User:Fermenter User/Notebook/MACG E3

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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

MACG E3 (F1/F2)

Day -1 (Feb 2 Mon)

  <Bioreactor headplate configuation>
   pH sensor: monitor or control pH
   dO2 sensor: monitor or control dO2
   Condenser: Gas outlet
   OD probe: monitor cell growth
   Triple: Addition of media, Acid or Base if necessary
   MeOH sensor: Monitor EtOH (byproduct)
   <Sung-Hye's Note>
    MeOH sensors also detects ethanol! :See reference #6]
Volume of F2: keep as same 1-L
Media (F2): 1-L Additional blade (higher than normal):no need Harvest time will be quicker when activity is Max. <Sung-Hye> 2/2/2009 (13:20) Pour media 2/2/2009 (13:20) pH calibration 2/2/2009 (13:40) Autoclave bioreactor 3 empty bottles/fermenter Carbon source, Inoculum, Feeding bottle 2/2/2009 (15:30) Antifoam added:added later! 2/2/2009 (15:35) Start Temp Control 2/2/2009 (15:45) Add Carbon sources using pumps 2/2/2009 (15:46) Start OD software 2/2/2009 (15:50) Start F1 software 2/2/2009 (15:52) Start F2 software

Day 0 (Feb 3 Tue)

    Sung-Hye Class 9:30-11:00
   <Sung-Hye>            2/3/2009 (8:15)   Calibrate dO2
                         2/3/2009 (8:15)   Set dO2
   <Sung-Hye and Isis>   2/3/2009 (9:00)  Overnight culture KLABDM3 OD: 60.8, no contamination 
                         2/3/2009 (10:00)  Fermentation 0:00 
                             Inoculation 30 ml each
   <Isis>                2/3/2009 (17:00)   Fermentation 7:00 
                             F1: Sampling: OD (??) Contamination? (N)
                             F2: Sampling: OD (??) Contamination? (N)

Day 1 (Feb 4 Wed)

   <Isis>                2/4/2009 (8:00)   Fermentation 22:00 
                             F1: OD (62.4) Contamination? (N)budding well, no vacuoles.
                             F2: OD (49.1) Contamination? (N)budding well, no vacuoles, clumpy.
   5 am - 9:15 am : O2 tanks ran out 
   <Sung-Hye>            2/4/2009 (9:15)  Fermentation 23:15 
                             Open O2 tank
   <Isis>                2/4/2009 (10:00)   Fermentation 24:00 
                             F1: OD (65.3) Contamination? (N)
                             F2: OD (35.0) Contamination? (N)
                                 Cells turning pink!!- not sure if 8am reading was anomalously high?
   <Isis>                2/4/2009 (13:00)   Fermentation 27:00 
                             F1: OD (62.8) Contamination? (N)
                             F2: OD (49.5) Contamination? (N)
   <Sung-Hye and Isis>   2/4/2009 (15:00)  Fermentation 29:00 
                            F1: Connect Pump to 500 ml of Feeding media:  Induction 0:00 
                            F2: Connect Pump to 500 ml of Feeding media (Wait till tomorrow)
                                Watson pump: 10 rpm ==> Same as MACG E1 (F1)
   <Isis>                2/4/2009 (16:00)   Fermentation 30:00   Induction 1:00 
                            F1: OD (86.9) Contamination? (N)no vacuoles, filter change due to pressure buildup. 
                            F2: OD (55.6) Contamination? (N)no vacuoles
   <Sung-Hye>            2/4/2009 (16:30)   Fermentation 30:30   Induction 1:30  
                            F1: 1uM Pepstatin added to F1 to limit activity of aspartyl proteases (Paper 8)
   <Isis>                2/4/2009 (17:00)   Fermentation 31:00 
                            F1: OD (112.9) Contamination? (N)  Induction 2:00 
                            F2: OD (59.1) Contamination? (N)


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Day 2 (Feb 5 Thu)

     Sung-Hye Class 9:30 - 11:00
   <Isis>                2/5/2009 (8:00)  Fermentation 46:00 
                            F1: OD (131.4) Contamination? (N) 
                                1-5 vacuoles present. Pellet cream color with pink  patches  Induction 17:00 
                            F2: OD (52.8) Contamination? (N) 
                                Cell pellet visibly pink now. One large vesicle in approximately 1/2 cells, 
                                Takes much much of the periplasmic space. 
   <Sung-Hye and Isis>   2/5/2009 (9:00)  Fermentation 47:00 
                           1uM Pepstatin added to F2 to limit activity of aspartyl proteases (Paper 8)
                            F2: Connect Pumps to 300 ml of Feeding media:  Induction 0:00 
                                Watson pump: 8 rpm ==> Same as MACG E1
<Isis> 2/5/2009 (11:00) Fermentation 49:00 F1: OD (139.4) Contamination? (N) Induction 20:00 Pellet is cream colored again, not much pink remains. F2: OD (60.5) Contamination? (N) Induction 2:00 Active budding, vacoules still present. Filter change due to pressure buildup. <Isis> 2/5/2009 (13:00) Fermentation 51:00 F1: OD (??) Contamination? (Y/N) Induction 22:00 F2: OD (??) Contamination? (Y/N) Induction 4:00 <Isis> 2/5/2009 (15:00) Fermentation 53:00 F1: OD (128.3) Contamination? (N) Induction 24:00 Harvest <Isis> 2/5/2009 (16:00) Fermentation 54:00 F2: OD (57.0) Contamination? (N) Induction 7:00 <Isis> 2/5/2009 (19:00) Fermentation 57:00 F2: OD (66.0) Contamination? (N) Induction 10:00

Day 3 (Feb 6 Fri)

   <Isis>                2/6/2009 (6:00)  Fermentation 68:00 
                            F2: OD (62.2) Contamination? (N) lots of budding. Induction 21:00
   <Isis>                2/6/2009 (7:00)  Fermentation 69:00 
                            F2: OD (64.2) Contamination? (N) Induction 22:00
                         2/6/2009 (7:30)  Fermentation 69:00 
                            Picture of bioreactor (F2) was taken Induction 22:30
   <Isis>                2/6/2009 (8:00)  Fermentation 70:00 
                            F2: OD (68.7) Contamination? (N) Induction 23:00
   <Isis>                2/6/2009 (9:00)  Fermentation 71:00 
                            F2: Harvest Induction 23:00


  1. Korz DJ, Rinas U, Hellmuth K, Sanders EA, and Deckwer WD. Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol. 1995 Feb 21;39(1):59-65. PubMed ID:7766011 | HubMed [Paper1]
  <Sung-Hye's Note>
    This is a good reference paper to cite for "growth limiting feeding" stragety.  
    Also Monod equation.
  1. Chung BH, Seo DJ, Nam SW, Na JG, and Chang YK. Optimization of Feeding Strategy for Overproduction of Human Lipocortin-I in Saccharomyces cerevisiae Controlled by the GAL10 Promoter. J Ferment. Bioeng. 1997 84(5) 466-470. (Not available in PubMed) ISSN 0922-338X [Paper2]
 1. Host stain and target: S. cerevisiae 2805 and Human Lipocortin-I  
 2. Plasmid and Promoter: YEG α-LC. GAL10 promoter
 3. Media: Start-up, Medium I, Medium II, and Medium III
    Glucose (not in Start-up), Galactose, Yeast extract, Casamino acid, and others.. 
 4. Inducible expression:
    Start of induction (feeding of MI-MIII: dO2 spike)
    Feeding of Media I-III: Keep Glucose (<1g/l), Ethanol (<10g/l)
    Feeding rate: 
F = μXV/(SF-S)Yx/s
≈ μXV/SFYx/s
F: feeding rate (l/h) μ(m or [mi]): specific growth rate (/h) ==>0.123 (glucose), 0.044 (galactose) SF: Carbon source concentration Yx/s: Cellular yield coefficient based on the carbon source consumption (g cell/g carbon source) ==> 0.436 (glucose), 0.722 (galactose)
<Sung-Hye's Note: There is no description what the X and V are. But from other reference (Paper#1)> X: Biomass concentration (g/l) V: Cultivation volume (l)
5. Batch Fermentation: Temp: 30°C, dO2: >10% pH: 5.5
  1. Note from New England Biolab [Paper3]

  1. Merico A, Capitanio D, Vigentini I, Ranzi BM, and Compagno C. How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis. J Biotechnol. 2004 Apr 8;109(1-2):139-46. DOI:10.1016/j.jbiotec.2003.10.031 | PubMed ID:15063622 | HubMed [Paper4]
 1. Host strain and target: K. lactis and Glucoamylase
 2. Plasmid and Promoter: pTS32x-GAA, GAP promoter
 3. Media: Minumum Media + (uracil), adenine + tryptophane + 2% of different carbon sources 
   (glucose, galatose, lactose, starch)
 4. Constitutive expression
 5. Batch Fermentation: 
      Temp: 30°C, 
      dO2: >20% (1L/min constant air purge, constant rpm at 1200)
      pH: 6.0 (Base, 2M KOH)
      Antifoam added
  1. Alberghina L, Porro D, Martegani E, and Ranzi BM. Efficient production of recombinant DNA proteins in Saccharomyces cerevisiae by controlled high-cell-density fermentation. Biotechnol Appl Biochem. 1991 Aug;14(1):82-92. PubMed ID:1910586 | HubMed [Paper5]
  1. [Paper6]
  1. Martegani E, Forlani N, Mauri I, Porro D, Schleuning WD, and Alberghina L. Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter. Appl Microbiol Biotechnol. 1992 Aug;37(5):604-8. PubMed ID:1368914 | HubMed [Paper7]
  1. Swaim CL, Anton BP, Sharma SS, Taron CH, and Benner JS. Physical and computational analysis of the yeast Kluyveromyces lactis secreted proteome. Proteomics. 2008 Jul;8(13):2714-23. DOI:10.1002/pmic.200700764 | PubMed ID:18601269 | HubMed [Paper8]

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