A protocol for chemical competence in phytopathogenic Ralstonia

Abstract

Here, we present a protocol for the transformation of Ralstonia solanacearum species complex (RSSC) strains by calcium chloride-induced chemical competence. We include a step-by-step protocol with media and solution recipes, quantitative results of the protocols efficacy for four of five tested RSSC strains, and an explanation of the logic underlying the protocol development. The protocol involves sequentially treating overnight cultures with a cold calcium chloride solution and a cold calcium chloride/magnesium chloride/glycerol solution, transforming through alternating heat shocks and ice incubations, and recovering cells via an outgrowth in rich medium. Data summarySupplemental File S1 is the preliminary version of this chemical competence protocol. Supplemental Figures S1 and S2 show transformation efficiency data for pUFR80 knockout plasmids, and heat shock number and temperature variation, respectively.