Hi Abdur, does it have to be a protoplast transformation? Which organism are you working with?

Hello, see below and attached info may be useful for you.

Article Transmating: Conjugative transfer of a new broad host range ...

https://www.mobitec.com/cms/products/bio/04_vector_sys/bacillus_megaterium_t7.html

Pascal Sartor yes, its Protoplast transformation. I am working with Bacillus isolates.

Dear Abdur,

I am also trying to transform native isolates of Bacillus. It is not an easy task, I tried almost all methodologies and I was not successful.

It seems that the problem is methylation. If foreign DNA is not methylated with methylases from your native strain, then it will be degraded.

Fortunately, some scientists designed a vector containing some methylases from Bacillus amyloliquefaciens and related species. So you need to get that E. coli strain containing that plasmid and in a first instance methylate your plasmid before introducing into Bacillus.

Please have a look at the following manuscript: A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. PLoS Genet 8(9): e1002987. doi:10.1371/journal.pgen.1002987

Best wishes,

Edgardo