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Published July 21, 2010 | Published + Supplemental Material
Journal Article Open

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

Abstract

Background: 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. 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. Results: 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. Conclusion: 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.

Additional Information

© 2010 Cox et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 20 January 2010; Accepted: 21 July 2010; Published: 21 July 2010. We thank R. Phillips, M. Fontes, J. Young, C. Dalal, H. Garcia, F. Tan, L. Cai, G. Suel, J. Huang, R. Murray, J. Garcia-Ojalvo, U. Alon, R. Kishony, N. Rosenfeld, D. Morris, B. Wold, P. Sternberg, C. Smolke, E. Winfree, J. Leadbetter, and B. Shraiman for helpful discussions. M. Surette helped with the DNA design, G. Seelig assisted with the control experiments in Table 1, A. Eldar invented the data representation shown in Figure 2. This research was supported by US National Institutes of Health grants R01GM079771, P50GM068763, National Science Foundation CAREER Award 0644463 and the Packard Foundation. RSC was supported by the Caltech Center for Biological Circuit Design. MJD was supported by the Institute for Collaborative Biotechnologies through grant DAAD19-03-D-0004 from the US Army Research Office. Authors' contributions: RSC designed the DNA sequences, performed the experiments and drafted the manuscript. MJD carried out the time-lapse data analysis and mathematical modeling, and helped write the manuscript. MBE conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Competing interests: The authors declare that they have no competing interests.

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Created:
August 19, 2023
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October 25, 2023