Team:Groningen/Notebook/Peter

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Bascillus Subtilus, easy info:  
Bascillus Subtilus, easy info:  
 +
 +
<br>
http://www.mobitec.de/int/products/bio/04_vector_sys/index.php?bac_sub.html <br>
http://www.mobitec.de/int/products/bio/04_vector_sys/index.php?bac_sub.html <br>
http://subtiwiki.uni-goettingen.de/wiki/index.php/SubtiPathways <br>
http://subtiwiki.uni-goettingen.de/wiki/index.php/SubtiPathways <br>
http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page <br>
http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page <br>
 +
 +
<br>
09/08
09/08
 +
 +
<br>
Cell wall isolation according to protocol: Cell disruption, samples are frozen.
Cell wall isolation according to protocol: Cell disruption, samples are frozen.
 +
 +
<br>
Culture Bascillus over night, all four strains: Rok, DegU, SigF, Sp0A
Culture Bascillus over night, all four strains: Rok, DegU, SigF, Sp0A
 +
 +
<br>
10/08
10/08
 +
 +
<br>
Cell wall isolation according to protocol: Cell disruption again, continue according to protocol
Cell wall isolation according to protocol: Cell disruption again, continue according to protocol
 +
 +
<br>
Dry-freeze over night
Dry-freeze over night
 +
 +
<br>
Culture Bascillus over a extra night, all four strains: Rok, DegU, SigF, Sp0A
Culture Bascillus over a extra night, all four strains: Rok, DegU, SigF, Sp0A
 +
 +
<br>
11/08
11/08
 +
 +
<br>
Chaplin isolation:
Chaplin isolation:
-
4x approximately 15 mg dry cell wall (180 ul TFA),  
+
<br>
-
4x approximately 10 mg dry cell wall (100 ul TFA),  
+
 
-
8x approximately 5 mg dry cell wall (100 ul TFA),  
+
4x approximately 15 mg dry cell wall (180 ul TFA), <br>
 +
4x approximately 10 mg dry cell wall (100 ul TFA), <br>
 +
8x approximately 5 mg dry cell wall (100 ul TFA), <br>
 +
 
 +
<br>
Cultured bascillus strains are taken out of the stove, medium will be used for degredation experiment.
Cultured bascillus strains are taken out of the stove, medium will be used for degredation experiment.
 +
<br>
 +
 +
Pellicle: ROK + SigF
 +
 +
<br>
DEGREDATION EXPERIMENT:
DEGREDATION EXPERIMENT:
 +
<br>
 +
3x appr. 5 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium) <br>
 +
3x appr. 5 mg samples + ROK, DegU, SigF lysed medium (+ 1 control lysis normal medium) <br>
 +
3x appr. 15 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium) <br>
 +
3x appr. 10 mg samples + ROK, DegU, SigF medium (+ 1control normal medium) <br>
 +
 +
normal medium is with 2% glucose for lysis
 +
 +
<br>
 +
 +
normal medium is TY for the rest
 +
 +
<br>
 +
 +
12.45: lysis at 37 degrees for 1 hour, 20 mg/ml lysozyme (control also)
 +
 +
<br>
 +
 +
13.45: incubation with bacillus and control media
 +
 +
<br>
 +
 +
12-08-2010
 +
 +
<br>
 +
 +
Hydrophobin samples were prepared for sds-gel at 14.00
 +
 +
<br>
 +
 +
Lysis samples were spinned off (13000 rpm/4 min), the samples were not boiled, the samples were prepared in 05,x SDS loading buffer
 +
 +
<br>
 +
 +
25 uL of sample was loaded on the SDS gel in the following order:
 +
 +
<br>
 +
 +
on gel 1:
 +
 +
<br>
 +
 +
5ulmarker-15ctrl-15sigf-15degu-15rok-rokl-sigfl-degu-l-ctrll-10ulmarker
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 +
<br>
 +
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on gel 2:
 +
 +
<br>
 +
 +
5ulmarker-10ctrl-10sigf-10degu-10rok-5ctrl-5sigf-5degu-5rok-10ulmarker
 +
 +
<br>
 +
 +
The 1 mm thick gel 1 was prepared using a 0,75 mm comb: Might be bad
 +
 +
<br>
 +
 +
Gel was runned on 60 v for 15 min
 +
 +
<br>
 +
 +
Then gel was runned on 120 v
 +
 +
<br>
 +
 +
Expression experiments:
 +
 +
<br>
 +
 +
Numero dos
 +
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<br>
 +
 +
1 growing: Thursday 26/08/2010
-
3x appr. 5 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium)
+
<br>
-
3x appr. 5 mg samples + ROK, DegU, SigF lysed medium (+ 1 control lysis normal medium)
+
-
3x appr. 15 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium)
+
2: analasis friday 27/08 (supernatant preperation - 4: ne1t=4, 1,1t=4, tasat=2, deltat=2)) and sunday, 29/08/2010, 01/09/2010: rest supernatant
-
3x appr. 10 mg samples + ROK, DegU, SigF medium (+ 1control normal medium)
+

Latest revision as of 12:48, 1 September 2010

Bascillus Subtilus, easy info:


http://www.mobitec.de/int/products/bio/04_vector_sys/index.php?bac_sub.html
http://subtiwiki.uni-goettingen.de/wiki/index.php/SubtiPathways
http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page


09/08


Cell wall isolation according to protocol: Cell disruption, samples are frozen.


Culture Bascillus over night, all four strains: Rok, DegU, SigF, Sp0A


10/08


Cell wall isolation according to protocol: Cell disruption again, continue according to protocol


Dry-freeze over night


Culture Bascillus over a extra night, all four strains: Rok, DegU, SigF, Sp0A


11/08


Chaplin isolation:


4x approximately 15 mg dry cell wall (180 ul TFA),
4x approximately 10 mg dry cell wall (100 ul TFA),
8x approximately 5 mg dry cell wall (100 ul TFA),


Cultured bascillus strains are taken out of the stove, medium will be used for degredation experiment.


Pellicle: ROK + SigF



DEGREDATION EXPERIMENT:
3x appr. 5 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium)
3x appr. 5 mg samples + ROK, DegU, SigF lysed medium (+ 1 control lysis normal medium)
3x appr. 15 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium)
3x appr. 10 mg samples + ROK, DegU, SigF medium (+ 1control normal medium)

normal medium is with 2% glucose for lysis


normal medium is TY for the rest


12.45: lysis at 37 degrees for 1 hour, 20 mg/ml lysozyme (control also)


13.45: incubation with bacillus and control media


12-08-2010


Hydrophobin samples were prepared for sds-gel at 14.00


Lysis samples were spinned off (13000 rpm/4 min), the samples were not boiled, the samples were prepared in 05,x SDS loading buffer


25 uL of sample was loaded on the SDS gel in the following order:


on gel 1:


5ulmarker-15ctrl-15sigf-15degu-15rok-rokl-sigfl-degu-l-ctrll-10ulmarker


on gel 2:


5ulmarker-10ctrl-10sigf-10degu-10rok-5ctrl-5sigf-5degu-5rok-10ulmarker


The 1 mm thick gel 1 was prepared using a 0,75 mm comb: Might be bad


Gel was runned on 60 v for 15 min


Then gel was runned on 120 v


Expression experiments:


Numero dos


1 growing: Thursday 26/08/2010


2: analasis friday 27/08 (supernatant preperation - 4: ne1t=4, 1,1t=4, tasat=2, deltat=2)) and sunday, 29/08/2010, 01/09/2010: rest supernatant

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