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Guschinskaya et al. Sci Rep 2016

Random mutagenesis in Thermococcales

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Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. We have developped a novel approach to perform random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. One of the many advantages of this strategy is its simplicity. Intact gDNA can be mutagenized in vitro and transformed into the strain at high efficiency without prior selection of transposon-containing genomic DNA fragments. A small-scale transposition reaction routinely generates up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion (see figure). This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.
Keywords : random mutagenesis ; natural transformation ; Archaea ; Pyrocuccus furiosus ; mariner ; in vitro mutagenesis

Voir en ligne : Guschinskaya et al. Sci Rep 2016