:00)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 3.5-L of BMXY (Fermenter #1,#2) 
                                 5.0-L of Basal Salts (Fermenter #3):  
                              3. 1-ml Antifoam 
<Workshop> 8/10/2010 (11:30) Prepare bottles 1) Acid, 1-L (tubing separate) x3 2) Base, 500-ml (tubing connected) x3 3) MeOH, 1-L (tubing connected) x3 4) Additives/Inoculum, 1 x 500-ml (tubing connected) x3 5) Water, 2-L

<Workshop> 8/10/2010 (12:50) Autoclave <Sung-Hye> 8/10/2010 (14:30) Autoclave completed! <Sung-Hye> 8/10/2010 (14:35) Adjust pH to 5.0 with 140 ml NH4OH <Sung-Hye> 8/10/2010 (14:40 -16:00) Add Adjutives (Fermenter #1 and #2) 4. 500 ml of 10x Potassium Phosphate 5. 500 ml of 10x YNB 6. 10 ml of 500x Biotin 7. 500 ml of 10x Glycerol

      <Raymond>          8/10/2010 (11:50 - 12:00)
                               Spind down cells and resuspend in fresh 150-ml BMGY media
                               Incubator in Fermentation Suite used
Starter Culture in 150 ml of BMGY
Starter Culture in 150 ml of BMGY
Bioreactors and bottles in Autoclave
Bioreactors and bottles in Autoclave
pH Adjustment with NH4OH, Fermenter #3
pH Adjustment with NH4OH, Fermenter #3
Media Feeding, Fermenter #2
Media Feeding, Fermenter #2
Media Feeding, Fermenter #1
Media Feeding, Fermenter #1


Day 0 (Aug 11 Wed): INOCULATION DAY

      <Workshop>         8/11/2010 (10:18)      Start Fermenter #1 software 
                         8/11/2010 (10:20)      Start Fermenter #2 software 
                         8/11/2010 (10:22)      Start Fermenter #3 software 
                         8/11/2010 (10:30)      Set Temperature 
                                                Purge Air 1 L/min at 600 rpm
8/11/2010 (11:00) Calibrate dO2 probes 8/11/2010 (11:00) Strt dO2 control
<Workshop> 8/11/2010 (11:30) Check contamination of starter culture
<Workshop> 8/11/2010 (12:00) Fermentation 0:00 Flask OD: ?? Inoculate 45 ml/bioreactor <Workshop> 8/11/2010 (12:00) Start pH control F1: Acid (500 ml) Base (400 ml) F2: Acid (500 ml) Base (400 ml) F3: No Acid Base (400 ml) <Sung-Hye> 8/11/2010 (16:30) Add 22 ml of PTM1 Salt into Fermenter#3

Does anybody has picture of inoculation today? If so, feel free to upload at Inoculation-20100811.jpg.

Pichia pastoris, starter culture x100 optical
Pichia pastoris, starter culture x100 optical
Contaminated samples, x 100 optical (did not use)
Contaminated samples, x 100 optical (did not use)
Inoculation
Inoculation

Day 1 (Aug 12 Thu):

      <Sung-Hye>         8/12/2010 (10:00)
                            F2: water leakage from heat exchange water out. repaired
                            F1: Temp control was not working (cooling was not sufficient to maintain temp at 28.5°C)
                                Ice was used to bring down temp to SP and removed.
      <Workshop>         8/12/2010 (12:00)      Fermentation 24:00  
                            F1 Acid(310 ml) Base(390 ml) OD(19.8) Contamination(N) Pressure(N) Change Filter(N)
                            F2 Acid(300 ml) Base(380 ml) OD(6.94) Contamination(N) Pressure(N) Change Filter(N)
                            F3 No Acid     Base(400 ml) OD(0.6) Contamination(N) Pressure(N) Change Filter(N)


Day 2 (Aug 13 Fri): MeOH Induction Day 1

      <Sung-Hye>         8/13/2010 (09:00 - 12:00)
                         Glycerol + PTM1 Salt Feed : 90 ml/hr, Final voluem: 180 ml
      <Workshop>         8/13/2010 (11:00)      Fermentation 48:00  
                            F1 Acid(130 ml) Base(380 ml) OD(22.15)Contamination(N) Pressure(N) Change Filter(N)
                            F2 Acid(150 ml) Base(380 ml) OD(22.3) Contamination(N) Pressure(N) Change Filter(N)
                            F3 No Acid      Base(300 ml) OD(38.2) Contamination(N) Pressure(N) Change Filter(N)
      <Workshop>         8/13/2010 (12:00)      MeOH sensor calibration 
                         8/13/2010 (12:00)      Fermentation 48:00  MeOH induction 00:00 
                            Connect MeOH
                              7. 800 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. 
      <Sung-Hye>         8/13/2010 (12:00)
                            Harvest F3 500 ml
      <Sung-Hye>         8/13/2010 (15:12)
                            F1 Acid (110 ml) + Add (500 ml) = (610 ml)
                            F2 Acid (100 ml) + Add (500 ml) = (600 ml)


Sample from Fermenter #1
Sample from Fermenter #1
Calibration curves for MeOH sensors
Calibration curves for MeOH sensors

Day 3 (Aug 14 Sat): MeOH Induction Day 2

      <Sung-Hye>         8/14/2010 (10:00)    
                            F3:  Set MeOH to 9000 mV (Max allow)
      <Dennis>           8/14/2010 (12:00)      Fermentation 72:00  MeOH induction 24:00  
                            F1: Acid(590 ml) Base(390 ml) MeOH(25 ml injection) OD(22.45) 
                                Contamination(N but I did observe one bacterium, possibly from dirty syringe?) Pressure(N) Change Filter(N) 
                                Activity assay (4.02 units/100ul), (0.94 mg/5-L)
                            F2: Acid(560 ml) Base(375 ml) MeOH(790 ml) OD(20.2) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (1.03 units/100ul), (0.24 mg/5-L)
                            F3: NO Acid     Base(275 ml) MeOH(775 ml) OD(42.25) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (6.91 units/20ul so 34.55 units/100ul), (8.08 mg/5-L) <-- WOW!

Day 4 (Aug 15 Sun): MeOH Induction Day 3

      <Dennis>           8/15/2010 (12:00)      Fermentation 96:00  MeOH induction 48:00  
                            F1: Acid(560 ml) Base(390 ml) MeOH(25 ml injection) OD(23.2) Contamination(N) Pressure(N) Change Filter(N) 
                                Activity assay (7.20 units/100ul), (1.68 mg/5-L)
                            F2: Acid(540 ml) Base(375 ml) MeOH(755 ml) OD(21.3) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (1.91 units/100ul), (0.45 mg/5-L)
                            F3: NO Acid     Base(275 ml) MeOH(755 ml) OD(44.3) 
                                Contamination(N there were a few bacteria but not enough for me to claim contamination) Pressure(N) Change Filter(N)
                                Activity assay (2.132/5ul so 42.64 units/100ul), (9.98 mg/5-L)

Day 5 (Aug 16 Mon): MeOH Induction Day 4

      <Workshop>         8/16/2010 (12:00)      Fermentation 112:00  MeOH induction 72:00  
                            F1: Acid(?? ml) Base(?? ml) MeOH(25 ml injection) OD(23.75) Contamination(N) Pressure(Y/N) Change Filter(Y/N) 
                                Activity assay (13.26 units/100ul), (?? mg/5-L)
                            F2: Acid(525 ml) Base(380 ml) MeOH(750 ml) OD(19.5) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (3.66 units/100ul), (0.856 mg/5-L)
                            F3: NO Acid     Base(300 ml) MeOH(750 ml) OD(41.35) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (3.74 units/5ul so 74.8 units/100ul), (17.5 mg/5-L)

(WORKSHOP ENDS HERE)

Day 6 (Aug 17 Tue): MeOH Induction Day 5

      <Dennis>           8/17/2010 (12:00)      Fermentation 136:00  MeOH induction 96:00  
                            F1: Acid(480 ml) Base(390 ml) MeOH(25 ml injection) OD(22.5) Contamination(N) Pressure(N) Change Filter(N) 
                                Activity assay (14.68 units/100ul), (3.44 mg/5-L)
                            F2: Acid(500 ml) Base(375 ml) MeOH(700 ml) OD(25.6) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (5.74 units/100ul), (1.34 mg/5-L)
                            F3: NO Acid     Base(275 ml) MeOH(725 ml) OD(48.7) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (3.72 units/5ul so 74.4 units/100ul), (17.4 mg/5-L)

Day 7 (Aug 18 Wed): MeOH Induction Day 6 : HARVEST

      <Dennis>           8/18/2010 (12:00)      Fermentation 160:00  MeOH induction 120:00  
                            F1: Acid(460 ml) Base(390 ml) MeOH(25 ml injection) OD(25.1) Contamination(N) Pressure(N) Change Filter(N) 
                                Activity assay (15.91 units/100ul), (3.72 mg/5-L)
                            F2: Acid(500 ml) Base(375 ml) MeOH(670 ml) OD(23.85) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (4.87 units/100ul), (1.14 mg/5-L)
                            F3: NO Acid     Base(275 ml) MeOH(705 ml) OD(46.05) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (2.59 units/5ul so 51.8 units/100ul), (12.12 mg/5-L)
                            HARVEST

Discussion 1: Zeocin

  Question: How Zeocin works? *Sung-Hye Grieco 11:34, 11 August 2010 (EDT):

Zeocin (copper chelated glycopeptide antibiotic) belongs to the bleomycin/ phleomycin family of antibiotics. It acts on mammalian, insect cells, yeast, bacteria and plants via intercalating into DNA and cleaving it, thereby causing cell death. The Sh ble gene gives resistance to Zeocin. The gene product binds to Zeocin and therefore inhibits its intercalation into the DNA.

  1. Grieco SH, Lee S, Dunbar WS, MacGillivray RT, and Curtis SB. . pmid:19221805. PubMed HubMed [Paper1]

Discussion 2: Media-related

  Question: Why we use MD media in the begining and then change to BMGY? *Sung-Hye Grieco 12:53, 11 August 2010 (EDT):

Apparently minimal media selects for double auxotrophs ? Maybe rich media loosens the selection criteria and allows one of the picZ insertions on the diploid chromosome to be dumped ? so by selecting on MD you are pickingdouble transformants in which chromosomal integration has taken place on both genomes ? Just a thought ... (Raj)*Sung-Hye Grieco 13:54, 17 August 2010 (EDT):

Discussion 3: Media-related

  Question: Why would Fermenter #3 (the Defined Medium) have more optimal growth than Fermenter #2
            again? Fermenter #2 is predicted to perform better than #1 because it has the feedback.*Shiau-Yun Tang 20:27, 11 August 2010 (EDT):

Becasue Fermentation strategy using define medium (Fermenter #3) includs Fed-Batch with Glycerol. If Glycerol is fed in growth-limiting rate, it increases biomass and also primes methanol-utilization pathway enzymes before induction. Feed rate and feed time will depend on the desired cell density and the end of the glycerol fed batch phase, which may vary from 150 to 450 WCW/L, and should be optimized based on teh production time course. Reference: Pichia Protocols, 2nd edition, p47 *Sung-Hye Grieco 19:12, 13 August 2010 (EDT):


Follow up:

 Recently, Project BROM-A19, produced a record-breaking amount of human Cathepsin K!  
 We procuded roughly 160 mg from 5-L Defined media.  
 The protocol was same but with slight modification of base and it seems that timing of Fed-Batch plaied a quite important role. 
 *Sung-Hye Grieco 17:16, 15 February 2011 (EST):
Fermentation Profile of BROM A19
Fermentation Profile of BROM A19
Production Profile of BROM A19
Production Profile of BROM A19

Announcement

Workshop Time Schedule

References

Invitrogen Pichia Expression Kit
Invitrogen Pichia Fermentation Process Guidelines
Video demonstration about Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris in Hallam lab at UBC


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