AbstractTransformation techniques used to genetically manipulate Borrelia burgdorferi, the agent of Lyme disease, play a critical role in generating mutants that facilitate analyses of the role of genes in the pathophysiology of this bacterium. A number of borrelial mutants have been successfully isolated and characterized since the first electrotransformation procedure was established 25 years ago (Samuels, 1995). This article is directed at additional considerations for transforming infectious B. burgdorferi to generate strains retaining the plasmid profile of the parental strain, enabling analysis of transformants for in vitro and in vivo phenotypes. These methods are built on previously published protocols and are intended to add steps and tips to enhance transformation efficiency and recovery of strains amenable for studies involving colonization, survival, and transmission of B. burgdorferi during the vector and vertebrate phases of infection. © 2021 Wiley Periodicals LLC.This article was corrected on 20 July 2022. See the end of the full text for details.Basic Protocol 1: Preparation of stock cultures, propagation of spirochetes, and analysis of plasmid profilesBasic Protocol 2: Preparation of plasmid and linear DNA templates for transformationBasic Protocol 3: Transformation of B. burgdorferiBasic Protocol 4: Antibiotic selection of borrelial transformantsBasic Protocol 5: Isolation of borrelial transformants in agar overlaysBasic Protocol 6: Complementation of mutant borrelial strains in cis or in trans