Project description:For expression analysis of wild-type V. cholerae, hapR, and rpoS deletion mutants in mid-exponential or stationary phase, the strains were grown to either OD600 of 0.3 or for 11 h in LB media at 37 0C, and bacteria from 2-ml culture were quickly pelleted, resuspended in Trizol reagent (GIBCO/BRL, San Diego, California, United States), and frozen on dry ice. RNA was isolated from the Trizol agent, treated with DNaseI (Ambion, Austin, Texas, United States), and cleaned by using the RNeasy kit (Qiagen, Valencia, California, United States). Labeling of cDNA and microarray hybridizations were performed as described [Yiliz et al. 2001, Mol. Micro. 53: 497-515]. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. At least four microarray experiments were performed for each of two biological replicates for the tested strains. Gene expression of V. cholerae, rpoS, and hapR deletion mutants in stationary phase LB cultures was analyzed and compared to the wild-type parent under identical conditions. Gene expression of the wild-type parent during stationary phase after 11 h growth in LB was analyzed using RNA from an exponentially growing culture as a reference.
