Fig. 3

Multi-round multiplex genome editing with the assistance of clmPheS. (A) Colony PCR screening of deletion mutants yielded in the first round of multiplex genome editing simultaneously targeting the recFOR genes. Predicted sizes of PCR products were indicated. -, PCR amplification using chromosomal DNA of Z. mobilis ZM4 as a DNA template; M, DNA size marker. (B) Percentages of the correct colonies of single, double, and triple deletions verified in (A). (C) Examination of the counterselection effect of 4-CP on curing of the genome editing plasmid. (D) Colony PCR screening of deletion mutants yielded in the second round of multiplex genome editing simultaneously targeting the recJQ₁Q₂ genes. Predicted sizes of PCR products were indicated. -, PCR amplification using chromosomal DNA of Z. mobilis ZM4 as a DNA template; M, DNA size marker. (E) Percentages of the correct colonies of single and double deletions verified in (D)