Fig. 5: Design and characterization of C:G to T:A and A:T to G:C dual type mutators.
a Design of three types of dual mutators by fusing PmCDA1-UGI and TadA8e to MmP1 RNAP in different orders or co-expressing PmCDA1-UGI-MmP1 and TadA8e-MmP1 (opt) within the same plasmid. The term “opt” refers to codon optimization. b The mutation frequencies of C:G to T:A and A:T to G:C types for three dual mutators were assessed by erythromycin resistance frequency (n = 3 independent experiments). c Construction of a sacB-based single PMmP1 promoter selection method in the genomic locus G7 of H. bluephagenesis TD01 (TD01-G7-sacB-PMmP1). The expression of the sacB gene was placed under the PPorin42 constitutive promoter, with a PMmP1 promoter positioned downstream in the reverse direction. d–f Mutation frequency (n = 4 independent experiments), mutation types, and the average number of mutations (n = 8 independent experiments) in sacB were determined using the selection method described in (c). g Distribution of mutations on sacB gene using selection method described in (c) (n = 8 independent experiments). h Construction a of sacB-based dual pMmP1 promoters selection method at the genomic locus G7 of H. bluephagenesis TD01 (TD01-G7-PMmP1-sacB-PMmP1). The expression of the sacB gene was placed under the PPorin42 constitutive promoter, with two PMmP1 promoters placed both upstream and downstream of the sacB gene. i–k Mutation frequency (n = 3 independent experiments), mutation type, and average number of mutations (n = 8 independent experiments) in sacB were determined using the selection method described in (h). l Distribution of mutations on sacB gene using selection method described in (h) (n = 8 independent experiments). Data are presented as mean values (bars), standard errors (error bars), and individual values (black or blue dots). Statistical analyses were conducted using two-tailed Student’s t-tests. A p-value < 0.05 was considered significant. Source data are provided as a Source Data file.
