We know that everybody has wetlab problems, just like us. And we actually spend most of the summer looking to standardize the Bacillus subtilis transformation protocol. And we did it! So we are presenting you our new standard:

Escherichia coli MC1061 competent cells protocol Please click this link to see the complete protocol in our notebook protocols

Two-step Bacillus subtilis transformation procedurePlease click this link to see the complete protocol in our notebook protocols

We notice that you not only need a plasmid to transform into B. subtilis, but a plasmid that has form multimers in a RecA+ E. coli and should be flanked by AmyE 5' & 3', so that when the plasmid is transformed into Bacillus, it does not get degradated, and it makes an integration into the genome.

We transform the 97 promoter plasmid (Heavy Metal AND) to the MC1061 RecA+ E. coli cells, and then we transform them again in the PY79 Bacillus subtilis strain and we integrated them successfully to the genome with a double recombination with the AmyE sequences.

Bacillus subtilis genome integration success !!!

Please see our wetlab notebook in the clicking the following image:

Bacillus subtilis Notebook