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

Uppal, Sheetal ; Jawali, Narendra ; Misra, Hari Sharan

Abstract

In vitro transcription (IVT) assay is a useful tool to monitor the transcriptional activity of a specific promoter under defined conditions in vitro. 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 in vitro 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 E. coli promoter.


Keyword(s)

CyclicAMP Receptor Protein (CRP), IVT assay

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