Generating HducCdtB Protein Mutants

HducCdtB mutations were generated with a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) using the pRSF-DUET1 HducCdtB WT plasmid as a template [34 (link)] and the primers (Sigma) listed in Table S1. All HducCdtB sequences were subcloned in the mammalian expression vector pmCherry-C1, in frame with mCherry fluorescent protein, using XhoI and EcoRI restriction enzymes (New England Biolabs). The HducCdtB sequence was amplified with Q5 High-Fidelity DNA polymerase (New England Biolabs) and pmCherry subcloning primers presented in Table S1. PCR products were purified with the GFX PCR DNA and gel band kit (Illustra-GE Healthcare, Chicago, IL, USA), and ligation was performed with Instant Sticky-end Ligase Master Mix (New England Biolabs). For several pmCherry-C1 HducCdtB constructions, the chromatibody (Cb) sequence was cloned in frame with the CdtB-mCherry. The chromatibody came from pmCherry-C1 chromatibody-RNF8 [57 (link)], and the cloning was performed with EcoRI and AgeI restriction enzymes (New England Biolabs). All plasmids were purified with EZ-10 Spin Column plasmid DNA Minipreps Kit (BioBasic, Markham, ON, Canada), and all constructs were analyzed by DNA sequencing (Eurofins Scientific, Luxembourg).