In vivo and in vitro characterization of σ70 constitutive promoters by real-time PCR and fluorescent measurements

Abstract

The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ70 transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction. © 2013 Springer Science+Business Media, New York.