In vivo toxicity assay for itaT and its variants

Escherichia coli strain MG1655, purchased from NBRB E. coli strain (NIG, Japan) was transformed with pBAD33-ItaT or its variants, inoculated in LB containing 50 μg/ml chloramphenicol in the presence of 0.5% (w/v) glucose, and cultured at 37°C. To evaluate the toxicities of ItaT in liquid medium, the overnight cultures were diluted to an OD₆₆₀ of 0.03 in fresh liquid LB containing 50 μg/ml chloramphenicol, supplemented with either 0.1% (w/v) glucose or 0.1% (w/v) arabinose. The cultures were continued at 37°C, and the OD₆₆₀ values of the cultures were measured. To evaluate the toxicities of ItaT and its variants by the spot assay, the overnight LB cultures were serially diluted, and 3 μl portions were spotted on LB agar plates containing 50 μg/ml chloramphenicol, supplemented with either 0.1% (w/v) glucose or 0.1% (w/v) arabinose, and then the plates were incubated overnight at 37°C.