User:Fermenter User/Notebook/MACG E4

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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 E4 (F2)

Day -1 (Feb 16 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>            2/16/2009 (10:30)  Pour media
                         2/16/2009 (11:00)  pH calibration
                         2/16/2009 (11:42)  Antifoam added
                         2/16/2009 (12:36)  Autoclave bioreactor
                                            3 empty bottles/fermenter
                                            Carbon source, Inoculum, Feeding bottle

2/16/2009 (14:15) Start Temp Control 2/16/2009 (15:00) Add Carbon source using pump at 30°C 2/16/2009 (15:15) Start F2 software

Day 0 (Feb 17 Tue)

   
    Sung-Hye Class 9:30-11:00
   
   <Sung-Hye>            2/17/2009 (7:35)   Connect MeOH sensor
                         2/17/2009 (7:50)   Calibrate dO2
                         2/17/2009 (7:50)   Set dO2

<Sung-Hye and Isis> 2/17/2009 (8:40) Fermentation 0:00 Inoculation 30 ml each using a pump. <Isis> 2/17/2009 (18:00) Fermentation 8:00 F2: OD (18.9) Contamination? (N)Budding well, clumping.

Day 1 (Feb 18 Wed)

   <Isis>                2/18/2009 (9:00)   Fermentation 24:00 
                             F2: OD (51.2) Contamination? (N)  Still budding, no vacuoles.
   <Isis>                2/18/2009 (13:00)   Fermentation 28:00 
                             F2: OD (49.8) Contamination? (N)                                   
   <Isis>                2/18/2009 (17:00)   Fermentation 31:00 
                             F2: OD (52.1) Contamination? (N)

Day 2 (Feb 19 Thu)

   
     Sung-Hye Class 9:30 - 11:00
   
   <Isis>                2/19/2009 (08:00)   Fermentation 47:00 
                             F2: OD (52.2) Contamination? (N) still budding, no vacuoles. Equivalent culture in flask OD 38.0
   <Isis>                2/19/2009 (11:00)   Fermentation 50:00 
                             F2: OD (54.0) Contamination? (N) 
   <Sung-Hye and Isis>   2/19/2009 (13:20)  Fermentation 52:20 
                         1uM Pepstatin added to F2 to limit activity of aspartyl proteases (Paper 8)
                             F2: Connect Pump to 290 ml of Feeding media  Induction 00:00 
                                Watson pump: 9 rpm (10rpm = 12.8 ml/h)
Flowate calibration of Watson pump for feeding. Click it to see large
Flowate calibration of Watson pump for feeding. Click it to see large
   <Isis>                2/19/2009 (14:00)   Fermentation 53:00 
                             F2: OD (55.4) Contamination? (N)  Induction 01:00      
   <Isis>                2/19/2009 (18:30)   Fermentation 57:30 
                             F2: OD (76.3) Contamination? (N)  Induction 5:30  Vacuoles forming, actively budding
   <Isis>                2/19/2009 (23:50)  Fermentation 62:50 
                            F2: OD (68.08) Contamination? (N)  Induction 10:50 

Day 3 (Feb 20 Fri)

   <Isis>                2/20/2009 (8:00)  Fermentation 71:00 
                            F2: OD (132.1) Contamination? (N)vacuoles approx. 4/cell Induction 19:00
   <Isis>                2/20/2009 (09:00)  Fermentation 71:00 
                            F2: OD (132.4) Contamination? (N) Induction 20:00
   <Sung-Hye and Isis>   2/20/2009 (10:00)  Fermentation 72:00 
                            F2: Harvest Induction 21:00

References

Error fetching PMID 7766011:
  1. Error fetching PMID 7766011: [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]


Error fetching PMID 15063622:
  1. Error fetching PMID 15063622: [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
Error fetching PMID 1910586:
  1. Error fetching PMID 1910586: [Paper5]
Media:ReferencePaper5.pdf
  1. www.ravenbiotech.com/product.html [Paper6]
Error fetching PMID 1368914:
  1. Error fetching PMID 1368914: [Paper7]
Error fetching PMID 18601269:
  1. Error fetching PMID 18601269: [Paper8]



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