Abstract
BackgroundCurrent methods for analyzing the dynamics of natural regulatory networks, and quantifying synthetic circuit function, are limited by the lack of well-characterized genetic measurement tools. Fluorescent reporters have been used to measure dynamic gene expression, but recent attempts to monitor multiple genes simultaneously in single cells have not focused on independent, isolated measurements. Multiple reporters can be used to observe interactions between natural genes, or to facilitate the 'debugging' of biologically engineered genetic networks. Using three distinguishable reporter genes in a single cell can reveal information not obtainable from only one or two reporters. One application of multiple reporters is the use of genetic noise to reveal regulatory connections between genes. Experiments in both natural and synthetic systems would benefit from a well-characterized platform for expressing multiple reporter genes and synthetic network components.ResultsWe 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.ConclusionWe 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.
Highlights
Current methods for analyzing the dynamics of natural regulatory networks, and quantifying synthetic circuit function, are limited by the lack of well-characterized genetic measurement tools
Regulatory networks Synthetic biology requires the assembly of regulatory networks encoded in DNA [1,2,3,4]
We minimized the microscope spectral crosstalk to be less than 0.1% and correctable to within 0.01%. (3) Independence: We wished to detect both strong and weak genetic signals simultaneously, with the ability to watch them change over time in single cells
Summary
Current methods for analyzing the dynamics of natural regulatory networks, and quantifying synthetic circuit function, are limited by the lack of well-characterized genetic measurement tools. One application of multiple reporters is the use of genetic noise to reveal regulatory connections between genes Experiments in both natural and synthetic systems would benefit from a well-characterized platform for expressing multiple reporter genes and synthetic network components. Regulatory networks Synthetic biology requires the assembly of regulatory networks encoded in DNA [1,2,3,4] Such networks are designed from qualitative or empirically fitted models of the individual genetic components [5,6,7,8,9,10,11], because detailed quantitative measurements [12,13] of these components and their in vivo interactions are often lacking. We show that interactions between the reporters are minimal when controlled by different transcription factors This tool can measure multiple network properties in parallel to characterize multicomponent systems
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