E coli pulser

We electroporated 1 μl of each ligation reaction into 19.5 μl of 10-Beta electrocompetent E. coli (NEB, C3020K) with a Bio-Rad E. coli Pulser (2.0 kV, 200 ohms, 25 μF) and 0.1-cm gap cuvettes (Bio-Rad Gene Pulser MicroPulser cuvette, 1652089). Then, we immediately added 980 μl of super optimal medium with catabolic repressor (SOC) medium and incubated the mixture at 37°C with 220 rpm shaking for 1 hour. Then, the electroporated bacteria were plated on LB agar plates with ampicillin at 100 μg/ml using glass beads and incubated overnight at 37°C.

The constructed plasmids (6.6 ng) were electroporated into E. coli cells with a Bio-Rad E.coli pulser set at 1.8 kV, 200 Ω and 25 µF. Plasmids from the overnight culture were extracted with a QIAprep Spin Miniprep Kit (Qiagen). To quantitate the fraction of the strand used as a template, plasmids were first linearized with ScaI, except for plasmids synthesized with X-1407/X-1394 (which were linearized with HindIII), and the linearized fragments were further digested with XhoI. The complete digestion with XhoI was confirmed by independent digestion with another restriction endonuclease, of which the recognition sequence was located opposite the XhoI recognition site in the constructed plasmid. After digestion with the restriction enzymes, plasmids were electrophoresed in an agarose gel, and the relative intensities of the ethidium bromide-stained DNA fragments were measured with a gel imaging system (FluoroChem Imager, ProteinSimple, Tokyo, Japan). The fraction of the strand used as a template was estimated based on the intensity of the uncut fragment relative to the intensity of all of the fragments (both cut and uncut fragments). Transformation was repeated three times for each plasmid construct, and the statistical significance of the differences in the fractions of uncut fragments between the plasmids was determined with the two-tailed t-test.

For protein secretion assay, S. enterica cells were grown at 30 °C until the cell density reached 1.0–1.2 at an absorbance of 600 nm. Cells were spun down and culture supernatant fractions were collected separately. Proteins were precipitated by adding trichloroacetic acid at a final concentration of 10% (v/v) and spun down by centrifugation. Pellets were completely suspended in 1 M unbuffered Tris-base before mixing with Tris/SDS gel loading buffer. Proteins were detected by immunoblotting with polyclonal anti-FlgD antibody as described elsewhere.
For motility assay, S. enterica cells were transformed by electroporation using E. coli Pulser (Biorad) with plasmids. Fresh transformants were selected on an LB plate containing 100 µg ml⁻¹ of ampicillin and inoculated onto 0.4% (w/v) soft-tryptone agar plate containing 100 µg ml⁻¹ of ampicillin and incubated at 30 °C for 5–6 h.

Recombinant plasmids were introduced into E. coli DH5α using an E. coli pulser (BioRad) set at 2.5 kV, 200 Ω and 25 μF and transformants were selected on Luria-Bertani (LB) medium with ampicillin (100 μg mL^-1) (10). 4Q7 and HD1 competent cells were prepared as described previously [28 (link)]. Approximately 3 μg of the recombinant plasmids were mixed with 300 μl of competent cell suspension, held on ice for 10 min followed by electroporation using a BTX ECM630 electro cell manipulator (San Diego CA, USA) set at 2.3 kV, 475 Ω and 25 μF. After the pulse, the suspension was added to 3 ml of brain heart infusion (BHI) (Bioxon México) and incubated with gentle shaking for 1 h at 37°C. Transformants were selected on BHI supplemented with 25 μg mL^-1 of erythromycin.

Agrobacterium strains used for experiments are listed in Table 1. Both GV [17 (link)] and LBA [53 (link)], were transformed by electroporation (Bio-Rad, E.coli Pulser) with the T-DNA vector pCP60-35S-DsRed2 source described in [10 (link)]. This vector harbours a 35S-promotor driven expression cassette expressing untagged DsRed2, and confers resistance to kanamycin in bacteria. A. tumefaciens strains were grown on YEB agar plates or YEB liquid medium according to [54 ] and were supplied with the respective antibiotics (rifampicin 100 μg ml-1; gentamycin 20 μg ml-1; kanamycin 100 μg ml-1; streptinomycin 100 μg ml-1) (Duchefa Biochemie, Haarlem, Netherlands).