Fig. 4

Native pZM33 plasmid editing with the assistance of clmPheS. (A) Schematic showing design of the deletion of the dctA gene located on the pZM33 native plasmid of Z. mobilis ZM4. The pPE-dctA plasmid harbors a MS block consisting of the chloramphenicol resistance gene, an artificial CRISPR with a spacer targeting a sequence in the dctA gene, and two arms (L-arm and R-arm) for homologous recombination. While transformants with the integration of the MS block into pZM33 are selected on chloramphenicol; the expected deletant is selected on 4-CP upon recombination between two L-arms, resulting in a sequence junction of the L- and R-arms. P₀₀₉, promoter of the ZMOp33x009 gene; cmr, chloramphenicol resistance gene. (B) PCR screening of dctA::MS recombinants using the primer set of Fwd-009 and Rev-011 indicated in (A). PCR products amplified from the transformants carrying the pZM33 plasmid with or without the integration of the MS block are indicated as int. and wt, respectively. -, PCR amplification using the total DNA of Z. mobilis ZM4 as a DNA template. M, DNA size marker. (C) PCR amplification verifying the ΔdctA mutant. The predicted sizes of PCR products in the dctA::MS recombinant (+) and ΔdctA mutant (D) are indicated with arrows. M, DNA size marker. (D) Representative chromatograph of Sanger sequencing result. The junction of the sequences immediately upstream and downstream of the dctA gene corresponding to the L- and R-arms pictured in (A) is showcased. (E) Statistical analysis of expression levels of the native plasmids-borne genes based on 3 RNA-Seq transcriptomic data. Gene expression levels and the corresponding gene counts are indicated. The expression value of ZMOp33x009 and the percentage and counts of genes with expression levels below this value are shown at the bottom