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From 2010.igem.org

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