User:Fermenter User/Notebook/MACG E9R

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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 E9R

Day -1 (May 19 Tue)

  <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>            4/13/2009 (11:00)  Pour media
                         4/13/2009 (11:00)  pH calibration  
                         4/13/2009 (11:00)  Add antifoam 0.5 ml/fermenter
                         4/13/2009 (11:30)  Autoclave bioreactor ==> 3 bioreactors
                                            3 empty bottles/fermenter (no need this time!)
                                            Carbon source, Inoculum, Feeding bottle

4/13/2009 (14:00) Start Temp Control

Day 0 (May 20 Wed)

                         4/14/2009 (8:47)  Start F1 software
                         4/14/2009 (8:48)  Start F2 software
                         4/14/2009 (8:49)  Start F3 software
                         4/14/2009 (8:45)  Connect Base (400 ml)
                         4/14/2009 (9:20)  Add Carbon source using pump at 28.5°C
                         4/14/2009 (9:25)  Calibrate dO2
                         4/14/2009 (9:25)  Set dO2
                         4/14/2009 (9:25)  Set pH
   <Sung-Hye and Isis>   4/14/2009 (9:30)  Fermentation 00:00 
                             Inoculation 30 ml each using a pump. Sample in YP Dex stored for SDSPaGE
   <Isis>                no readings taken today

Day 1 (May 21 Thu)

   <Sung-Hye>            4/15/2009 (7:00)  Fermentation 21:30 
                             F1: Base (380 ml)
                             F2: Base (395 ml)
                             F3: Base (370 ml)
   <Isis>                4/15/2009 (10:30)   Fermentation 25:00 
                             F1: OD (71.0) Contamination? (N)white, stationary, large ovoid cells with large vesicle. 
                             F2: OD (65.0) Contamination? (N)white, stationary, large ovoid cells with large vesicle. 
                             F3: OD (58.4) Contamination? (N)white, stationary, large ovoid cells with large vesicle.
                                 High pressure released and new filter used.
   <Sung-Hye and Isis>   4/15/2009 (13:30)   Fermentation 28:00  Induction 00:00 
                             Pepstatin added (13:30)
                             Stop dO2 control
                             F1: Watson pump: 20 rpm (2rpm pump was used, 10rpm = 12.8 ml/h)
                                 OD (67.5)Connect Pump to 250 ml of Feeding media (400g/l Gal)Cell viability: 99.5%
                             F2: Watson pump: 20 rpm (2rpm pump was used, 10rpm = 12.8 ml/h)
                                 OD (64.3.0)Connect Pump to 250 ml of Feeding media (1/10th YP; 400g/l Gal)Cell viability: 99.5%
                             F3: Watson pump: 1 rpm (32rpm pump was used, 10rpm = 209.3 ml/h)
                                 OD (54.9)Connect Pump to 250 ml of Feeding media (1xYP; 400g/l Gal)Cell viability: 99.3%
                              

Flowate calibration of Watson pump (2 rpm) for feeding. Click it to see large
Flowate calibration of Watson pump (2 rpm) for feeding. Click it to see large
Flowate calibration of Watson pump (32 rpm) for feeding. Click it to see large
Flowate calibration of Watson pump (32 rpm) for feeding. Click it to see large
    <Isis>                4/15/2009 (15:30)   Fermentation 30.50 Induction 02.50 
                             F1: OD (86.9) Contamination? (N)white, large ovoid cells with large vesicle, budding. 
                             F2: OD (85.5) Contamination? (N)white, large ovoid cells with large vesicle, budding. 
                             F3: OD (56.4) Contamination? (N)white, large ovoid cells with large vesicle, budding.F2 AND F3 smell like IAA, F1 smells nasty. 
                                 High pressure released and new filter used.
<Isis>                4/15/2009 (23:30)   Fermentation 38.5 Induction 10.0 
                             F1: OD (106.0) Contamination? (N)white, budding. 
                             F2: OD (107.8) Contamination? (N)white, budding. 
                             F3: OD (81.8) Contamination? (N)white, budding. all smell like IAA

Day 2 (May 22 Fri)

   <Sung-Hye>            4/16/2009 (7:00)Fermentation 46.5  Induction 18.0 
                             F1: Base (360 ml)
                             F2: Base (380 ml)
                             F3: Base (370 ml)
   <Isis>                4/16/2009 (7:30)   Fermentation 46.5  Induction 18.0 
                             F1: OD (119.6) Contamination? (N) white, cells, some fat large buds still, lots of small vesicles in cells, 99.99% viability
                             F2: OD (107.0) Contamination? (N) white, cells, some fat large buds still, lots of small vesicles (approx 5/cell), >99.99% viability
                             F3: OD (105.5) Contamination? (N) white, cells, still budding, small and large buds seen, some small vesicles in cells, >99.99% viability
   <Isis>                4/16/2009 (10:00)   Fermentation 48:50  Induction 20.5 
                             F1: OD (122.1) Contamination? (N) 
   <Isis>                    F2: OD (124.8) Contamination? (N) 
                             F3: OD (115.2) Contamination? (N) all still budding a little, and lots of vacuoles. 
   <Isis>                4/16/2009 (11:30)   Fermentation 50:00  Induction 22.0   
                             F1: OD (125.2) Contamination? (N) 
                             F2: OD (126.6) Contamination? (N) 
                             F3: OD (113.0) Contamination? (N) lots of vacuoles.
   <Isis>                4/16/2009 (18:00)   Fermentation 55.00  Induction 28.5 
                             F1: OD (120.6) Contamination? (N) 3 dead cells
                             F2: OD (122.2) Contamination? (N) 5 dead cells
                             F3: OD (113.8) Contamination? (N) 2 dead cells 
   <Isis>                4/16/2009 (19:30)   Fermentation 54:50  Induction 30.0 
                             no ODs taken- 50ml samples only.
   <Sung-Hye>            4/17/2009 (00:00) Fermentation ??  Induction ?? 
                             Terminate Fermentation

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]
  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)
Error fetching PMID 9548779:
  1. Error fetching PMID 9548779: [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]
Error fetching PMID 17577998:
  1. Error fetching PMID 17577998: [Paper13]
Error fetching PMID 11470515:
  1. Error fetching PMID 11470515: [Paper14]
Error fetching PMID 7764684:
  1. Error fetching PMID 7764684: [Paper15]

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