User:Fermenter User/Notebook/MACG E2

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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 E2 (F1/F2)

Day -1 (Jan 25 Sun)

   <Isis>               1/25/2009 (00:00) Prepare small culture for inoculum.

Day 0 (Jan 26 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
   <Sung-Hye>            1/26/2009 (10:30)  Pour media
                         1/26/2009 (10:30)  Antifoam added
                         1/26/2009 (10:30)  pH calibration
                         1/26/2009 (10:30)  Autoclave bioreactor
                                            4 empty bottles/fermenter
                                            Acid, Base, Carbon source+Inoculation, Nut feeding
                         1/26/2009 (10:45)  Autoclave
                         1/26/2009 (12:43)  Start OD software
                         1/26/2009 (13:00)  Start Temp Control
                         1/26/2009 (13:30)  Add Carbon sources using pumps
                         1/26/2009 (13:35)  Start pH control
                         1/26/2009 (13:35)  Start F1 software
                         1/26/2009 (13:37)  Start F2 software
                         1/26/2009 (14:00)  Calibrate dO2
                         1/26/2009 (14:00)  Set dO2
   <Sung-Hye and Isis>   1/26/2009 (14:00)   Fermentation 0:00 
                             Inoculation 30 ml each using pump.

Day 1 (Jan 27 Tue)

    Sung-Hye: CHBE Class Auditing 9:30 - 11:00
   <Sung-Hye>            1/27/2009 (8:30) Meausre Acid, Base  Fermentation 18:30 
                             F1: Acid (460 ml) Base (315 ml)
                             F2: Acid (485 ml) Base (350 ml) 
   <Isis>                1/27/2009 (11:00)   Fermentation 21:00 
                             F1: Sampling: OD (55.1) Contamination? (N)no budding, clumpy
                             F2: Sampling: OD (61.9) Contamination? (N)no budding.
   <Isis>                1/27/2009 (12:00)   Fermentation 22:00 
                             F1: OD (52.1) Contamination? (N)
                             F2: OD (53.4) Contamination? (N)
   <Sung-Hye and Isis>   1/27/2009 (13:00)  Fermentation 23:00
                             F1: Connect Pump to 400 ml of Feeding media:  Induction 0:00 
                             F2: Connect Pump to 400 ml of Feeding media:  Induction 0:00 
                           1uM Pepstatin added to F1 and F2 to limit activity of aspartyl proteases (Paper 8)
                             Connect feeding bottles
                             F1: 2 rpm
                             F2: 2 rpm

   <Discussion about feeding>
    1. When?: Check OD to see if it has stabilized on F1 and start feeding.
    2. How? : Use Monad equation
       1 OD600 unit corresponds to a dry cell weight of 0.233mg/ml (Paper4) 
       Please see bottom of this page to see more.

   <Isis>                1/27/2009 (16:00)   Fermentation 26:00 
                             F1: OD (57.9) Contamination? (N)  Induction 3:00 
                             F2: OD (59.2) Contamination? (N)  Induction 3:00  

    We decided to double the feed rate to 4 rpm as the cells are not growing and O2 consumption is not changing. 

   <Sung-Hye>            1/27/2009 (16:25>  Fermentation 26:30  
                             Change feeding rate 
                             F1: 4 rpm  Induction 3:30 
                             F2: 4 rpm  Induction 3:30 
   <Isis>                1/27/2009 (20:00)   Fermentation 30:00 
                             F1: OD (83.0) Contamination? (N)  Induction 7:00 
                             F2: OD (54.1) Contamination? (N)  Induction 7:00  
   <Isis>                1/27/2009 (24:00)   Fermentation 34:00 
                             F1: OD (79.5) Contamination? (N)  Induction 11:00  not many buds
                             F2: OD (78.9) Contamination? (N)  Induction 11:00  Budding still

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Day 2 (Jan 28 Wed)

   <Isis>                1/28/2009 (7:00)   Fermentation 41:00 
                             F1: OD (112.4) Contamination? (N) Induction 18:00 
                             F2: OD (110.0) Contamination? (N) Induction 18:00 
   <Sung-Hye>            1/28/2009 (8:30)   Fermentation 42:30 
                             F1: Acid (420 ml)   Base (275 ml) Feed (305 ml)
                             F2: Acid (470 ml)   Base (315 ml) Feed (305 ml)
   <Isis>                1/28/2009 (10:00)  Fermentation 44:00 
                             F1: OD (92.7) Contamination? (N) Induction 21:00 
                             F2: OD (104.5) Contamination? (N) Induction 21:00 
   <Isis>                1/28/2009 (14:00)   Fermentation 48:00 
                             F1: OD (103.2) Contamination? (N) Induction 25:00 
                             F2: OD (117.4) Contamination? (N) Induction 25:00 

Day 3 (Jan 29 Thu)

   Sung-Hye: CHBE Class Auditing 9:30 - 11:00
   <Sung-Hye>            1/29/2009 (8:30)   Fermentation 66:30 
                            F1: Acid (395 ml)   Base (200 ml) Feed (200 ml)
                            F2: Acid (480 ml)   Base (290 ml) Feed (200 ml)
                            Picture of bioreactors taken
   <Isis>                1/29/2009 (11:30)  Fermentation 69:00 
                            F1: OD (109.3) Contamination? (N)  vacuoles present approx 2/ cell  Induction 43:30 
                            F2: OD (124.0) Contamination? (N) still actively budding!!  Induction 43:30 
   <Isis>                1/29/2009 (4:30)   Fermentation 74:00 
                            F1: OD (108.8) Contamination? (N)  Induction 48:30 
                            F2: OD (130.5) Contamination? (N)  Induction 48:30 
   Expected power outrage 6pm-9pm
   No access to building
   All fermenters and computer is connected to UPS
   Computer was off!! 

   <Isis>                1/29/2009 (22:30)  Fermentation 81:00 
                            F1: OD (-) Contamination? (N)  Induction 55:30 
                            F2: OD (-) Contamination? (Y)  Induction 55:30 

Day 4 (Jan 30 Fri)

   <Isis>                1/30/2009 (8:30)  Fermentation 91:00 
                            F1: OD () Contamination? (N)  Induction 65:30 
                            F2: OD () Contamination? (N)  Induction 65:30 
                       The SN was turbid when warm and became clear again as the temperature decreased. 
                       SH thinks it could be antifoam or cell secretions reacting with antifoam. 
   <Sung-Hye>            1/30/2009 (14:30)  Fermentation 97:00 
                            EtOH calibration</font>
                            F1: Induction 71:30 Acid (380 ml) Base (130 ml)
                            F2: Induction 71:30 Acid (460 ml) Base (280 ml)


  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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