This study examined the genes under the control of sigma32 in E. coli by moderate induction of a plasmid-borne rpoH gene under defined steady-state growth condition. Samples were taken from culture at mid log phase (OD=0.2) before or 5 minutes, 10 minutes or 15 minutes after induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com). Meanwhile, we measured the protein level of sigma32 during the time course. The changes of some sigma32?dependent genes were also determined in a DnaK null mutant to examine the anti-sigma function of the gene. This sample is taken from a seperate culture subject to 10 minutes induction by anhydrotetracycline.
