User:Fermenter User/Notebook/BROM J1

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==Project Description/Abstract==
 
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* Place short description of project or notes regarding this project
 
-
* Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Donec porta commodo tellus. Nam a est eget libero mollis tincidunt. Aliquam purus. Quisque nulla ligula, facilisis in, pulvinar sed, molestie a, quam. Vestibulum at pede. In in sem eget odio eleifend placerat. Phasellus ultricies felis quis sapien. Etiam molestie volutpat quam. Praesent pulvinar scelerisque mi. Nam mi urna, fringilla eu, mattis sed, venenatis id, nunc. Maecenas velit eros, congue ut, placerat in, ornare vel, sem. Aenean porta enim sit amet felis gravida posuere. Phasellus faucibus nibh et orci.
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<font size=3>Background color codes:</font><br>
 +
<font style=background-color:#00FF00> Pastel Green: Major change of setup this time </font><br>
 +
<font style=background-color:yellow> yellow: Sung-Hye's correction or question to user </font><br><br>
 +
 
 +
<font size=3>Letter color codes:</font><br>
 +
Black: Basic comment<br>
 +
<font color=red> Red: Emergency Information (Power shutdown, building access, Manager's absence etc) </font><br>
 +
<font color=blue> Blue: Fermentation Setup information </font><br>
 +
<font color=green> Green: Discussion </font><br>
 +
<font color=orange> Orange: Fermentation hrs </font><br>
 +
<font color=purple> Purple: Induction hrs </font><br><br>
 +
 
 +
 
 +
<font size=5>'''BROM H1'''</font>
 +
 
 +
== Day -4 (Jun 3 Fri): ==
 +
 
 +
== Day -3 (Jun 4 Sat): ==
 +
 
 +
== Day -2 (Jun 5 Sun): ==
 +
      <Jason>          6/19/2011 (13:00)
 +
                              Thaw glycerol stock and grow in 5-ml MD media
 +
                              <b>Incubator in Fermentation Suite used</b>
 +
      <Jason>          6/19/2011 ()
 +
                              Preparing 5-L of Basal Medium (Defined media)
 +
                              PTM1 Salt is from BROM-A16
 +
 
 +
== Day -1 (Jun 20 Mon):<font color=red> MEDIA PREPARATION DAY </font> ==
 +
 
 +
      <Jason>            6/20/2010 (??:?? - ??:??)
 +
                                Bring powder to make 5-L basal salts
 +
 
 +
      <Sung-Hye>        6/20/2011 (14:00)    <font color=blue>Assemble bioreactors: </font> 
 +
                              1. Calibrate pH sensors                                       
 +
                              2. 5.0-L of Basal Salts
 +
                              3. 1-ml Antifoam <br>
 +
      <Sung-Hye>        6/20/2011 (14:00)  Prepare bottles
 +
                                            1) no Acid need
 +
                                            2) Base, 500-ml (tubing connected) x1
 +
                                            3) MeOH, 1-L (tubing connected) x1
 +
                                            4) Additives/Inoculum, 500-ml (tubing connected) x1
 +
                                            5) No Water, 2-L
 +
                                            6) Fed-Batch, 500-ml (tubing connected) x1<br><br>
 +
      <Sung-Hye>        6/20/2011 (15:13)  Autoclave
 +
 
 +
      <Jason>            6/20/2011 (15:00)
 +
                                1.Transfer cells (5ml MD media) to 50ml BMGY media.
 +
                                  <b>Incubator in Fermentation Suite used</b>
 +
                                2.Prepare sterilized Methanol by filtration
 +
                                3.Prepare sterilized NaOH
 +
 
 +
== Day 0 (Jun 21 Tue): <font color=red> INOCULATION DAY </font>==
 +
 
 +
      <Jason>            6/21/2011 (10:30)  Check contamination of starter culture
 +
                                            Flask OD: 32.10
 +
 
 +
      <Sung-Hye>        6/21/2011 (11:00)  <font color=blue>Set temperature to 28.5°C </font>
 +
      <Sung-Hye>        6/21/2011 (11:00)  <font color=blue>Purge Air 1L/min at 900 rpm </font>
 +
      <Jason>            6/21/2011 (11:10)  Add 21.75 ml of PTM1 Salt/5-L media <-- Prepared at BROM A16
 +
                                            Re-filtered by Jason
 +
                                            (4.35 ml PTM1 salt per 1-L media = 21.75 ml in 5-L))
 +
      <Jason>            6/21/2011 (11:30)  <font color=blue>Adjust pH to ~3.0 (with ~100 ml NH4OH)</font>
 +
      <Jason>            6/21/2011 (11:40)  <font color=blue>Adjust pH to 5.0 with **2M NaOH** <-- 4M is better!
 +
                                            (borrowed from Bromme lab, 500 ml)
 +
                                            150 ml used</font>
 +
      <Sung-Hye>        6/21/2011 (11:45)  <font color=blue>Start pH control </font>
 +
                                            F1: No Acid Base, 2M (350 ml)<-- 4M is better!
 +
      <Sung-Hye>        6/21/2011 (11:45)  <font color=blue>Calibrate dO2 probes </font>
 +
                          6/21/2011 (11:45)  <font color=blue>Strt dO2 control </font>
 +
      <Sung-Hye>        6/21/2011 (11:46)  <font color=blue>Start Fermenter #1 software </font>
 +
      <Jason&Sung-Hye>  6/21/2011 (12:00)  <font color=orange>Fermentation 0:00 </font>
 +
                                            Inoculate 55 ml/bioreactor
 +
                                            Cells were transferred using pumps.
 +
 
 +
      <Sung-Hye>        6/21/2011 (15:00)  <font color=blue> Change dO configuration </font>
 +
 
 +
== Day 1 (Jun 22 Wed): ==
 +
 
 +
      <Jason>          6/22/2011 (13:20)    <font color=orange> Fermentation 25:20 </font>
 +
                            F1:  Base(315 ml) OD(4.70) Contamination(N) Pressure(N) Change Filter(N)
 +
 
 +
== Day 2 (Jun 23 Thu): <font color=#FF00FF> Fed-Batch and MeOH Induction Day 1 </font>== 
 +
      <Jason>            6/23/2011 (9:00)    <font color=orange> Fermentation 45:00 </font>
 +
                            F2: Base(30 ml) OD(31.75) Contamination(n) Pressure(n) Change Filter(n)
 +
      <Sung-Hye>        6/23/2011 (9:40 - 12:40) <font color=orange> Fermentation 44:40 - 47:40</font>
 +
                            F2: Fed-Batch
 +
                            400 ml 50% Glycerol + 4.8 ml PTM1 at 90.75ml/hr/5-L (RPM 65 w/ 2RPM pump)
 +
      <Sung-Hye>        6/23/2011 (11:00)
 +
                            F2: Take out 500-ml media
 +
      <Sung-Hye>        6/23/2011 (11:07)
 +
                            Manually inject ~5 ml of conc. NH4OH via septum (pH: 4.75 → 5.20)
 +
      <Jason>            6/23/2011 (11:50)
 +
                            F2: Refill Base (0 ml +500 ml = 500 ml): pH reading: 5.0
 +
      <Jason>            6/23/2011 (12:40)    <font color=orange> Fermentation 48:00 </font>
 +
                            F1: Base(480 ml) OD(44.30) Contamination(n) Pressure(n) Change Filter(n)
 +
      <Sung-Hye>        6/23/2011 (13:30)    <font color=orange> Fermentation 49:30 </font><font color=#FF00FF> MeOH induction 00:00 </font>
 +
                            Calibrate MeOH sensor 
 +
                            F2: 6231 mV = 0.5% (MeOH 750 ml)<-- Mix with PTM1
 +
                            Start MeOH induction
 +
      <Sung-Hye>        6/23/2011 (17:00)
 +
                            F2: Take out 500-ml media
 +
      <Jason>            6/23/2011 (17:40)
 +
                            F2: Replace Base (2M, 350 ml) → Base (4M, 500 ml)
 +
 
 +
== Day 3 (Jun 24 Fri): <font color=#FF00FF> MeOH Induction Day 2 </font>==
 +
      <Jason>          6/24/2011 (13:30)    <font color=orange> Fermentation 73:30 </font><font color=#FF00FF> MeOH induction 24:00 </font>
 +
                            F2: Base(390 ml) MeOH( 480 ml) OD(58.70) Contamination(N) Pressure(N) Change Filter(N)
 +
                                Activity assay (3.12 units/100 ul), (0.73 mg/5-L)
 +
      <Sung-Hye>        6/24/2011 (14:45)
 +
                            F2: Take out 500-ml media
 +
      <Jason>          6/24/2011 (16:30)
 +
                            F2: Refill MeOH (450 ml +500 ml = 950 ml)
 +
 
 +
== Day 4 (Jun 25 Sat): <font color=#FF00FF> MeOH Induction Day 3 </font>==
 +
      <Jason>          6/25/2011 (13:30)    <font color=orange> Fermentation 97:30 </font><font color=#FF00FF> MeOH induction 48:00 </font>
 +
                            F2: Base(380 ml) MeOH(650 ml) OD(65.05) Contamination(N) Pressure(N) Change Filter(N)
 +
                                Activity assay (18.72 units/100ul), (4.38 mg/5-L)
 +
 
 +
== Day 5 (Jun 26 Sun): <font color=#FF00FF> MeOH Induction Day 4 </font> == 
 +
      <Jason>            6/26/2011 (13:30)    <font color=orange> Fermentation 121:30 </font><font color=#FF00FF> MeOH induction 72:00 </font>
 +
                            F2: Base(370 ml) MeOH(350 ml) OD(68.10) Contamination(N) Pressure(N) Change Filter(N)
 +
                                Activity assay (15.08 units/100ul), (3.529 mg/5-L)
 +
                            F2: Refill MeOH (350 ml +650 ml = 1000 ml)
 +
 
 +
== Day 6 (Jun 27 Mon): <font color=#FF00FF> MeOH Induction Day 5</font> ==
 +
      <Jason>          6/27/2011 (12:00)    <font color=orange> Fermentation 144:00 </font><font color=#FF00FF> MeOH induction 94:30 </font>
 +
 +
                            F1: Base(370 ml) MeOH(800 ml) OD(70.05) Contamination(N) Pressure(Yes) Change Filter(Yes)
 +
                                Activity assay (18.87 units/100ul), (4.41 mg/5-L)
 +
      <Sung-Hye>        HARVEST
 +
 
 +
== Suggestion for next trials: ==
 +
1.  Add PTM1 salt into MeOH (12 ml/L of MeOH)
 +
2.  Even add Glycerol together with MeOH
 +
3.  Extend fed-batch hours (from 3 hrs to overnight) before start MeOH induction
 +
4.  Measure samples twice a day
 +
 
 +
== References ==
 +
<font color=green>   
 +
    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]]
 +
    </font>
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==Notes==
 
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* Place some notes here for visitors
 
-
* Example: This project is currently on hold until further notice.
 
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__NOTOC__
__NOTOC__

Revision as of 11:23, 8 July 2011

Customize your entry pages

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


BROM H1

Day -4 (Jun 3 Fri):

Day -3 (Jun 4 Sat):

Day -2 (Jun 5 Sun):

      <Jason>           6/19/2011 (13:00)
                              Thaw glycerol stock and grow in 5-ml MD media
                              Incubator in Fermentation Suite used
      <Jason>           6/19/2011 ()
                              Preparing 5-L of Basal Medium (Defined media)
                              PTM1 Salt is from BROM-A16

Day -1 (Jun 20 Mon): MEDIA PREPARATION DAY

      <Jason>            6/20/2010 (??:?? - ??:??)
                               Bring powder to make 5-L basal salts
      <Sung-Hye>         6/20/2011 (14:00)    Assemble bioreactors:   
                              1. Calibrate pH sensors                                         
                              2. 5.0-L of Basal Salts
                              3. 1-ml Antifoam 
<Sung-Hye> 6/20/2011 (14:00) Prepare bottles 1) no Acid need 2) Base, 500-ml (tubing connected) x1 3) MeOH, 1-L (tubing connected) x1 4) Additives/Inoculum, 500-ml (tubing connected) x1 5) No Water, 2-L 6) Fed-Batch, 500-ml (tubing connected) x1

<Sung-Hye> 6/20/2011 (15:13) Autoclave
      <Jason>            6/20/2011 (15:00)
                               1.Transfer cells (5ml MD media) to 50ml BMGY media.
                                 Incubator in Fermentation Suite used
                               2.Prepare sterilized Methanol by filtration
                               3.Prepare sterilized NaOH

Day 0 (Jun 21 Tue): INOCULATION DAY

      <Jason>            6/21/2011 (10:30)  Check contamination of starter culture 
                                            Flask OD: 32.10
      <Sung-Hye>         6/21/2011 (11:00)  Set temperature to 28.5°C 
      <Sung-Hye>         6/21/2011 (11:00)  Purge Air 1L/min at 900 rpm 
      <Jason>            6/21/2011 (11:10)  Add 21.75 ml of PTM1 Salt/5-L media <-- Prepared at BROM A16
                                            Re-filtered by Jason
                                            (4.35 ml PTM1 salt per 1-L media = 21.75 ml in 5-L))
      <Jason>            6/21/2011 (11:30)  Adjust pH to ~3.0 (with ~100 ml NH4OH)
      <Jason>            6/21/2011 (11:40)  Adjust pH to 5.0 with **2M NaOH** <-- 4M is better!
                                            (borrowed from Bromme lab, 500 ml)
                                            150 ml used
      <Sung-Hye>         6/21/2011 (11:45)  Start pH control 
                                            F1: No Acid Base, 2M (350 ml)<-- 4M is better!
      <Sung-Hye>         6/21/2011 (11:45)  Calibrate dO2 probes 
                         6/21/2011 (11:45)  Strt dO2 control 
      <Sung-Hye>         6/21/2011 (11:46)  Start Fermenter #1 software 
      <Jason&Sung-Hye>   6/21/2011 (12:00)  Fermentation 0:00 
                                            Inoculate 55 ml/bioreactor
                                            Cells were transferred using pumps.
      <Sung-Hye>         6/21/2011 (15:00)   Change dO configuration 

Day 1 (Jun 22 Wed):

      <Jason>           6/22/2011 (13:20)      Fermentation 25:20  
                            F1:   Base(315 ml) OD(4.70) Contamination(N) Pressure(N) Change Filter(N)

Day 2 (Jun 23 Thu): Fed-Batch and MeOH Induction Day 1

      <Jason>            6/23/2011 (9:00)      Fermentation 45:00  
                            F2: Base(30 ml) OD(31.75) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         6/23/2011 (9:40 - 12:40)  Fermentation 44:40 - 47:40 
                            F2: Fed-Batch
                            400 ml 50% Glycerol + 4.8 ml PTM1 at 90.75ml/hr/5-L (RPM 65 w/ 2RPM pump)
      <Sung-Hye>         6/23/2011 (11:00)
                            F2: Take out 500-ml media
      <Sung-Hye>         6/23/2011 (11:07) 
                            Manually inject ~5 ml of conc. NH4OH via septum (pH: 4.75 → 5.20)
      <Jason>            6/23/2011 (11:50)
                            F2: Refill Base (0 ml +500 ml = 500 ml): pH reading: 5.0
      <Jason>            6/23/2011 (12:40)      Fermentation 48:00  
                            F1: Base(480 ml) OD(44.30) Contamination(n) Pressure(n) Change Filter(n)
      <Sung-Hye>         6/23/2011 (13:30)      Fermentation 49:30  MeOH induction 00:00 
                            Calibrate MeOH sensor  
                            F2: 6231 mV = 0.5% (MeOH 750 ml)<-- Mix with PTM1
                            Start MeOH induction
      <Sung-Hye>         6/23/2011 (17:00)
                            F2: Take out 500-ml media
      <Jason>            6/23/2011 (17:40)
                            F2: Replace Base (2M, 350 ml) → Base (4M, 500 ml)

Day 3 (Jun 24 Fri): MeOH Induction Day 2

      <Jason>           6/24/2011 (13:30)      Fermentation 73:30  MeOH induction 24:00  
                            F2: Base(390 ml) MeOH( 480 ml) OD(58.70) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (3.12 units/100 ul), (0.73 mg/5-L)
      <Sung-Hye>        6/24/2011 (14:45)
                            F2: Take out 500-ml media
      <Jason>           6/24/2011 (16:30)
                            F2: Refill MeOH (450 ml +500 ml = 950 ml)

Day 4 (Jun 25 Sat): MeOH Induction Day 3

      <Jason>           6/25/2011 (13:30)      Fermentation 97:30  MeOH induction 48:00  
                            F2: Base(380 ml) MeOH(650 ml) OD(65.05) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (18.72 units/100ul), (4.38 mg/5-L)

Day 5 (Jun 26 Sun): MeOH Induction Day 4

      <Jason>            6/26/2011 (13:30)      Fermentation 121:30  MeOH induction 72:00 
                            F2: Base(370 ml) MeOH(350 ml) OD(68.10) Contamination(N) Pressure(N) Change Filter(N)
                                Activity assay (15.08 units/100ul), (3.529 mg/5-L)
                            F2: Refill MeOH (350 ml +650 ml = 1000 ml)

Day 6 (Jun 27 Mon): MeOH Induction Day 5

      <Jason>           6/27/2011 (12:00)      Fermentation 144:00  MeOH induction 94:30 

                            F1: Base(370 ml) MeOH(800 ml) OD(70.05) Contamination(N) Pressure(Yes) Change Filter(Yes)
                                Activity assay (18.87 units/100ul), (4.41 mg/5-L)
      <Sung-Hye>         HARVEST

Suggestion for next trials:

1.  Add PTM1 salt into MeOH (12 ml/L of MeOH)
2.  Even add Glycerol together with MeOH
3.  Extend fed-batch hours (from 3 hrs to overnight) before start MeOH induction
4.  Measure samples twice a day

References

     
    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
   



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