A synthetic three-color scaffold for monitoring genetic regulation and noise

We describe such a plasmid-based platform for the design and optimization of synthetic gene networks, and for analysis of endogenous gene networks. This network scaffold consists of three distinguishable fluorescent reporter genes controlled by inducible promoters, with conveniently placed restriction sites to make modifications straightforward. We quantitatively characterize the scaffold in Escherichia coli with single-cell fluorescence imaging and time-lapse microscopy. The three spectrally distinct reporters allow independent monitoring of genetic regulation and analysis of genetic noise. As a novel application of this tool we show that the presence of genetic noise can reveal transcriptional co-regulation due to a hidden factor, and can distinguish constitutive from regulated gene expression.We have constructed a general chassis where three promoters from natural genes or components of synthetic networks can be easily inserted and independently monitored on a single construct using optimized fluorescent protein reporters. We have quantitatively characterized the baseline behavior of the chassis so that it can be used to measure dynamic gene regulation and noise. Overall, the system will be useful both for analyzing natural genetic networks and assembling synthetic ones.Synthetic biology requires the assembly of regulatory networks encoded in DNA [1-4]. Such networks are designed from qualitative or empirically fitted models of the individual genetic components [5-11], because detailed quantitative measurements [12,13] of these components and their in vivo interactions are often lacking. In many cases, the behavior of designed genetic networks differs significantly from initial model predictions.It would be helpful to have a tool for characterizing the quantitative behavior of both natural and synthetic genetic networks. Here we present a three-color genetic reporter that can be used to monitor dynamic gene expression in single bacterial cells. This "three-color