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. 2018 Dec 5;9:3024. doi: 10.3389/fmicb.2018.03024

FIGURE 2.

FIGURE 2

Scheme for one gene deletion using the single-plasmid system in L. casei BL23. (A) Diagram showing in-frame hicD3 deletion and cat marker excision. Nisin induced LCABL_13040-50-60 mediated allelic replacement resulted in disruption of the hicD3 gene and insertion of the cat marker, while lactose induced Cre subsequently excised the marker. (B) Two single colonies were randomly picked and verified by PCR with the forward primer of the up homology (uF) and the reverse primer of the down homology (dR). The PCR fragment of the strain in which the cat gene has replaced the hicD3 gene was about 3.0 kb. (C) Ten colonies were randomly selected to tested by PCR with the forward primer of the up homology (uF) and the reverse primer of the down homology (dR) after Cre recombinase induction and incubation. The expected PCR fragment from the excision of the cat fragment was about 2.0 kb while the wild-type PCR fragment was about 3.0 kb.