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Rapid and Robust RCA-based Approach for Bacillus DNA Transformation

          Bacillus DNA transformation still represents a bottleneck in Bacillus gene cloning, expression and library construction. Current methods introducing foreign DNA into Bacillus strains, e.g. phage transduction, electroporation, protoplast transformation and protoplast electroporation, are generally laborious and less efficient. Recently, the ComK competent Bacillus cells, combined with PCR-generated foreign DNA multimer, significantly improves Bacillus DNA transformation efficiency. The comK overexpression in Bacillus, however, can disturb the expression pattern of the entire host genome. Furthermore, the PCR-based DNA multimer generation needs special PCR primers for certain DNA fragments, along with all concern of PCR technology, such as template DNA GC content, secondary structure and repeat sequence etc.

          We developed PicoPhi Rolling Circle Amplification (RCA)-based approach to produce DNA multimers. The resulting DNA multimer contains hundreds of tandem repeats, offerring the highest transformation efficiency for either comK or natural Bacillus competent cells. Whole approach including the cell-free cloning and subsequent DNA multimer production, was tailored in a streamlined, one-tube workflow, and can be finished in one day. Any circle template DNA (as less as 1 ng), such as the in vitro assembled or ligated circular DNA, plasmid, or the extended linear DNA (genomic DNA), with special or short random DNA or RNA primers, can be efficiently amplified into DNA multimer within hours, regardless of all concern of PCR technology.


          PicoPhi RCA-based approach represents the best method for Bacillus DNA transformation. Below is ​workflow of PicoPhi RCA-based Bacillus DNA transformation. The whole procedure, from DNA fragments to transformation,  can be finished in one-tube workflow and on-day fashion. 

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