Fig. 2. Overview of the SLICER method. (IMAGE)

Nanjing Agricultural University The Academy of Science

Fig. 2. Overview of the SLICER method.

Caption

Step 1: Transformation of the seamless deletion (SD) cassette, containing a neomycin resistance gene (nptII) and β-galactosidase (lacZ) gene for antibiotic selection and visual screening, into D. radiodurans. Homologous recombination of the 1-kb homology 1 (H1) and homology 2 (H2) regions with the D. radiodurans genome results in integration of the SD cassette replacing the gene of interest (GOI). Step 2: Conjugation of the pSLICER plasmid into D. radiodurans where it expresses the codon-optimized I-SceI endonuclease that cuts at the 18-bp I-SceI restriction site within the SD cassette. This double-strand break prompts a second homologous recombination event between H1 and the duplicated 3′ 80 bp of H1, removing the nptII and lacZ markers. Step 3: Finally, plasmid curing to remove pSLICER results in a marker-free D. radiodurans ∆GOI strain. Created with BioRender.com.

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