Genetic switchboard for synthetic biology applications

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 15; pp. 5850 - 5855
• Main Authors: Callura, Jarred M, Cantor, Charles R, Collins, James J
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 10.04.2012; National Acad Sciences
• Subjects: Base Sequence; Biological Sciences; Biotechnology; Carbon; Cells; Cellular metabolism; Circuits; DNA damage; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene expression regulation; Genes; Genetic Engineering; Iron; Magnesium; Messenger RNA; metabolic flux; Metabolism; metabolome; Molecular Sequence Data; mRNA; Mutation - genetics; pentose phosphate cycle; Pentose phosphate pathway; pentoses; Protein metabolism; quorum sensing; RNA; Sensors; Starvation; synthetic biology; Synthetic Biology - methods
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1203808109

A key next step in synthetic biology is to combine simple circuits into higher-order systems. In this work, we expanded our synthetic riboregulation platform into a genetic switchboard that independently controls the expression of multiple genes in parallel. First, we designed and characterized riboregulator variants to complete the foundation of the genetic switchboard; then we constructed the switchboard sensor, a testing platform that reported on quorum-signaling molecules, DNA damage, iron starvation, and extracellular magnesium concentration in single cells. As a demonstration of the biotechnological potential of our synthetic device, we built a metabolism switchboard that regulated four metabolic genes, pgi, zwf, edd, and gnd, to control carbon flow through three Escherichia coli glucose-utilization pathways: the Embden–Meyerhof, Entner–Doudoroff, and pentose phosphate pathways. We provide direct evidence for switchboard-mediated shunting of metabolic flux by measuring mRNA levels of the riboregulated genes, shifts in the activities of the relevant enzymes and pathways, and targeted changes to the E. coli metabolome. The design, testing, and implementation of the genetic switchboard illustrate the successful construction of a higher-order system that can be used for a broad range of practical applications in synthetic biology and biotechnology.