A novel method for assessment of transcription activation in vitro for bacterial promoter

Abstract

<em>In vitro</em> transcription (IVT) assay is a useful tool to monitor the transcriptional activity of a specific promoter under defined conditions <em>in vitro.</em> Conventional IVT assay involves use of radiolabeled probes which makes it tedious to perform and limits its utility for large scale applications. Here, a reverse-transcription-PCR (RT-PCR) based transcript detection method has been developed for bacterial <em>in vitro</em> transcription assay. Unlike conventional radiolabeling approach, this method is simple, fast, needs smaller reaction volumes, does not require special infrastructure and could be potentially used for any large-scale screening applications. The present study demonstrates the feasibility of this method by showing Cyclic AMP Receptor Protein (CRP) mediated activation of a CRP dependent <em>E. coli</em> promoter.