Expression analysis of stress-specific responsive genes in two-stage continuous cultures of Escherichia coli using cDNA microarray and real-time RT-PCR analysis
For the continuous cultivation of the three Escherichia coli strains, which harbor luciferase operon under control of promoter recA (DPD2794), katG (DK1), and their wild type host RFM443, multi-channel two-stage continuous mini-bioreactor system was employed. Stressful conditions were initiated by pulse-injections of mitomycin C (MMC) and hydrogen peroxide (HP) into the second mini-bioreactors of each channel in order to analyze specific gene expression along with bioluminescence emission. When stressed, the recombinant strains responded through changes in their bioluminescence levels via an activation of the promoters from specific stress-responsive genes (recA or katG). Likewise, changes of expression levels of target genes (recA and katG) and other relevant genes in host E. coli RFM443 were also observed by using cDNA microarray and further quantified with real-time RT-PCR assay. The target gene expression of the host strain analyzed by using cDNA microarray and real-time RT-PCR assays were matched well with bioluminescent profiles at the conditions triggered by different toxic chemicals. The results from this study indicated that changes in bioluminescence level the continuous water toxicity monitoring system using recombinant strains are due to the exact activation of the stress-specific target genes controlled by the stress-specific promoter. In addition, DNA microarray and real-time RT-PCR techniques can be implemented to make system performance validated.
Journal: Enzyme and Microbial Technology - Volume 39, Issue 3, 3 July 2006, Pages 440–446