Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.
Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.
Liberati
Nicole T
NT
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
Urbach
Jonathan M
JM
Thurber
Tara K
TK
Wu
Gang
G
Ausubel
Frederick M
FM
D004251:DNA Transposable Elements / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D020043:Genes, Essential / Q000502:physiology*; D016680:Genome, Bacterial; D015698:Genomic Library; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics*
D004251:DNA Transposable Elements