The overarching goal of this project is to enable the postdoctoral student to investigate if attenuation of the pathogen Francisella noatunensis subsp. orientalis (Fno) through the insertional mutagenesis of a fluorescent gene markers is plausible. Specifically, we aim to produce a mutant strain of Fno by insertional mutagenesis of a gene encoding the green fluorescent protein or the m-cherry protein, resulting in inactivation of the iglD gene from a wild type Fno strain isolated at the IP Fisheries Institute laboratory (IPFNO0012018) from cultured tilapia (Oreochromis spp.) in Brazil. Experiments will be conducted in collaboration with the University of California, Davis. The iglD is located on the Francisella Pathogenicity Island (FPI) and is suspected to plays a role in the survival and intracellular proliferation of Fno. The mutation will be confirmed by PCR, gene sequencing and in-vitro fluorescence studies. The virulence of the generated mutant strain will be compared to the wild-type strain in vivo using tilapia fingerlings as model of infection. Briefly, the lethal dose 50 of the wild-type and mutant strains will be investigated using immersion challenges during a period of 30d.
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