Targeted chromosomal knockouts in Mycoplasma pneumoniae.
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.
Krishnakumar
Radha
R
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. rkrishna@jcvi.org
Assad-Garcia
Nacyra
N
Benders
Gwynedd A
GA
Phan
Quang
Q
Montague
Michael G
MG
Glass
John I
JI
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D055786:Gene Knockout Techniques / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008969:Molecular Sequence Data; D009177:Mycoplasma pneumoniae / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial