Dpni

Site-directed mutagenesis of CagL was performed using the pAD1 vector as DNA template [9] (link). As shown in Figure 1B, CagL of GC strains contain the amino acids Tyr-Glu-Ile-Gly-Lys (YEIGK) at position 58–62 of the protein, while strain 26695 has the amino acids Asn-Glu-Met-Gly-Glu (NEMGE) at this position. To generate a CagL 26695^YE mutant (Figure 1B, bottom), we performed PCR reactions with the following primers: 628F 5′-GGTAAAGAAGATGCTCTAAACATC and 628R 5′-GATTTCATAATTAGCACTAGGGCTAG to open and amplify the entire construct. For amplification, Phusion^® High-Fidelity DNA Polymerase (NEB, Ipswich, USA) was used, followed by PCR purification (MinElute PCR Purification Kit, Qiagen, Hilden, Germany), digestion with DpnI (Promega, Madison, USA), and ligation using T4 DNA Ligase (Promega). Re-sequencing and Western blotting of E. coli or H. pylori lysates, respectively, verified the appropriate expression of CagL mutant variants from the resulting plasmids.

In the first step, the galactokinase (GalK) cassette was amplified by PCR from the pGalK plasmid using primers that carried 45–base pair homology sequences directly up- or downstream of the protein V coding region (see table S2, primers GalK_f and GalK_r). The PCR product was purified by gel extraction and digested with Dpn I (Promega) for 1 hour to remove residual template DNA before a second round of purification. Electrocompetent Escherichiacoli SW102 cells harboring the AdV-C5 containing bacmid (pKSB2) were then electroporated with the purified PCR construct. Positive clones were verified by sequencing and underwent a second electroporation reaction. In the case of AdV-C5-ΔV, this was done with a dsDNA oligonucleotide (dV_f and dV_r; table S2) consisting of the left and right homology sequences. For the AdV-C5-V-KR mutant, the recombination substrate was a synthesized modified protein V DNA sequence in which all lysine codons were replaced by arginine codons flanked by the left and right homology arms (table S2; synthesized by Thermo Fisher Scientific). The resulting AdV-C5-V-KR bacmid then served as the template for constructing the AdV-C5-V-KRrev* mutant. Bacmid DNA from positive clones was extracted, digested with Pac I to release the AdV genome, and transfected into HER911 cells. Rescued viruses were plaque-purified, expanded, and verified by sequencing.

A series of mutations were made at sites affecting the structure of human TRPM8 or TRPA1 (Figures 1a and 3a). Oligonucleotides were purchased from Eurofins-MWG-Operon. Mutated TRP channel cDNAs were created in plasmid pcDNA3.1neo(+) using quick-change PCR mutagenesis with velocity DNA polymerase (Bioline)-mediated amplification. Methylated template DNA was removed by digestion with DpnI (Promega) and mutated plasmids were cloned and isolated following transformation of competent Escherichia coli (Bioline). DNA sequences were confirmed using automated sequencing (Eurofins Genomics) with appropriate primers.