A novel and versatile dual fluorescent reporter tool for the study of gene expression and regulation in multi- and single copy number

To unravel intricate mechanisms of gene regulation it is imperative to work in physiologically relevant conditions and therefore preferentially in single copy constructs, which are not always easy to manipulate. Such in vivo studies are generally based on enzymatic assays, microarrays, RNA-seq, qRT-PCR, or multicopy reporter gene systems, frequently with β-galactosidase, luciferase or a fluorescent protein as reporter. Each method has its advantages and shortcomings and may require validation. Enzyme assays are generally reliable but may be quite complex, time consuming, and require a (expensive) substrate. Microarrays and RNA-seq provide a genome wide view of gene expression but may rapidly become expensive and time consuming especially for detailed studies with large numbers of mutants, different growth conditions and multiple time points. Multicopy reporter gene systems are handy to generate numerous constructs but may not provide accurate information due to titration effects of trans-acting regulatory elements. Therefore and in spite of the existence of various reporter systems, there is still need for an efficient and user-friendly tool for detailed studies and high throughput screenings. Here we develop and validate a novel and versatile fluorescent reporter tool to study gene regulation in single copy mode that enables real-time measurement. This tool bears two independent fluorescent reporters that allow high throughput screening and standardization, and combines modern efficient cloning methods (multicopy, in vitro manipulation) with classical genetics (in vivo homologous recombination with a stable, self-transmissible episome) to generate multi- and single copy reporter systems. We validate the system with constitutive and differentially regulated promoters and show that the tool can equally be used with heterologous transcription factors. The flexibility and versatility of this dual reporter tool in combination with an easy conversion from a multicopy plasmid to a stable, single copy reporter system makes this system unique and attractive for a variety of applications. Examples are in vivo studies of DNA-binding transcription factors (single copy) or screening of promoter and RBS libraries (multicopy) for synthetic biology purposes.