Fig. 2: Design and characterization of C:G to T:A type single mutators. | Nature Communications

Fig. 2: Design and characterization of C:G to T:A type single mutators.

From: An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo

Fig. 2

a Construction of MmP1-based C:G to T:A type mutators by fusing cytosine deaminases to MmP1 RNAP via the XTEN linker. The sfGFP was used as the reporter to determine the transcriptional activity of C:G to T:A type single mutators. Erythromycin-resistant mutant ErmC Y104S was used to assess on-target mutation rate, as C:G to T:A correction at position 104 restored the antibiotic function (S104 TCT to F104 TTT). b Transcriptional activity of C:G to T:A type single mutators tested using flow cytometry after 12 h with 200 mg/L IPTG. c–e Analysis of the on-target mutation rate (erythromycin resistance frequency, EM resistance frequency), off-target rate (rifampicin resistance frequency, RIF resistance frequency), and cell viability (colony forming CFU/mL) of C:G to T:A type single mutators. f–h Analysis of the on-target mutation rate, off-target rate, and cell viability of pMT2-MmP1 (PmCDA1-UGI-MmP1) at different inducer concentrations. Data are presented as mean values (bars), standard errors (error bars), and individual values (black dots). n = 3, which represents three independent replicates of the experiment. 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.

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