Skip to main content
. 2020 Sep 24;11:563491. doi: 10.3389/fgene.2020.563491

FIGURE 3.

FIGURE 3

Construction of different gene modifications of cpxRgene. (A) Schematic diagram of the gene structure of different cpxRgene modifications. JSCpxRD51A and JSCpxRM199A mutants include the point mutation at the 51st amino acid (D → A) and at 199th amino acid (M → A) of CpxR protein, respectively; the JSCpxRD51A/M199A mutant include double-point mutation at 51st and 199th amino acids of the CpxR protein; the JSCpxRSD mutant was seamlessly deleted in the major region of the CpxR protein. The JSCpxRN–3×FLAG strain expresses N-terminal 3 × FLAG-tagged CpxR protein. (B) Primer design for constructing a mutagenic substitution fragment. The universal primers are CpxR-UF/DR and CpxR-F*/R*, and the mutation primers are CpxR-D51A-DF/UR, CpxR-M199A-DF/UR, CpxR-SD-DF/UR, and CpxR-N3F-DF/UR. The overlapping region of CpxR-D51A-DF/UR includes the point mutation at codon 51 (GAC → GCC) of CpxR gene used to create the CpxRD51A point mutation fragment. The overlapping region of CpxR-M199A-DF/UR includes the point mutation at codon 199 (ATG → GCG) of the CpxR gene used to create the CpxRM199A point mutation fragment. Both primer pairs CpxR-D51A-DF/UR and CpxR-M199A-DF/UR were used to create the CpxRD51A/M199A mutation fragment. The overlapping region of CpxR-SD-DF/UR lacks the great mass of codons (34ATG → 206CGC) used to create the CpxRSD deletion mutation fragment. The primers 3 × FLAG-F/R are two complementary oligonucleotides used to create 3 × FLAG tag fragment. The primers CpxR-N3F-DF and CpxR-N3F-DF both include the overlapping region with the 3 × FLAG tag fragment used to create the N-terminal tagging CpxR fragment. (C) PCR analysis of CpxR multiple mutants. Band sizes: WT, 1.88 kb; intermediate strain (JSCpxR:sacBKan), 4.40 kb; final strain (JSCpxRD51A), 1.88 kb; final strain (JSCpxRM199A), 1.88 kb; final strain (JSCpxRD51A/M199A), 1.88 kb; final strain (JSCpxRSD), 1.32 kb; final strain (JSCpxRN–3 ×FLAG), 1.94 kb. Molecular size markers (250 bp DNA ladder marker, Takara) are indicated. WT, wild strain.