TraDIS-Xpress: Assaying Essential Genes

Although the original TraDIS protocol has proved to be hugely powerful in assaying the role of the majority of genes of any given bacteria in surviving any given stress, essential genes cannot be assayed owing to the lethality of any insertions within them. To allow essential genes to be assayed for roles in any given stress, we included the use of an inducible, outward facing promoter into the transposon cassette. This allows controlled induction of transcription from the transposon insertion site: Inserts immediately upstream of or downstream from an essential gene will either up- or down-regulate the gene. Changes in the abundance of these inserts can therefore be assayed by our new TraDIS-Xpress protocol. To enable this approach, we included an outward-oriented IPTG-inducible tac promoter at the end of the kanamycin cassette, which allows controlled induction of expression with IPTG in E. coli (for controlled expression from the tac promoter in the transposon cassette as assayed using a beta-galactosidase assay, see Supplemental Fig. S6). We used E. coli strain BW25113 as a model organism for these experiments as E. coli has been well studied for triclosan mechanisms of action and resistance, is compatible with the use of the tac promoter for conditional changes in gene expression, and is the parent strain for the defined Keio-mutant library, allowing access to independent inactivation mutants to validate TraDIS predictions. Supplemental Table 3 shows all strains and vectors used in this study.