Profiling the intragenic toxicity determinants of the hok/Sok toxin-antitoxin system

Here, we adapted and improved our FASTBAC-Seq method originally designed in Helicobacter pylori to investigate T1TAs in the model organism Escherichia coli. Our approach combines a life and death selection with deep-sequencing to assess the killing capability of a toxin and obtain an overview of single-nucleotide substitutions suppressing the toxin expression or activity in absence of its antitoxin. As a proof of concept, we revisited the regulation of the plasmidic hok/Sok T1TA system. Overall design: Amplicon DNA sequencing of bacterial strains transformed with mutated PCR libraries of the hok/Sok toxin-antitoxin system, either a wild-type hok/Sok locus, a hok/Sok locus with a mutated Sok promoter, or a hok/Sok locus with mutated hok and Sok promoters. Five biological replicates were sequenced for each PCR libraries (15 samples in total).