User:Fermenter User/Notebook/MACG F7

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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 F7R

Day -1 (May 12 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 #1 on the bottom of the page
   
   <Ann>                 5/12/2009 (9:10)  Prepare small culture for inoculum (10mL)
   <Ann/Sung-Hye>        5/12/2009 (11:30)  Pour media
                         5/12/2009 (11:30)  pH calibration
                                            No Antifoam added
                         5/12/2009 (12:45)  Autoclave bioreactor
                                            3 empty bottles/fermenter
                                            Base, Carbon source+Inoculation, Feeding

5/12/2009 (14:30) Cooling & Start Temp Control 5/12/2009 (14:50) Add carbon source using pumps 5/12/2009 (15:05) Connect Base (400 ml) Prime the pumps Set pH 5/12/2009 (15:10) Start F1 software 5/12/2009 (15:11) Start F2 software 5/12/2009 (15:12) Start F3 software
   <Ann>                 5/12/2009 (12:45)  Prepare small culture for inoculum (20mL)
   <Ann>                 5/12/2009 (20:00)  Prepare small culture for inoculum (420mL)

Day 0 (May 13 Wed)

   <SungHye>            5/13/2009 (08:40)  Connect MeOH sensor
                        5/13/2009 (08:40)  Wrap bioreactors with foil
                        5/13/2009 (08:40)  Calibrate dO2
                        5/13/2009 (08:40)  Set dO2

   <Ann>                5/13/2009 (09:00)   Fermentation 00:00 
                             Inoculation 50 ml (F1 and F2) and 250 ml (F3) using pump.
   <Ann>                5/13/2009 (09:00)  Fermentation 00:00 
                             F1: OD (0.150) Contamination? (N)
                             F2: OD (0.135) Contamination? (N)
                             F3: OD (0.209) Contamination? (N)
   <Ann>                5/13/2009 (10:00)  Fermentation 01:00  
                             F1: 380 ml 
                             F2: 380 ml
                             F3: 360 ml
   <Ann>                5/13/2009 (14:00)  Fermentation 05:00 
                             F1: OD (0.633) Contamination? (N)
                             F2: OD (0.635) Contamination? (N)
                             F3: OD (0.610) Contamination? (N)
   <Ann>                5/13/2009 (17:30)  Fermentation 08:30 
                             F1: OD (1.086) Contamination? (N)
                             F2: OD (1.123) Contamination? (N)
                             F3: OD (1.089) Contamination? (N)


  Expected Power Outrage 
                  7:00 pm - 10:00 pm
                  UPS (Undisrupted power supply) will be running 

Day 1 (May 14 Thu)

   <Ann>                5/14/2009 (07:00-08:00)  Fermentation 22:00-23:00 
                             F1: Spin down cells and re-suspend with fresh media (1-L)
                                 Pour culture back into fermenter
   <Ann>                5/14/2009 (09:00)  Fermentation 24:00  
                             F1: 290 ml 
                             F2: 300 ml
                             F3: 30 ml (replaced with 400 mL base at 10x concentration)
   <Ann>                5/14/2009 (09:00)  Fermentation 24:00 
                             F1: OD (7.7) Contamination? (N)
                             F2: OD (10.5) Contamination? (N)
                             F3: OD (8.9) Contamination? (N)
   <Ann>                5/14/2009 (09:00)  Fermentation 24:00 Induction 00:00 
                             F1: Induce with Galactose (55 mL)  
                                 Start feeding: (10 rpm)
                             F2: Induce with Galactose (55 mL)                                     
                                 Start feeding: (10 rpm)
                             F3: Induced with Galactose (275 mL)  
                                 Start feeding: (2 rpm with 32 rpm pump)                              
   File:Watsonpump.tif
   File:Watsonpump2.tif

<Ann> 5/14/2009 (10:00) Fermentation 25:00 F3: 500 mL was taken out to avoid overflow / clogging the filter F3: Induction 01:00 <Ann> 5/14/2009 (11:00) Fermentation 26:00 F1: OD (8.8) Contamination? (N)F1: Induction 02:00 F2: OD (9.5) Contamination? (N)F2: Induction 02:00 F3: OD (8.4) Contamination? (N)F3: Induction 02:00 <Ann> 5/14/2009 (13:00) Fermentation 28:00 F1: OD (9.8) Contamination? (N)F1: Induction 04:00 F2: OD (9.0) Contamination? (N)F2: Induction 04:00 F3: OD (8.4) Contamination? (N)F3: Induction 04:00 <Ann> 5/14/2009 (15:00) Fermentation 30:00 F1: OD (10.7) Contamination? (N)F1: Induction 06:00 F2: OD (9.4) Contamination? (N)F2: Induction 06:00 F3: OD (8.7) Contamination? (N)F3: Induction 06:00 <Ann> 5/14/2009 (17:00) Fermentation 32:00 F1: OD (15.6) Contamination? (N)F1: Induction 08:00 F2: OD (11.0) Contamination? (N)F2: Induction 08:00 F3: OD (8.9) Contamination? (N)F3: Induction 08:00 <SungHye> 4/24/2009 (00:00) Harvest

References

  1. www.ravenbiotech.com/product.html [Paper1]


  1. High Cell Density Fermentation of Saccharomyces cerevisiae JUL3 in Fed-batch Culture for the Production of β-Glucan (2007) 13, 1, 153-158 [Paper2]
  File:IE13-1-0153.pdf
  Fermentation condition: 30°C, 200 rpm, no pH control, 1 vvm
  
  1. Optimization of enterokinase fermentation using a recombinant Saccharomyces cerevisiae. (2005) 40, 717-722 [Paper3]
  File:PB-40-717.pdf
  Fermentation condition: 30°C, 500 rpm, no pH control, 2 vvm
  1. Alakomi HL, Paananen A, Suihko ML, Helander IM, and Saarela M. Weakening effect of cell permeabilizers on gram-negative bacteria causing biodeterioration. Appl Environ Microbiol. 2006 Jul;72(7):4695-703. DOI:10.1128/AEM.00142-06 | PubMed ID:16820461 | HubMed [Paper4]
  1. Peamiability test: Aliquots (100 µl) of this cell suspension were pipetted into fluoroplate wells, 
     which contained NPN (10 µM) and, as test substances, either EDTA (1.0 and 0.1 mM), PEI (10 µg ml–1), 
     DMSA (1 mM), AOT (1 mM), or HEPES buffer (control) to make up a total volume of 200 µl. If desired, 
     MgCl2 was added to the cell suspension before addition of NPN. Fluorescence was monitored within 
     3 min from four parallel wells per sample (excitation, 355 nm; half bandwidth, 38 ± 3 nm; emission, 
     402 nm; half bandwidth, 50 ± 5 nm). Each assay was performed at least three times. 
  1. Hounsa CG, Brandt EV, Thevelein J, Hohmann S, and Prior BA. Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress. Microbiology. 1998 Mar;144 ( Pt 3):671-80. DOI:10.1099/00221287-144-3-671 | PubMed ID:9534237 | HubMed [Paper5]
  1. OD600 unit is equivalent to 0.64 +/- 0.02 g cells (dry weight)/L
  1. Hao Q, Hong SH, and Maret W. Lipid raft-dependent endocytosis of metallothionein in HepG2 cells. J Cell Physiol. 2007 Feb;210(2):428-35. DOI:10.1002/jcp.20874 | PubMed ID:17111383 | HubMed [Paper6]
  1. To demonstrate the localization of the proteins relative to the cells, cell contours were visualized 
     with Alexa 647-concanavalin A.
  1. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, and Iwata S. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc. 2008;3(5):784-98. DOI:10.1038/nprot.2008.44 | PubMed ID:18451787 | HubMed [Paper7]
  1. Newstead S, Kim H, von Heijne G, Iwata S, and Drew D. High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):13936-41. DOI:10.1073/pnas.0704546104 | PubMed ID:17709746 | HubMed [Paper8]
  1. Kostov Y, Albano CR, and Rao G. All solid-state GFP sensor. Biotechnol Bioeng. 2000 Nov 20;70(4):473-7. PubMed ID:11005931 | HubMed [Paper8]
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