Abstract
Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can also cause healthcare-associated infections with severe consequences. Understanding the specific genes responsible for its virulence through genetic manipulation is crucial for potential therapeutic interventions. However, manipulating K. pneumoniae presents challenges due to its exopolysaccharide capsule. This article presents a comprehensive collection of protocols designed to facilitate the genetic manipulation of K. pneumoniae. By following these protocols, researchers will acquire the necessary skills to prepare electrocompetent cells, utilize electroporation for efficient plasmid DNA introduction, construct isogenic mutants using the λ Red recombinase system, and generate a complementation vector for restoring the phenotypic traits of knockout strains. These protocols provide valuable tools and techniques to navigate the intricacies associated with studying and modifying K. pneumoniae. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing electrocompetent K. pneumoniae cells Alternate Protocol 1: Preparing electrocompetent K. pneumoniae cells for recombineering Basic Protocol 2: Transforming K. pneumoniae using electroporation Basic Protocol 3: Constructing isogenic mutants in K. pneumoniae using the λ Red recombinase system Support Protocol 1: Confirming a knockout via colony PCR Support Protocol 2: Verifying absence of secondary mutations Basic Protocol 4: Generating unmarked knockout mutants in K. pneumoniae using the pFLP plasmid Basic Protocol 5: Constructing a complementation vector for K. pneumoniae.