In Vivo Screening Method for the Identification and Characterization of Prokaryotic, Metabolite-Responsive Transcription Factors

In prokaryotes, transcription factors (TFs) are of uttermost importance for
the regulation of gene expression. However, the majority of TFs are not
characterized today, which hampers both the understanding of fundamental
processes and the development of TF-based applications, such as
biosensors, used in metabolic engineering, synthetic biology, diagnostics,
etc. One way of analyzing TFs is through in vivo screening, enabling the
study of TF-promoter interactions, ligand inducibility, and ligand specificity
in a high-throughput fashion. Here, an approach is described for the
selection and cloning of TF-promoter pairs, the development of a reporter
system, and the measurement and analysis of fluorescent reporter assays. Furthermore, the importance of a suitable inducible plasmid system is
illustrated together with prospective adaptations to modify a reporter
system’s output signal. The given approach can be used for the
investigation of native, heterologous, or even artificially created TFs in
Escherichia coli, and can be extended toward use in other
microorganisms.