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

Cox, Robert Sidney; Dunlop, Mary J.; Elowitz, Michael B.

doi:10.1186/1754-1611-4-10

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

Journal Article · Wed Jul 21 00:00:00 EDT 2010 · Journal of Biological Engineering

DOI:https://doi.org/10.1186/1754-1611-4-10· OSTI ID:1629614

Cox, Robert Sidney ^[1]; Dunlop, Mary J. ^[2]; Elowitz, Michael B. ^[3]

California Institute of Technology (CalTech), Pasadena, CA (United States). Division of Biology; California Institute of Technology (CalTech), Pasadena, CA (United States). Dept. of Engineering and Applied Science; RIKEN Systems and Structural Biology Center, Yokohama (Japan); DOE/OSTI
California Institute of Technology (CalTech), Pasadena, CA (United States). Dept. of Engineering and Applied Science; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint BioEnergy Inst.
California Institute of Technology (CalTech), Pasadena, CA (United States). Division of Biology; California Institute of Technology (CalTech), Pasadena, CA (United States). Dept. of Engineering and Applied Science; California Institute of Technology (CalTech), Pasadena, CA (United States). Howard Hughes Medical Inst.

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1629614

Journal Information:: Journal of Biological Engineering, Journal Name: Journal of Biological Engineering Journal Issue: 1 Vol. 4; ISSN 1754-1611

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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