Detektion av mRNA från en cytokrom c gen belägen i genklustret för kloratmetabolism i Ideonella dechloratans
Abstract:In previous experiments with the bacterium Ideonella dechloratans a probable sequencecoding for a cytochrome c protein (cyt c) was observed, the sequence is assumed to have afunction in the bacterial respiratory chain. Detection attempts have been made to try to findthis protein, but have not succeeded. The purpose of this project is to detect the expressionof mRNA associated with the cytochrome c. And if it is detected, it will also be investigated ifthere is any difference in expression of the mRNA due to aerobic or anaerobic environment.Total RNA was purified from cultures grown under aerobic and anaerobic conditions. cDNAwas then synthesized using reverse transcriptase and subsequently amplified with thespecific primers in a qRT-PCR.qRT-PCR testing showed a distinct amplification of primer product, confirming that I.dechloratans expresses mRNA coding for the cyt c protein. Since the protein has not beenobserved in attempts to purify c cytochromes from I. dechloratans this indicates that thedetection efforts of the protein failed or that the regulation of the protein is stronglyinhibited after the transcription step. The tests suggest that a possible difference inexpression between aerobic and anaerobic culture conditions may occur, but more and better-optimized tests are needed to confirm this.