Analysis of rap overexpression and phr deletion on gene expression in Bacillus subtilis

We monitored changes in Bacillus subtilis global gene expression in response to overexpression of rapF, rapH, rapJ, or rapK or deletion of phrC, phrF, or phrK. Keywords: genetic modifications For monitoring changes in gene expression in response to rap overexpression, we grew cells that contained an IPTG-inducible copy of rapF, rapH, rapJ, or rapK in S750 minimal media. Treated and untreated cultures were grown in parallel. At OD600 ~ 0.5, we added IPTG (1 mM final volume) to induced cultures. We collected samples from induced and uninduced cultures ~30 min. later. We extracted RNA, reverse transcribed, labeled, and co-hybridized induced and uninduced samples to arrays containing PCR products amplified from >99% of B. subtilis genes. We performed three independent replicates of each experiment. For monitoring changes in gene expression in response to deletion of each phr, we grew wild-type and phrC, phrF, and phrK null cells in minimal media to OD600~1. We collected samples and extracted RNA, reverse transcribed, labeled, and co-hybridized each RNA along with a labeled reference pool of RNAs to arrays containing 65-mer oligonucleotides complementary to >99% of B. subtilis genes. We performed three independent replicates of each experiment. For final analysis of these samples, we then made ratios of each phr null mutant to a paired wild-type sample.