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Cellular functions are often controlled by small molecular weight molecules such as metabolites. Microorganisms, mainly prokaryotes, have evolved sensing and regulatory mechanisms based on transcriptional regulators (TRs) that are able to activate gene expression in response to changes in intra- and extracellular metabolite (ligand) concentrations. To understanding control mechanisms and cell factory development in synthetic biology applications, high throughput analytical procedures are required. In this chapter, we outline a methodological pipeline to design and build reporter constructs enabling the characterization of metabolite-responsive inducible gene expression systems. As an example, we present the design, cloning and characterization of the itaconate-inducible system which is composed of the LysR-type transcriptional regulator ItcR and the promoter Pccl from Yersinia pseudotuberculosis. Fluorescence-based plate reader and flow cytometry assays are described and the steps for performing data analysis are provided.
Pubmed ID: 31128776
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Collection of manually curated inferences of regulons in prokaryotic genomes. Database for capturing, visualization and analysis of transcription factor regulons that were reconstructed by comparative genomic approach in wide variety of prokaryotic genomes.
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