After electroporation, Bacillus subtilis was cultured with Recovery Media in shaker at 100rpm, 37°C for 3 hours. The Culture became clear, is it normal?
Growth Medium: LB + 0.5M Sorbitol
ETM: 0.5M Trehalose, 0.5M Sorbitol, 0.5M Mannitol, 10% Glycerin
Recovery Media: LB + 0.5M Trehalose, 0.5M Sorbitol, 0.38M Mannitol
Electric shock conditions:2200V/mm, 200Ω，25μf.
I have zero experience with Bacillus subtilis,
but sometimes E. coli Bl21 also shows decrease in turbidity (depends on the prep).
I guess it is normal since most of the cells die anyway.
- if you plate them and get positive colonies, I wouldn't worry.
I this repeats itself, I would remake the competent cells.
could be useful (works for E. coli):
1) Younger culture for the prep (0.3-0.5 OD)
2) Minimize the time/roughness/number of manipulations during the prep: spin "on dry" tubes to discharge the leftovers of the wash, mix them by hand not vortex, check after second wash if you can stop by re-suspending in "target" volume and electroporating the 50ul sample - if the pulse time is good then split into aliquotes and freeze. This way you don't do unnecessary steps that might decrease survival of bacteria.
3) Make sure that you don't keep them on ice for too long before electroporation, mix gently, recover them in the cuvette after thorough but gentle mixing with recovery media.
4) It happens very often that "no colonies" are due to some confusion with antibiotic markers - make sure that you have the right plates/plasmid etc.
I have had terrible luck trying electroporation with B. subtiils - especially with a 3610-like strain I have. Remember that they are prone to autolysis from interruptions to the electric/pH gradient across their membrane. I have had much better luck with transformation protocols that utilize natural competence
I am afraid that your experience with electroporation of Bacillus subtilis is very common. They tend to undergo massive lysis nearly synchronously about an hour or so after the electric pulse is applied. Part of the problem is osmotic shock, and you can mitigate the problem somewhat by using growth media with high osmolarity and incorporating glycine betaine as an osmoprotectant. See: Meddeb-Mouelhi F, Dulcey C, Beauregard M. High transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant. Anal Biochem. 2012 May 15;424(2):127-9 (PMID: 22387342). I have also attached their protocol in cook book form. But no matter what you do, electroporation is always going to be several orders of magnitude less efficient with B. subtilis than with E. coli.
http://www.ncbi.nlm.nih.gov/pubmed/?term=22387342
As I have experienced with electroporation, I didn't incubate it with shaking conditions because of the fact that the cells were very sensitive because of the voltage applied during the process. When I did without shaking, I found colonies the following day when I plated it after three hours of incubation.
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