The catalytic DPPY motifs of both M.BceJIII and M.EcoGIX (Supplementary Figure S1 ) were converted into catalysis-defective mutant variants by introduction of an APPA substitution mutation using reverse PCR. Amplicons were obtained using plasmid templates carrying the wild type MTase allele. Primers P4 and P5 (M.BceJIII) and P10 and P11 (M.EcoGIX) introduced the mutations. The wild type plasmid templates in the amplification reaction were destroyed by DpnI digestion of the Dam-modified (G(m6A)TC) modified backbone to enrich for mutant variants.
Plasmids from candidate colonies were purified using Monarch miniprep kits (NEB, MA) and analyzed by sequencing and restriction digestion. Correct plasmids were identified, and retransformed into strain ER2796, which is deficient in all resident MTases as well as restriction systems (41 (link)), selecting Ampr or Cmr. Plasmid preparations from this background allowed us to use the RSII Pacific Biosciences sequencing instrument for modification detection and motif deduction.
Plasmids from candidate colonies were purified using Monarch miniprep kits (NEB, MA) and analyzed by sequencing and restriction digestion. Correct plasmids were identified, and retransformed into strain ER2796, which is deficient in all resident MTases as well as restriction systems (41 (link)), selecting Ampr or Cmr. Plasmid preparations from this background allowed us to use the RSII Pacific Biosciences sequencing instrument for modification detection and motif deduction.
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