An E. coli cell-free transcription- translation system: modeling gene expression and characterizing CRISPR elements and gene circuits

Abstract

Cell-free transcription-translation systems are versatile tools for rapid prototyping and characterization of biological systems and processes. Proteins can be expressed and measured in a matter of hours, whereas in vivo experiments often take days to weeks because they require protein purification or live cell transformations and cultures. TXTL systems, however, are still lacking in simple models that quantitatively describe the behavior of reactions. Here, we present an model of the all E. coli TXTL system using ordinary differential equations, encompassing the limited concentrations of transcription and translation machineries, capturing the linear and saturated regime of gene expression. Many biochemical constants are determined through experimental assays. We then show how this TXTL system was used to characterize CRISPR technologies. CRISPR-Cas systems have huge potential to be used as tools for genome engineering, as well as gene silencing and regulation. We characterize a set of sgRNAs, CRISPR nucleases, anti- CRISPR proteins, and determine protospacer-adjacent motifs. Finally, we use the TXTL system to execute gene circuits, including an IFFL and an integral controller.