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

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.

Pubmed ID: 20646328 RIS Download

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Cold Spring Harbor Laboratory

A non-profit, private research and education institution that performs molecular and genetic research used to generate methods for better diagnostics and treatments for cancer and neurological diseases. This lab has done specific research of cancer-causing genes and their respective signaling pathways. They have also researched mutations and structural variations of the human genome that could cause neurodevelopmental and neurodegenerative illnesses such as autism, schizophrenia, and Alzheimer's and Parkinson's diseases. This laboratory is also involved in plant genetics and quantitative biology.

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