Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis.
We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement.
Horzempa
Joseph
J
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Shanks
Robert M Q
RM
Brown
Matthew J
MJ
Russo
Brian C
BC
O'Dee
Dawn M
DM
Nau
Gerard J
GJ
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D005604:Francisella tularensis / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014406:Tularemia / Q000382:microbiology
D001426:Bacterial Proteins; D003850:Deoxyribonuclease I