Journal: bioRxiv

Article Title: Introduction of CG methylation in E. coli induces mutagenesis at AT base pairs

doi: 10.64898/2026.01.24.701492

Figure Lengend Snippet: A) WT and Δ alkB C2523 E. coli with active and inactive M.SssI were plated on both rifampicin and LB media. The number of rifampicin-resistant mutant colonies was compared to total colonies on LB media to calculate a rifampicin-resistant mutation rate. Each individual point represents an independent biological replicate, each averaged over two technical replicates. DNA from mutant colonies was isolated and a 500bp region of rpoB amplified by PCR. PCR products were sequenced to determine mutational signatures in WT (B) and WT and Δ alkB (C) C2523 E. coli with active and inactive M.SssI. Signatures are categorised by substitution type and trinucleotide context, comprised of the 3’ and 5’ bases adjacent to the mutated base. This gives 96 possible signatures. The percentage of unique signature was multiplied by average mutation rate for each strain to give a qualitative contribution to overall mutation signature. Observed signatures occurring in the CpG context are indicated by black vertical arrows.

Article Snippet: PCR reactions were set up as follows: 1μl DNA template, 12.5μl 2x MasterMix (MedChemExpress, Cat No. HY-K0531), 1.25μl each of 10μM forward and reverse primers, 9μl dH 2 O.

Techniques: Mutagenesis, Isolation, Amplification