Team:Groningen/Notebook/Peter

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Bascillus Subtilus, easy info:  
Bascillus Subtilus, easy info:  
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<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>
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<br>
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09/08
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<br>
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Cell wall isolation according to protocol: Cell disruption, samples are frozen.
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<br>
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Culture Bascillus over night, all four strains: Rok, DegU, SigF, Sp0A
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<br>
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10/08
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<br>
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Cell wall isolation according to protocol: Cell disruption again, continue according to protocol
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<br>
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Dry-freeze over night
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<br>
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 +
Culture Bascillus over a extra night, all four strains: Rok, DegU, SigF, Sp0A
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<br>
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 +
11/08
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<br>
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Chaplin isolation:
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<br>
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4x approximately 15 mg dry cell wall (180 ul TFA), <br>
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4x approximately 10 mg dry cell wall (100 ul TFA), <br>
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8x approximately 5 mg dry cell wall (100 ul TFA), <br>
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<br>
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Cultured bascillus strains are taken out of the stove, medium will be used for degredation experiment.
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<br>
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Pellicle: ROK + SigF
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<br>
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DEGREDATION EXPERIMENT:
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<br>
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3x appr. 5 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium) <br>
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3x appr. 5 mg samples + ROK, DegU, SigF lysed medium (+ 1 control lysis normal medium) <br>
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3x appr. 15 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium) <br>
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3x appr. 10 mg samples + ROK, DegU, SigF medium (+ 1control normal medium) <br>
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 +
normal medium is with 2% glucose for lysis
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<br>
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normal medium is TY for the rest
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<br>
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12.45: lysis at 37 degrees for 1 hour, 20 mg/ml lysozyme (control also)
 +
 +
<br>
 +
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13.45: incubation with bacillus and control media
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<br>
 +
 +
12-08-2010
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<br>
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 +
Hydrophobin samples were prepared for sds-gel at 14.00
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<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
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<br>
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25 uL of sample was loaded on the SDS gel in the following order:
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<br>
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on gel 1:
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<br>
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5ulmarker-15ctrl-15sigf-15degu-15rok-rokl-sigfl-degu-l-ctrll-10ulmarker
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<br>
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on gel 2:
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<br>
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5ulmarker-10ctrl-10sigf-10degu-10rok-5ctrl-5sigf-5degu-5rok-10ulmarker
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<br>
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The 1 mm thick gel 1 was prepared using a 0,75 mm comb: Might be bad
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<br>
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Gel was runned on 60 v for 15 min
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<br>
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Then gel was runned on 120 v
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<br>
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Expression experiments:
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<br>
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Numero dos
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<br>
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1 growing: Thursday 26/08/2010
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<br>
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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

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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