Optimisation of DNA electroporation protocols for different plant-associated bacteria

Electroporation is a vital process that facilitates the use of modern recombineering and other high -throughput techniques in a wide array of microorganisms, including non -model bacteria like plant growth -promoting bacteria (PGPB). These microorganisms play a significant role in plant health by colonizing plants and promoting growth through nutrient exchange and hormonal regulation. In this study, we introduce a sequential Design of Experiments (DOE) approach to obtain highly competent cells swiftly and reliably for electroporation. Our method focuses on optimizing the three stages of the electroporation procedure-preparing competent cells, applying the electric pulse field, and recovering transformed cells-separately. We utilized a split -plot fractional design with five factors and a covariate to optimize the first step, response surface methodology (RSM) for the second step, and Plackett-Burman design for two categorical factors and one continuous factor for the final step. Following the experimental sequence with three bacterial models, we achieved efficiencies 10 to 100 times higher, reaching orders of 105 to 106 CFU/mu g of circular plasmid DNA. These results highlight the significant potential for enhancing electroporation protocols for non -model bacteria.