User:Fermenter User/Notebook/BROM F1 F1

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BROM F1 (F1) and (F2)

Day -8 (Oct 27th Mon):

                           10/27/2008 (5:00 - 8:00) HAVC system test, no access to building
      <Kevin>              10/27/2008 (14:00 - 15:00) Based on Western Blooting results, Y98C colony 9 was chosen for fermentation. 
                                                      Based on activity screening, D5 (double horns) colony 5 was chosen for fermention.

Day -7 (Oct 28th Tue):

      <Kevin>              10/28/2008 (14:00)     Decided not to plate the yeast to reselect colonies for both Y98C and D5 
                                                  because fresh stocks were recently made.

Day -6 (Oct 29th Wed):

    Discussion about Feeding strategy for Mutants (Mut+/Muts) 
 :There are couple of referenes I recommend you to read. References regarding Feeding: Media:DAnjou_(2000)_Biotech._Bioeng._(72).pdf Media:Loewen_(1997)_Appl._Microbiol._Biotechnol._48_p480.pdf Effect of Ethanol or Acetate on Protein Expression in Pichia pastoris: Media:Meagher_(2001)_J._Bioscience._Bioeng._92_p337.pdf

                           10/29/2008 (19:30 - 19:40)  Expected Power Outrage, No access to building
                           10/29/2008 (19:45 - 22:45)  Multiple stop/start for breif duration 

      <Kevin>              10/29/2008 (18:45 - 19:45)  The papaers above suggested two valuable points for Picha fermentation. 
                        1. Because Mut+ strains also utilize MeOH as a carbon sourse, it would be better to limit the cells growth to a 
                           relatively lower O.D. (O.D. = 6) before passage to BMMY from BMGY. 
                        2. Acetic acid was suggested to reduce protein production in Pichia. 
                           Therefore, we decided to use  1M  HCl to regulate the pH.

Day -5 (Oct 30th Thu):

      <Kevin>              10/30/2008 (12:00)     Prepared 500mL MD media for first stage cell culturing and 
                                                  500mL BMGY for second stage culturing before fermentation for 
                                                  each mutant. 
                           10/30/2008 (13:00)     Decided with Sung-Hye to set up glycerol pump for O.D. Control. 
      <Sung-Hye>           10/31/2008 (13:00)     Leaving early

Day -4 (Oct 31st Fri):

      <Kevin>              10/31/2008 (12:00 - 18:00)  Autoclaved two 100ml flasks and two 500ml flasks for culturing. 
                                                       Prepared all necessary media and media components for fermentation, including
                                                          1. 1.5L 10x YNB (filtered, excess amount)
                                                          2. 1.5L Phosphate buffer, pH 6.0 (autoclaved, excess amount)
                                                          3. 1L 10x Glycerol (filtered)
                                                          4. 100mL 500x Biotin (filtered, excess amount)
                                                          5. 1.5L 10x Methanol (5%):wrong! (filtered, excess amount)

Day -3 (Nov 1st Sat):

      <Kevin>              11/1/2008 (17:00 - 18:00)  culturing 20uL stocks of Y98C and D5 in 5mL MD media at 30C over night.

Day -2 (Nov 2nd Sun):

      <Kevin>              11/2/2008 (17:00 - 18:00)  O.D.(both mutants)= 6.0. 
                                                      Spin down the cells (Y98C and D5) at 2000g for 5 minutes and resuspend them in 10mL BMGY. 
                                                      Transfer 100uL of the resuspended culture of each mutant to 50mL BMGY for over night culturing.

Day -1 (Nov 3rd Mon):

      <Sung-Hye>           11/3/2008 (11:00)     Prepared bottles for Acid/Base, MeOH, Glycerol, Label for each fermenter.             
      <Kevin>              11/3/2008 (12:00)     Gave all the necessary media components, as well as two 4L BMXY (non-sterile) media to 
      <Kevin>              11/3/2008 (13:00)     Both mutant cultures (Y98C and D5) reached O.D =6.        
                                                 Take 1mL of each culture and regrew them in 500mL 
                                                 BMGY for next day fermentation.

<Sung-Hye> 11/3/2008 (13:25) Assemble bioreactors: 1. 2 x 3.5-L of BMXY: 2. 2 ml of antifoam/ fermenter Autoclave

<Sung-Hye> 11/3/2008 (15:40) Cooling bioreactors 11/3/2008 (15:40) Purge Air (1 L/min) at 100 rpm 11/3/2008 (15:48) Start OD probe software 11/3/2008 (15:50) Start Fermenter #1 software 11/3/2008 (15:53) Start Fermenter #2 software 11/3/2008 (16:00 - 17:30) Transfer nutrients into bioreactors: Transfer using autoclave bottle/tube and pumps. 3. 2 x 500 ml of 10x Potassium Phosphate: Fermenter #2 was added using syringe/niddle at first and then switched to pump. 4. 2 x 500 ml of 10x YNB 5. 2 x 10 ml of 500x Biotin

Day 0 (Nov 4th Tue):

      <Sung-Hye>           11/4/2008 (7:45)        Autoclave bottles/tube for transfer inoculum and Glycerol     
      <Sung-Hye>           11/4/2008 (7:55)        Calibrate dO2 probe 
      <Sung-Hye>           11/4/2008 (7:55)        Set rpm at 900 
      <Sung-Hye>           11/4/2008 (8:52)        Start dO2 control (O2 valve Max) 
      <Sung-Hye>           11/4/2008 (8:55)        Start pH control 
                              6. 2 x 500 ml of 10x Glycerol: Glycerol feeding bottles (2) were provided from fermenter suite.  
                                                             Trasfered glycerol into the feeding bottles.
                              7. 2 x 500 ml of MeOH: MeOH feeding bottles (2) were provided from fermenter suite.  
                                                     Tasfer MeOH into MeOH feeding bottles. 
<Sung-Hye and Kevin> 11/4/2008 (10:00) Fermentation 0:00 Inoculate cells (O.D: 4.5 Volume: 2 fermenters x 500 ml) Cells were transferred to bottles/tube and inoculated using pumps.

<Sung-Hye and Kevin> 11/4/2008 (12:00) Fermentation 2:00 Sampling Y98C (F1)=(O.D:0, AU:-0.004) D5 (F2)=(O.D:0, AU:-0.033) [DO NOT USE THESE FOR O.D Correlation] It would expect the starting O.D to be approximately 0.45 because the 500mL O.D= 4.5 culture was diluted in 5L BMXY. Mistakes could have happened during O.D measuring for the over night 500mL cultures. <Sung-Hye and Kevin> 11/4/2008 (12:30) Fermentation 2:30 Feeding Glycerol 100 ml <Sung-Hye> 11/4/2008 (15:30) Fermentation 5:30 Gave to Paul. Sampling Y98C (F1)=(OD:0, AU:0.007) (F2)=(OD:0, AU:-0.023) ----suspected that the starting culture has much lower O.D than 4.5. <Sung-Hye and Kevin> 11/4/2008 (15:35) Fermentation 5:35 Feeding Glycerol 100 ml <Sung-Hye and Kevin> 11/4/2008 (17:00) Fermentation 7:00 MeOH sensor calibration <Sung-Hye and Kevin> 11/4/2008 (18:00) Reconfiguration of pH control (no Base) <Sung-Hye and Kevin> 11/4/2008 (18:00) Fermentation 8:00 MeOH Induction 0:00 Connect MeOH pump and Start MeOH control <Kevin> 11/4/2008 (19:53) Fermentation 9:53 MeOH Induction 1:53 Sampling Y98C (F1)=(O.D:0.56, 0.152) D5 (F2)=(O.D:0.51, 0.224) <Kevin wrote> 11/4/2008 (20:00) Fermentation 10:00 MeOH Induction 2:00 Glycerol feeding was completed <Kevin> 11/4/2008 (21:00) Fermentation 11:00 MeOH Induction 3:00 Sampling Y98C (F1)=(O.D:0.705, 0.190) D5 (F2)=(O.D:0.661, 0.329)

Day 1 (Nov 5th Wed):

                           11/5/2008 (8:00)       Fermentation 22:00  MeOH Induction 14:00 
                                                   MeOH Consumption Started       
      <Sung-Hye>           11/5/2008 (8:10)       Reconfiguration of dO2 control for F1 and F2 (No Glycerol) 
      <Sung-Hye>           11/5/2008 (8:25)       Fermentation 22:25  MeOH Induction 14:25 
                                                   Reconfiguratoin of pH control (Base included) 
                                                   Start level for F2 
                                                   Acid: 250 ml Base: 380 ml
      <Sung-Hye>           11/5/2008 (8:30)       Fermentation 22:30  MeOH Induction 14:30 
                                                   Reconfiguration of pH control (Base included) 
                                                   Start level for F1 
                                                   Acid: 280 ml Base: 370 ml 
      <Sung-Hye>           11/5/2008 (9:08)        Add antifoam (1.5 ml) to F2 
      <Sung-Hye>           11/5/2008 (13:00)       Add antifoam (3.0 ml) to F2 
      <Kevin>              11/5/2008 (14:30)      Fermentation 28:30  MeOH Induction 20:30 
                                                   Sampling  Y98C (F1)=(O.D:12.2, 0.771)  
                                                               D5 (F2)=(O.D:13.47, 1.388, Activity= 0.0125 slope units/ul)
      <Sung-Hye and Kevin> 11/5/2008 (17:20)      Fermentation 31:20  MeOH Induction 23:20 
                                                   Refill MeOH bottle of F2 (Add 150 ml) 
      Safty cut-off for MeOH feeding for both F1 and F2  Don't know when 

Day 2 (Nov 6th Thu):

      <Sung-Hye>           11/6/2008 (8:32)        Resume MeOH sensors for F1 and F2   
      <Sung-Hye>           11/6/2008 (8:32)       Fermentation 46:32  MeOH induction 38:32 
                                                   Measure Acid and Base
                                                   F1 Acid (260 ml) Base (365 ml)
                                                   F2 Acid (140 ml) Base (380 ml)  
      <Kevin>              11/6/2008 (12:15)      Fermentation 50:15  MeOH induction 38:32 
                                                   Sampling  Y98C (F1)=(O.D:17.06, 0.789)  
                                                               D5 (F2)=(O.D:17.96, 1.441)
      <Sung-Hye and Kevin> 11/6/2008 (13:18)      Fermentation 51:18  MeOH induction 43:15   <--- Terminate F#2                              
                                                   Terminate F#2: (Acid 140 ml) (Base 370 ml) (MeOH 400 ml)
      <Sung-Hye and Kevin> 11/6/2008 (14:00)      Fermentation 52:00  MeOH induction 44:00       
                                                   Change feeding speed of MeOH pump for F1 (prop Max buttom with toothpick)
      Safty cut-off for MeOH feeding for F1       Don't know when 

Day 3 (Nov 7th Fri):

      <Sung-Hye>           11/7/2008 (8:30)        Resume MeOH pump for F1 
                           11/7/2008 (8:30)        Acid (250 ml)  Base (370 ml) MeOH (200 ml)    

    Discussion about MeOH Concentration 
 : It turned out that MeOH concentration used for BROM F1 F1/F2 was 20 times diluted. That means that MeOH calibration is off and concentration of MeOH is 20 times lower than we think. (It also explains why MeOH pumps went "safty cut-off") Sung-Hye discussed with Kevin and decided to continue current (F1) fermentation by re-calibrating MeOH with concented sample and hope that cells weren't starved too much. Kevin's microscopic observation tells that cells are still deviding (budding) which is a good sign.
      <Kevin>              11/7/2008 (15:30)       Prepare 5x MeOH for refill (1050 ml): We did not use.
                                                   Brought pure MeOH 1-L for F#1
      <Kevin and Sung-Hye> 11/7/2008 (16:00 - 17:00)  Fermentation 78:00  MeOH induction 70:00  
                                                   Re-calibrate MeOH sensor with 35 ml of pure MeOH  
                                                   (In sterilized 50-ml falcon tube, pure MeOH was poured.
                                                   MeOH tube and pump was connected to feed) 
      <Sung-Hye>           11/7/2008 (17:30)     Fermentation 78:30  MeOH induction 70:30      
                                                   Re-set Setpoint of MeOH sensor 

Day 4 (Nov 8th Sat):

      <Kevin>              11/8/2008 (14:25)     Fermentation 100:25  MeOH induction 92:25 
                                                  No contamination. No Activity was detected. 
                                                  Did not record the amount of acid/base and MeOH used.

Day 5 (Nov 9th Sun):

      <Kevin>              11/9/2008 (14:25)     Fermentation 124:25  MeOH induction 116:25  
                                                  No contamination. No Activity was detected. 
                                                  Did not record the amount of acid/base and MeOH used.

Day 6 (Nov 10th Mon):

      <Kevin>              11/10/2008 (14:25)    Fermentation 148:25  MeOH induction 140:25 
                                                  No contamination. No Activity was detected. 
                                                  Did not record the amount of acid/base and MeOH used.
                                                  Western Blotting using samples collected from different days, and the results are expected to be on Nov 11th.

Day 7 (Nov 11th Tue):

      <Kevin>              11/11/2008 (14:25)    Fermentation 172:25  MeOH induction 164:25 
                                                  No contamination. No Activity was detected. 
                                                  Did not record the amount of acid/base and MeOH used.
                                                  Western Blotting results indicate that production was not as much as expected, 
                                                  and the amount of protein decreased from Nov. 7 (Day 4).

Day 8 (Nov 12th Wed):

      <Sung-Hye>           11/12/2008 (10:38)    Fermentation 192:38  MeOH induction 184:38  <--- Terminate Fermenter #1  

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