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

FIGURE 6.

FIGURE 6

Construction of different gene modifications of acrB gene. (A) Schematic diagram of the gene structure of different acrB gene modifications. The JSacrBD408A mutant includes the point mutation at the 408th amino acid (A → D) of the acrB protein. The JSacrBSD mutant was seamlessly deleted in the major region of the acrB protein. (B) Primer design for constructing the mutagenic substitution fragment. The universal primers are acrB-UF/DR and acrB-F*/R*, and the mutation primers are acrB-M408A-DF/UR and acrB-SD-DF/UR. The overlapping region of acrB-D408A-DF/UR includes the point mutation at codon 408 (GAC → GCC) of the acrB gene used to create the acrBD408A point mutation fragment. The overlapping region of acrB-SD-DF/UR lacks a great mass of codons (1ATG → 876CTG) used to create the acrBSD deletion mutation fragment. (C) PCR analysis of acrB multiple mutants. Band sizes: WT, 4.19 kb; intermediate strain (JSacrB:sacBKan), 4.78 kb; final strain (JSacrBD408A), 2.19 kb; final strain (JSacrBSD), 1.56 kb. Molecular size markers (250 bp DNA ladder marker, Takara) are indicated. WT, wild strain.