Akey 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 indepen- dently controls the expression ofmultiple 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- signalingmolecules, DNAdamage, iron starvation, and extracellular magnesium concentration in single cells. As a demonstration of the biotechnological potential of our synthetic device,webuilt ametab- olism 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–Dou- doroff, and pentose phosphate pathways. We provide direct evi- dence 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 imple- mentation of the genetic switchboard illustrate the successful con- structionof a higher-order systemthat canbeusedfor a broadrange of practical applications in synthetic biology and biotechnology.
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