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