Fig. 3. Seamless deletion of RM1-4 genes using SLICER in D. radiodurans. (IMAGE)
Caption
(A) Representative schematic of the multiplex PCR amplicons present in D. radiodurans strains: 1) wild type (WT), 2) following integration of the SD cassette at the RM locus (+SD), 3) following conjugation of pSLICER and excision of the SD cassette (+SLI), and 4) following curing of pSLICER (∆RM). Expected multiplex PCR amplicons are shown as green lines with the corresponding size in base pairs. Created with BioRender.com. (B) Spot plates of 10-fold serial dilutions of the same strains listed in (A). All plates contain X-Gal 40 μg ml−1. (C) Gel electrophoresis of multiplex PCR analysis (RM1-RM4 MPX) of a single D. radiodurans colony from each step in the creation of the 4 seamless R-M gene deletions in the order depicted in (A): WT, +SD +SLI, and following plasmid curing (∆RM1, ∆RM1-2, ∆RM1-3, and ∆RM1-4). Additional controls include the SD plasmid DNA extracted from E. coli (SD). Expected amplicon sizes are approximately 150 bp for the D. radiodurans gDNA control, 300 bp for nptII in the SD cassette, 500 bp for the R-M gene, and 650 bp for the pSLICER backbone. L, 1-kb plus ladder.
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BioDesign Research
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