Elsevier

Methods in Enzymology

Volume 497, 2011, Pages 275-293
Methods in Enzymology

Chapter thirteen - Use of Fluorescence Microscopy to Analyze Genetic Circuit Dynamics

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Abstract

The physiological processes and programs of cells are not typically determined by single genes, but are governed by the patterns of interactions between genes and proteins [Alon, U. (2007). An Introduction To Systems Biology: Design Principles of Biological Circuits. Chapman & Hall/CRC, Boca Raton.]. These interactions are commonly referred to as genetic circuits, and the pattern of these interactions is called the circuit's architecture [Sprinzak, D. and Elowitz, M.B. (2005). Reconstruction of genetic circuits. Nature438(7067), 443–448.]. Genetic circuits control diverse cellular processes, and each process requires specific dynamic behaviors to properly function. Biochemical evidence aids in the identification of interactions between genes and proteins, but the spatiotemporal dynamics of these interactions are more difficult to probe using conventional techniques. Fluorescence time-lapse microscopy is a powerful tool in the study of genetic circuit dynamics, allowing the measurement of circuit dynamics in single cells [Suel, G.M., et al. (2007). Tunability and noise dependence in differentiation dynamics. Science315(5819), 1716–1719.]. Uncovering the dynamics of genetic circuits allows verification of mathematical models of genetic circuits and aids in the design of forward experiments. By enabling the study of relationships between circuit architecture and dynamic behavior, fluorescence time-lapse microscopy opens new frontiers in synthetic biology, allowing for the alteration of genetic circuits to achieve novel behaviors [Cagatay, T., et al. (2009). Architecture-dependent noise discriminates functionally analogous differentiation circuits. Cell139(3), 512–522.], and even the generation of completely synthetic, purpose built genetic circuits [Elowitz, M.B. and Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature403(6767), 335–338.]. Perhaps more importantly, determination of genetic circuit dynamics can reveal the concepts and principles underlying the biological functions they regulate.

Section snippets

Fluorescent proteins

The discovery and cloning of green fluorescent protein (GFP) from the jellyfish Aequorea victoria marks the beginning of a revolution in cellular biology (Tsien, 1998). Fluorescent proteins are naturally occurring chromophores, capable of absorbing energy from a photon and returning to a lower energy state by emitting a photon of a different wavelength (Tsien, 1998). Today, various natural and engineered fluorescent proteins span the spectrum of visible light from far red to near violet (Heim

Transcriptional reporters

The construction of transcriptional reporters are a common use for fluorescent proteins. The goal is to measure promoter activity, allowing analysis of the dynamics of gene expression. To accomplish this, the fluorescent protein coding sequence is placed downstream of the promoter of interest. In most cases transcriptional reporters do not interfere with any biological functions of the cell, excluding side effects such as phototoxicity and overexpression of fluorescent proteins. The

A simple method for setting up a movie with bacteria

Time-lapse microscopy is one of the most suitable techniques for measuring the dynamics of genetic circuits in living cells. Below is a short protocol for the preparation of cells for time-lapse microscopy that has been optimized for bacterial cells and yeast, but the basic principles of the method are also applicable to cells of higher organisms such as mammalian cells.

Streak bacteria on an agar plate and grow overnight. Start a liquid culture from a single colony and grow to high optical

Measuring and Interpreting Dynamics

The previous sections discuss the technical aspects of designing and measuring fluorescent reporter constructs. This chapter describes how fluorescence time-lapse microscopy can be used as a tool to quantitatively measure and interpret the dynamics of genetic circuits in living cells.

Applications for Measurement of Circuit Dynamics

Information describing interactions among genes and proteins is accumulating rapidly; however, understanding of the operational principles of genetic circuits has not increased proportionally. Extracting simple concepts from vast amounts of available data is becoming a major challenge for biomedical researchers. Since measurement of dynamics provides critical information that can reveal the design principles of genetic circuits, there are many applications for fluorescence time-lapse

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