Engineered Salmonella Typhimurium Strains

The nirB and pagC promoters, as well as the SspH1 and Cathepsin B (codon-optimized for expression in S. Typhimurium) sequences, were utilized in this study^{8 (link),82 (link)}. The frr promoter was obtained from YS1646 S. Typhimurium by PCR. The pGP-Tn7-Cm plasmid backbone^{11 (link)} was digested using FastDigest restriction enzymes EcoRI and KpnI (Thermo Fisher Scientific). The promoter, secretory signal, and antigen sequences were inserted using the pEASY—Uni Seamless Cloning and Assembly kit (TransGen Biotech, Beijing, China). Following the construction of novel Tn7 plasmids, DAP⁻E. coli MGN-617 was transformed to generate 3 novel conjugative donor strains. The transformed E. coli strains were then used for conjugation experiments with a YS1646 strain containing the temperature-sensitive pSTNSK plasmid which encodes the Tn7 transposase system and confers Km resistance^{11 (link)}. Donor E. coli and recipient YS1646 were resuspended in LB supplemented with Km and DAP. About 100 μL of the donor strain and 50 μL of the recipient strain were centrifuged together and then resuspended in 10 μL at 30 °C for 5 h. Mixed culture was later grown on LB Km-Cm plates at 37 °C, and YS1646 colonies that grew in the absence of DAP, but that had lost resistance to antibiotic markers present on the pSTNSK plasmid but gained Cm resistance associated with the attTn7 targeting sequence were selected. Chromosomal integration at the attTn7 was then confirmed by PCR. The temperature-sensitive pCP20 plasmid, encoding the recombinase flippase (FLP) and conferring Amp and Cm resistance, was transformed into YS1646 strains. The Cm resistance cassette, integrated at the attTn7 site, was flanked by two FRT regions. Loss of the Cm resistance cassette is mediated by FLP-FRT recombination. Transformants were selected on LB-Amp plates at 30 °C to maintain pCP20 activity and then serially passaged on LB-Amp at 30 °C as well as LB-Cm and LB at 37 °C to screen for loss of the pCP20 plasmid and antibiotic susceptibility. Loss of Cm resistance was further confirmed by PCR. Following a similar strategy as outlined above, mCherry-expressing strains were generated for confocal microscopy experiments.