I am trying to purify some C. diff toxin from Bacillus megaterium, however, the protoplasts kit is super expensive (Mo Bi Tec pHIS1522 system). I have never worked with B. megaterium or B. subtilis, so I have few questions about these two bacteria.
1. Is it possible to store the B. megaterium glycerol stock at -80C for long term?
2. If 1 is not possible, is it possible to prepare protoplasts myself, and how to do it?
3. If 1 and 2 is not possible , is it possible to replace B. megaterium protoplasts with B. subtilis competent cells? How efficient it will be?
4. If 3 is possible, how different are the expression systems (B. megaterium vs B. sutilis)?
Any suggestions and thoughts are very appreciated! Thank you!
Dear Ji Zeng
Do You want prepare the full lenght toxin or a toxin domain (e.g the catalitic domain)?
Because several years ago i was in your situation, after many unduccesfull trials with e.coli i understood that i need a bacillius to express the catalitic domain of tcdA. After some preliminary trial of one my co-worker with b .megaterium we find out that propoplast step is critical and we decided to test brevibacillus chosinensis (takara) which is much more simple (trasformation can be performed by elettroporation similar to e.coli) and it worked quite well for us.
https://febs.onlinelibrary.wiley.com/doi/abs/10.1111/j.1742-4658.2012.08688.x
We were also able to use it in conbination with phis1522 and it works well.
https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-12
In this case the trasformation is little bit more complex because tetracline resistance not work very well in bravobacillus and is important optimize tetracline concentration and incubation time but we was able to obtain good colonies.
I'm not sure that with this sistem you can produce the full lenght toxin because it is really big protein and it expression could be challenging but
If you already have clones is phis1522 for b.megateriun you can buy the brevibacillus cells from takara and test it its quite simple and fast.
Good luck
Manuele
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