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Targeted Chromosomal Knockouts in Mycoplasma pneumoniae▿ †

By Radha Krishnakumar, Nacyra Assad-Garcia, Gwynedd A. Benders, Quang Phan, Michael G. Montague and John I. Glass


Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame

Topics: Methods
Publisher: American Society for Microbiology (ASM)
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Provided by: PubMed Central
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