AI Article Synopsis
- Cellular functions are regulated by small molecules like metabolites, with prokaryotic microorganisms using transcriptional regulators (TRs) to respond to changes in these metabolite levels.
- A methodological pipeline is proposed for designing reporter constructs to study how gene expression systems respond to metabolites, which is crucial for synthetic biology applications.
- The chapter details the creation and analysis of an itaconate-inducible system using a specific transcriptional regulator and promoter, with guidelines for fluorescence assays and data analysis.
Article Abstract
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 P from Yersinia pseudotuberculosis. Fluorescence-based plate reader and flow cytometry assays are described and the steps for performing data analysis are provided.
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| http://dx.doi.org/10.1016/bs.mie.2019.02.018 | DOI Listing |
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