User:Fermenter User/Notebook/MACG E7T

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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 E7T

Day -1 (Mar 30 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>            3/30/2009 (13:00)  Pour media
                         3/30/2009 (13:00)  pH calibration  
                         3/30/2009 (13:00)  Add antifoam 0.5 ml/fermenter
                         3/30/2009 (15:10)  Autoclave bioreactor ==> 3 bioreactors
                                            3 empty bottles/fermenter (no need this time!)
                                            Carbon source, Inoculum, Feeding bottle

3/30/2009 (17:00) Start Temp Control

Day 0 (Mar 31 Tue)

                         3/31/2009 (10:24)  Start F1 software
                         3/31/2009 (10:25)  Start F2 software
                         3/31/2009 (10:27)  Start F3 software
                         3/31/2009 (10:30)  Connect Base (400 ml) for Fer#7
                         3/31/2009 (11:20)  Add Carbon source using pump at 30°C
                         3/31/2009 (11:27)  Calibrate dO2
                         3/31/2009 (11:27)  Set dO2
                         3/31/2009 (11:30)  Set pH for Fer#7
   <Sung-Hye and Isis>   3/31/2009 (12:00)  Fermentation 00:00 
                             Inoculation 30 ml each using a pump.
   <Isis>                no readings taken today

Day 1 (Apr 1 Wed)

   <Sung-Hye>            4/1/2009 (7:00) 
                             F3: Base (370 ml)
   <Isis>                4/1/2009 (13:00)   Fermentation 25:00 
                             F1: OD (41.1) Contamination? (N)white, budding
                             F2: OD (59.4) Contamination? (N)white, budding
                             F3: OD (57.2) Contamination? (N)white, budding
<Sung-Hye and Isis>   4/1/2009 (13:00)   Fermentation 25:00 
                             Pepstatin added (13:00)
                             F5: Connect Pump to 500 ml of Feeding media (1xYP; 400g/l Gal)
                             Watson pump: 10 rpm (2rpm pump was used, 10rpm = 12.8 ml/h) 

Day 2 (Apr 2 Thu)

   <Sung-Hye>            4/2/2009 (07:00)
                             F3: Base (370 ml)
   <Isis>                4/2/2009 (9:00)   Fermentation 45:00  
                             F5: OD (113.3) Contamination? (N) F5:Induction 20:00 
                             F6: OD (60.6) Contamination? (N) F6:Induction 00:00 
                            F7: OD (57.8) Contamination? (N) F7:Induction 00:00 
   <Isis>                4/2/2009 (10:00)   Fermentation 46:00  
                             F5: OD (113.3) Contamination? (N) F5:Induction 21:00 HARVEST 
   <Sung-Hye and Isis>   4/2/2009 (10:30)   Fermentation 45:30 
                             Pepstatin added (00:00)
                             F6: Connect Pump to 250 ml of Feeding media (1xYP; 400g/l Gal)
                             Watson pump: 10 rpm (2rpm pump was used, 10rpm = 12.8 ml/h) 
   <Sung-Hye and Isis>   4/2/2009 (10:30)   Fermentation 45:30 
                             Pepstatin added (00:00)
                             F7: Connect Pump to 250 ml of Feeding media (1xYP; 400g/l Gal)
                             Watson pump: 10 rpm (2rpm pump was used, 10rpm = 12.8 ml/h)
   <Isis>                4/2/2009 (12:30)   Fermentation 47:30  
                             F6: OD (73.2) Contamination? (N) F6:Induction 2:00, 02 stopped as cells growing too fast
                             F7: OD (68.8) Contamination? (N) F7:Induction 2:00 02 stopped as cells growing too fast, pH was dropping, stopped at 6.0 by Base. 
   <Isis>                4/2/2009 (14:00)   Fermentation 49:00  
                             F6: OD (86.3) Contamination? (N) F6:Induction 3:50 pH 6.5 
                             F7: OD (73.9) Contamination? (N) F7:Induction 3:50 pH 6.6
   <Isis>                4/2/2009 (16:00)   Fermentation 51:00  
                             F6: OD (94.5) Contamination? (N) F6:Induction 5:50 
                             F7: OD (84.2) Contamination? (N) F7:Induction 5:50 
   <Isis>                4/2/2009 (18:00)   Fermentation 53:00  
                             F6: OD (99.4) Contamination? (N) F6:Induction 7:50 
                             F7: OD (82.2) Contamination? (N) F7:Induction 7:50 
   


File:Watsonpump.tif

Day 3 (Apr 3 Fri)

   <Sung-Hye>            4/3/2009 (07:00)
                             F3: Base (340 ml)
   <Isis>                4/3/2009 (07:30)  Fermentation 67:30 
                             F6: OD (124.4)  F6:Induction 21:30 
                            Contamination?(N)no budding, multiple vacuoles, cells cream color still  
                             F7: OD (111.5)  F3:Induction 21:30 
                            Contamination? (N) cells cream color still isoamyl acetate smell
   <Sung-Hye>            4/3/2009 (08:00)  Fermentation 68:00 
                             Harvest

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).  
  1. Enhanced Human Lysozyme Production by Kluyveromyces lactis, Eric Lu Huang and Ali Demirci,Food and Bioprocess Technology, Volume 2, Number 2 / June, 2009 [Paper12]
  1. Li ZF, Yu XL, Huang JL, Fang HQ, and Chen HP. [Recombinant batroxobin expressed highly in Pichia pastoris]. Sheng Wu Gong Cheng Xue Bao. 2007 May;23(3):483-6. PubMed ID:17577998 | HubMed [Paper13]


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