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. 2020 Sep 24;11:563491. doi: 10.3389/fgene.2020.563491

FIGURE 5.

FIGURE 5

Construction of different gene modifications of cpxA gene. (A) Schematic diagram of the gene structure of different cpxA gene modifications. The JSCpxAL38F mutant includes the point mutation at the 38th amino acid (L → F) of the CpxA protein. The JSCpxAΔ 92–104 mutant lacks a region including 13 amino acids encoding the periplasmic loop of the CpxA protein. The JSCpxAC–3×FLAG strain expresses the C-terminal 3 × FLAG-tagged CpxA protein. (B) Primer design for constructing the mutagenic substitution fragment. The universal primers are CpxA-UF/DR and CpxA-F*/R*, and the mutation primers are CpxA-L38F-DF/UR, CxpA-92-104-DF/UR, and CpxA-C3F-DF/UR. The overlapping region of CpxA-L38F-DF/UR includes the point mutation at codon 38 (CTG → TTT) of the CpxA gene used to create the CpxAL38F point mutation fragment. The overlapping region of CpxA-92-104-DF/UR lacks 13 codons (92GGA → 104ATC) used to create the CpxAΔ 92–104 deletion mutation fragment. The primers 3 × FLAG-F/R are two complementary oligonucleotides used to create the 3 × FLAG tag fragment. The primers CpxA-C3F-DF and CpxA-C3F-DF both include the overlapping region with the 3 × FLAG tag fragment used to create the C-terminal tagging CpxA fragment. (C) PCR analysis of CpxA multiple mutants. Band sizes: WT, 2.89 kb; intermediate strain (JSCpxA:sacBKan), 4.76 kb; final strain (JSCpxAL38F), 2.89 kb; final strain (JSCpxAΔ 92–104), 2.85 kb; final strain (JSCpxAC–3×FLAG), 2.95 kb. Molecular size markers (250 bp DNA ladder marker, Takara) are indicated. WT, wild strain.