Phusion system

To generate transposon mutants, the pSAM-Bt vector ^{42 (link)} was introduced to ATCC 43858 via conjugation with a donor E. coli S17 strain. Clones with transposon insertions were selected on BHIS agar containing gentamicin and erythromycin, used to inoculate BHIS liquid media containing antibiotics, and grown overnight in deep 96-well plates. 1:100 dilutions of culture supernatants in serum-free DMEM were used to treat semi-confluent HT-29 cell monolayers for 3 hours. Cells were fixed in 10% formalin, stained with 0.05% Crystal Violet for 5 minutes, washed with water, and screened visually. Screening PCR of transposon mutants was performed using the GoTaq system (Promega). Cloning and inverse PCR was performed using the Phusion system (ThermoFisher). For inverse PCR, genomic DNA was prepared using the Wizard Genomic DNA Extraction kit (Promega), digested with BstNI (NEB), and then treated with T4 ligase (NEB). PCR was performed on this DNA using primers that annealed in the transposon. PCR products were gel extracted and sequenced. Supplementary Table 2 describes all primers utilized for cloning and PCR-based analysis.