Chloramphenicol cm

The strains, plasmids and their relevant characteristics are shown in Supplementary Table S3 of the supporting information. E. coli strain was cultured using Luria-Bertini (LB, Lennox) broth (10 g tryptone, 5 g Yeast extract, 5 g NaCl per 1L). Solid medium (LB-agar) was prepared by addition of 1.5% agar [Nakalai tesque, Kyoto Japan]. If necessary, kanamycin (Km) and chloramphenicol (Cm) [FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan] was added to LB or LB-agar at 50 and 25 μg/ml, respectively. Brucella Broth (BB) [BD Bioscience, San Jose, CA, United States] supplemented with 10% fetal bovine serum (BB-FBS) [Cell Culture Laboratories, Cincinnati, OH, United States] was used for liquid culture of H. pylori. The solid media used were BB-FBS agar (BB-FBS, 1.5% agar), BB-HB agar (BB supplemented with 5% horse blood, 1.5% agar), or BD BBL Trypticase Soy agar with 5% Sheep Blood (TSA) [BD Bioscience]. If necessary, Km and Cm were added to BB-FBS agar at 15 and 5 μg/ml, respectively. Both liquid cultures and agar plates were incubated at 37°C in the presence of 10% CO₂ and 5% O₂.

The gpt assay was conducted following the previously published protocol [23 (link), 28 (link), 29 (link)]. Briefly, genomic DNA was extracted from the spleen using a RecoverEase DNA isolation kit (Agilent Technologies, Santa Clara, CA, USA). The λEG10 transgene was rescued from the genomic DNA by using a Transpack Packaging Extract (Agilent Technologies). The rescued λEG10 phage was incubated with Escherichia coli YG6020 at 37°C for 20 min. After incubation, E. coli was incubated at 37°C with vigorous agitation for 30 min. E. coli was mixed with 0.6% molten soft agar with or without 25 μg/ml 6-thioguanine (6-TG, Tokyo Chemical Industry Co., Tokyo, Japan) and the entire contents were poured onto M9 plates containing 25 μg/ml chloramphenicol (Cm, Wako Pure Chemical Industries, Osaka, Japan) and 25 μg/ml 6-TG (M9 + Cm + 6-TG plates) or M9 plates containing chloramphenicol only (M9 + Cm plates), respectively, and incubated at 37°C for 3–4 days. The number of colonies appearing on the M9 + Cm plate provided the total number of E. coli cells harboring the gpt-carrying plasmid, and the number of colonies on the M9 + Cm + 6-TG plates provided the number of E. coli cells harboring the mutant gpt-carrying plasmid. Colonies on the M9 + Cm + 6-TG plates were subjected to colony PCR for the gpt gene.