User:Fermenter User/Notebook/MACG E5

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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 E5 (F1 and F2)

Day -1 (Feb 23 Mon)

  <Bioreactor headplate configuation>
   pH sensor: monitor or control pH
   dO2 sensor: monitor or control dO2
   Condenser: Gas outlet
   OD probe: monitor cell growth ==> Not used! Replate it with septum
   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>            2/23/2009 (9:30)  Pour media
                         2/23/2009 (9:30)  pH calibration
                                            pH sensor #1 (F1)
                                            pH sensor #2 (F2)
                                            pH sensor #3 (F3)                                             
                         2/23/2009 (9:30)  Antifoam added
                         2/23/2009 (10:15)  Autoclave bioreactor ==> 3 bioreactors
                                            3 empty bottles/fermenter
                                            Carbon source, Inoculum, Feeding bottle

2/23/2009 (12:00) Start Temp Control 2/23/2009 (13:30) Add Carbon source using pump at 30°C 2/23/2009 (13:39) Start F1 software 2/23/2009 (13:40) Start F2 software

Day 0 (Feb 24 Tue)

   
    Sung-Hye Class 9:30-11:00
   
   <Sung-Hye>            2/24/2009 (08:00)   Connect MeOH sensor
                         2/24/2009 (08:00)   Calibrate dO2
                         2/24/2009 (08:00)   Set dO2

<Sung-Hye and Isis> 2/24/2009 (12:00) Fermentation 00:00 Inoculation 30 ml each using a pump. <Isis> no readings taken today

Day 1 (Feb 25 Wed)

   <Isis>                2/25/2009 (9:00)   Fermentation 21:00 
                             F1: OD (58.5) Contamination? (N)smaller, rounder cells, very clumpy
                             F2: OD (49.3) Contamination? (N)larger oval cells, budding well, pink color developing already. 
   <Isis>                2/25/2009 (10:00)   Fermentation 22:00 
                             F1: OD (61.5) Contamination? (N)
                             F2: OD (57.0) Contamination? (N)
   <Isis>                2/25/2009 (11:00)   Fermentation 23:00 
                             F1: OD (64.3) Contamination? (N)
                             F2: OD (59.7) Contamination? (N)
   <Isis>                2/25/2009 (12:00)   Fermentation 24:00 
                             F1: OD (63.5) Contamination? (N)
                             F2: OD (59.2) Contamination? (N)

------------------------------------ Induction of F1 (Transfer to F3) -------------------------------- <Sung-Hye and Isis> 2/25/2009 (13:40) Fermentation 25:40 Spin-down cell pellet and transfer to second bioreactor (13:48) Start software for F3 <Sung-Hye and Isis> 2/25/2009 (14:00) Fermentation 26:00 F3: Transfer cells + Carbon source (5ml of 40% Gal) to second bioreactor by pump Pepstatin and Benzamidine.HCl (1 mM Final) were added via septum F3: OD (53.2) Contamination? (N) <Sung-Hye and Isis> 2/25/2009 (15:00) Fermentation 27:00 F3: Connect Pump to ?? ml of Feeding media F3:Induction 00:00 Watson pump: 9 rpm (10rpm = 12.8 ml/h)

------------------------------------ Induction of F2 ------------------------------------------------ <Sung-Hye and Isis> 2/25/2009 (16:00) Fermentation 28:00 Spin-down cell pellet and pour back to same bioreactor F2: OD (59.0) Contamination? (N) <Sung-Hye and Isis> 2/25/2009 (16:00) Fermentation 28:00 F2: Transfer cells + Carbon source (5ml of 40% Gal) to second bioreactor by syringe Pepstatin and Benzamidine.HCl (1 mM Final) were added via septum <Sung-Hye and Isis> 2/25/2009 (17:00) Fermentation 29:00 F2: Connect Pump to ?? ml of Feeding media F2:Induction 00:00 Watson pump: 9 rpm (10rpm = 12.8 ml/h) ---------------------------------------------------------------------------------------------------------

File:Watsonpump.tif

Day 2 (Feb 26 Thu)

   
     Sung-Hye Class 9:30 - 11:00
   
   <Isis>                2/26/2009 (07:30)   Fermentation 55:30 
                             F3: OD (96.6) Contamination? (N)  F3:Induction 16:30  
                             Cells budding, small and round, clumping. Cell pellet cream/white. 
                             Smell from fermenter sweet like banana flavored milk. 
                          --> See Reference #9, #10 about "Banada Odor": It is [Isoamyl Acetate]

F2: OD (118.8) Contamination? (N) F2:Induction 14:30 Cells budding, larger, vacuoles present (or multiple buds). Cell pellet pink. <Isis> 2/26/2009 (09:00) Fermentation 57:00 F3: OD (-) Contamination? (N) Fe:Induction 18:00 F2: OD (-) Contamination? (N) F2:Induction 16:00 <Isis> 2/26/2009 (11:00) Fermentation 59:00 F3: OD (102.0) Contamination? (N) F3:Induction 20:00 F2: OD (130.0) Contamination? (N) F2:Induction 18:00 <Isis> 2/26/2009 (13:00) Fermentation 61:00 F3: OD (103.0) Contamination? (N) F3:Induction 22:00 Harvest 575 ml F2: OD (126.7) Contamination? (N) F3:Induction 20:00 <Isis> 2/26/2009 (15:00) Fermentation 63:00 F3: OD (-) Contamination? (N) F3:Induction 24:00 F2: OD (-) Contamination? (N) F2:Induction 22:00 Harvest 500 ml <Isis> 2/26/2009 (17:00) Fermentation 65:00 F3: OD (108.5) Contamination? (N) F3:Induction 26:00 F2: OD (135.2) Contamination? (N) F2:Induction 24:00

Day 3 (Feb 27 Fri)

   <Isis>                2/27/2009 (11:00)  Fermentation 83:00 
                             F3: OD (114.1) Contamination? (N)  F3:Induction 44:00 
                             F2: OD (124.8) Contamination? (N)  F2:Induction 42:00 
   <Isis>                2/27/2009 (15:00)  Fermentation 87:00 
                             F3: Harvest  F3:Induction 48:00 
                             F2: Harvest  F2:Induction 46:00 

References

  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]
Media:ReferencePaper5.pdf
  1. www.ravenbiotech.com/product.html [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]
  1. Banana odor generator BBa_J45200 from Registry of Standard Biological Parts, iGEM2006_MIT [Paper9]
 Acetic Acid + Isoamyl Alcohol (also called Amyl Alcohol, Isopentyl Alcohol, Pentanol)→ Isoamyl Acetate (Banana Odor)
  1. Fabre CE, Blanc PJ, and Goma G. Production of 2-phenylethyl alcohol by Kluyveromyces marxianus. Biotechnol Prog. 1998 Mar-Apr;14(2):270-4. DOI:10.1021/bp9701022 | PubMed ID:9548779 | HubMed [Paper10]
  1. Jiang J. Identification of flavour volatile compounds produced by Kluyveromyces lactis. Biotechnol Tech 1993 7(12) 863-6. [Paper11]
 Media:Jiang_J_(1993)_Biotechnol._Tech.pdf 
 One of predominant compounds from K. lactis are isoamyl alcohol (73 mg/L), 2-phenylethanol (72 mg/L), and acetoin (22 mg/L).  


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