Dear Zahra Sabouri

I worked with E. coli and Agro, and always using "Electroporation mediated transformation" showed higher efficiency compared to heat shock methods, which needs to prepare competent cells by 10% glycerol.

Between heat shock, "Inoue" method for preparation competent cells played more efficient to me.

You could find Inoue method below:

http://cshprotocols.cshlp.org/content/2020/6/pdb.prot101196.abstract

https://bio-protocol.org/pdf/bio-protocol143.pdf

Dont forgot that using SOB and SOC media instead of common LB would help to increase the rate of regenerating transformed cells.

Dear Zahra,

so far we used classical Spizizen's protocol which is based on development of natural competence of Bacillus subtilis. This protocol is easy and chip, while has low efficiency, but if you have enough pDNA - you will get transformants.

Alternatively, try protoplasts transformation, see https://link.springer.com/content/pdf/10.1007/BF00267940.pdf

While this protocol is efficient, it requires a lot of manipolations. As well, keep in mind that transformants can grow on 2nd-4th day of cultivation.

The electroporation works badly on Bacilli, as we learned, it requires extra-pure DNA and high voltage.