Transformation protocol for Bacillus subtilis needed - (Dec/13/2007 )
I am new to the field of Expression in Bacillus subtilis. And I hope this is the correct place to address my question Wink
At the moment I am struggling with the transformation of B.subtilis with my plasmid (pHT based). I wanted to use the method of Anagnostopoulos and Spizizen:
glucose, 5 μg/ml DL-tryptophane, 5 μg/ml uracil,
0.01% casein hydrolysate, 0.1% yeast extract, 1 mM MgS04,
2.5 mM MgCl2, 0.5 mM CaCl2) are inoculated with 1 ml of an 5 ml
overnight culture grown in HS medium (Spizizen’s medium*
supplemented with 0.5 % glucose, 50 μg/ml DL-tryptophane,
50 μg/ml uracil, 0.02% casein hydrolysate, 0.1% yeast extract, 8
μg/ml arginine, 0.4 μg/ml histidine, 1 mM MgSO4) at 37°C, shaking
slowly at 30°C for 3 to 4 hours.
2. Incubate 1 ml of this HS culture (late log/early stationary phase; OD578)
with 10 μl of 0.1 M EGTA at room temperature for 5 minutes and add 1
to 2 μg plasmid DNA.
3. After shaking at 37°C for 2 hours for development of antibiotic resistance,
the cells are plated on selective plates.
*Spizizen's medium: 2 g (NH4)2SO4, 14 g K2HPO4, 6 g KH2PO4, 1 g sodium citrate;
add 100 ml distilled water, autoclave, then add 0.1 ml 1 M MgSO4.
But Bacillus doesn´t even grow in Spizizen medium for inoculation of the ONC... Sad
Does anybody have other protocols for Bacillus transformation which are easy to use?
I would like to avoid protoblast generation and electroporation if possible.
Hi,
I spent the majority of my frst year trying to transform bacillus subtilis (TEB1030), its definitely not as easy as it seems in the protocols. One thing that was quite strange was that bacillus would not grow on a particular type of powedered agar (fsher)... if you are using powdered agar try switching to the granulated type.
Another problem we had was that we would get false positives on the selective plates, and we read ("Protein production by auto-induction in high density shaking cultures." Protein Expr Purif 41(1): 207-34) that phosphate can effect the efficacy of kanamycin so we started to up the concentration from 30 to 60 and sometimes 120 ug/ul. If you aren't doing this already its a good idea to get into the habbit of doing a blank transformation (say with water) and see how many colonies this give on these plates.
I was using the pBSmul-2 plasmid, which may have added to my problems as its quite big (size of plasmid can be a factor) and more importantly there is only one paper on it (and no citations) so its usefullness is questionable. I would say its important to use a series of plasmids that have been proven to work time and time again with a variety of inserts.
Anyway... methods.
The most successful method we used was the method of Botts and Wilson (Development of competence in the Bacillus subtilis transformation system)...
... and when making your media... autoclave your glucose seperately from everything else, find out which amino acids you need to add as these are strain specific.
hope this helps,
any questions then please ask
James.
At the moment I am struggling with the transformation of B.subtilis with my plasmid (pHT based). I wanted to use the method of Anagnostopoulos and Spizizen:
glucose, 5 μg/ml DL-tryptophane, 5 μg/ml uracil,
0.01% casein hydrolysate, 0.1% yeast extract, 1 mM MgS04,
2.5 mM MgCl2, 0.5 mM CaCl2) are inoculated with 1 ml of an 5 ml
overnight culture grown in HS medium (Spizizen’s medium*
supplemented with 0.5 % glucose, 50 μg/ml DL-tryptophane,
50 μg/ml uracil, 0.02% casein hydrolysate, 0.1% yeast extract, 8
μg/ml arginine, 0.4 μg/ml histidine, 1 mM MgSO4) at 37°C, shaking
slowly at 30°C for 3 to 4 hours.
2. Incubate 1 ml of this HS culture (late log/early stationary phase; OD578)
with 10 μl of 0.1 M EGTA at room temperature for 5 minutes and add 1
to 2 μg plasmid DNA.
3. After shaking at 37°C for 2 hours for development of antibiotic resistance,
the cells are plated on selective plates.
*Spizizen's medium: 2 g (NH4)2SO4, 14 g K2HPO4, 6 g KH2PO4, 1 g sodium citrate;
add 100 ml distilled water, autoclave, then add 0.1 ml 1 M MgSO4.
But Bacillus doesn´t even grow in Spizizen medium for inoculation of the ONC... Sad
Does anybody have other protocols for Bacillus transformation which are easy to use?
I would like to avoid protoblast generation and electroporation if possible.