Figure 7

RT-PCR analysis to validate expression of genes in P. putida WCS358. Total RNA isolations were carried out from bacterial cultures grown in minimal M9 medium using Ribopure RNA isolation kit (Ambion) and DNase treatment was carried out. cDNA synthesis was done using AMV Reverse Transcriptase (Promega) and second strand synthesis performed using Go Taq Flexi polymerase (Promega). RT-PCR analysis was performed with RNA obtained from two independent isolations and the figure shows results of one such experiment. (a) Agarose gel showing RT-PCR products for the genes PP_0170, PP_0233 and PP_0235. RT_PCR for 16S rRNA was carried out from the same RNA samples as control to ensure that equal amounts of RNA were taken. A. RT-PCR on RNA sample from P. putida WCS358 containing pBBR vector alone and B. RT-PCR on RNA sample from P. putida WCS358 containing pBBRPpoR. (b) Graph showing normalized fold difference of genes when compared to 16S rRNA expression levels. The gel image containing bands was analyzed by the ImageJ software and the bars indicate the fold increase in the intensity of the bands in PpoR++ strain (P. putida WCS358 containing pBBRPpoR) when compared to wild type (P. putida WCS358 containing pBBR vector alone).