The disciplines of Borrelia (Borreliella) burgdorferi microbiology and Lyme disease pathogenesis have come to depend on the genetic manipulation of the spirochete. Generating mutants in these recalcitrant bacteria, while not straightforward, is routinely accomplished in numerous laboratories, although there are several crucial caveats to consider. This chapter describes the design of basic molecular genetic experiments as well as the detailed methodologies to prepare and transform competent cells, select for and isolate transformants, and complement or genetically restore mutants.
Keywords: Antibiotic resistance; Borrelia burgdorferi; Complementation; Electroporation; Lyme disease; Molecular genetics; Mutagenesis; Shuttle vector; Spirochete; Transformation.