Pbad myc hisc

To construct plasmids that encoded for epitope-tagged fusion proteins, DNA fragments that contained the genes of interest were amplified by PCR with specific primers (Table 2), and cloned into pGEX-6P-1 (GE Healthcare) for N-terminal GST-tagged fusion proteins and pFLAG-CTC (Sigma-Aldrich), p2HA-CTC [27] (link), and pBAD-Myc-HisC (Invitrogen) for C-terminal FLAG, 2HA, and Myc-His₆-tagged fusion proteins, respectively.
To construct the complementing pSsaN plasmid that expressed SsaN-FLAG fusion proteins, the ssaN-flag gene was amplified from the pFLAG-SsaN plasmid using the primers FLAG-SphI-FW and FLAG-BamHI-RV (Table 2), and then ligated into a low-copy-number pMW119 vector (Nippon Gene).
A point mutation in the ssaN gene was created with a QuikChange Site-directed mutagenesis kit (Stratagene) using the primers SsaN-R192G-FW and SsaN-R192G-RV (Table 2) to replace arginine with glycine at position 192 in SsaN. This mutation was confirmed by DNA sequencing.

Overnight cultures of E. coli O157:H7 strain EDL933 were mechanically lysed by bead beating with 100 µl of 0.1 mm zirconia beads (Carl Roth, Germany) in a FastPrep-24 Instrument (MP Biomedicals). The genomic DNA was isolated with CTAB (Sigma Aldrich) and subsequent phenol-chloroform extraction using Roti®Phenol (Carl Roth, Germany) and Roti®Phenol plus chloroform/isoamylalcohol (Carl Roth, Germany). The RNA was subsequently removed using RNase A (20 mg/ml, Sigma Aldrich). The gene asa was amplified with PCR (primers: 8220+1F-NcoI, GAT CCA TGG GGA TGT TGC TGG TTT CAA ACA; 8220+245R-HindIII, GCC AAG CTT CTA TCT GTC TGC CGG AAT GG) by adding restriction enzyme cutting sites for NcoI and HindIII. For all PCRs, the Q5 High-Fidelity DNA Polymerase (New England Biolabs) was used. The vector pBAD-myc/His C (Invitrogen) and asa were double digested with NcoI and HindIII (Thermo Fisher Scientific) at 37 °C for 3.5 h and ligated using T4 ligase (Thermo Fisher Scientific). The ligation preparation was desalted by swimming filter dialysis and transformed in E. coli TOP-10 by electroporation. The successful cloning was verified by colony PCR (primers: pBAD-C+165F, CAG AAA AGT CCA CAT TGA TT; pBAD-C-R, TGA TTT AAT CTG TAT CAG GC) and Sanger sequencing (Eurofins, Ebersberg).