Genetic switchboard for synthetic biology applications

Abstract

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.

Fig. 1.

Engineered riboregulation and the genetic switchboard. (A) Overview of engineered riboregulation. Mfold-predicted secondary structures of a crRNA and taRNA, along with the proposed structure of the crRNA–taRNA complex that promotes gene expression. Important features are color-coded: the cis-repressive sequence (orange), RBS (blue), target gene (red), trans-activating sequence (green), and crRNA–taRNA recognition bases (gray). The ribosome is presented as yellow circles. (B) Genetic switchboard schematic. Our higher-order synthetic device is composed of orthogonal riboregulators that have unique cis-repressive and trans-activating sequences. All components are modular, including the two promoters (purple) that regulate crRNA and taRNA for a single riboregulator. The ellipses indicate the potential for further expansion.

Fig. 2.

Characterization of riboregulators RR42 and RR12y. (A) Mfold-predicted secondary structures of crR12, crR42, and crR12y. Mutations in variants crR42 and crR12y, relative to the parent crR12 variant, are outlined in purple. Important features are color-coded: cis-repressive sequence (orange), RBS (blue), target gene start codon (red), and taRNA recognition bases (gray). (B–D) Fold changes in GFP expression from fully induced crRNA cotranscribed with cognate and noncognate taRNA. (B) crR42. (C) crR12y. (D) crR12. All values were normalized by OFF state (no crRNA or taRNA induction). Graphs depict the triplicate mean ± SEM.

Fig. 3.

Switchboard sensor. (A) Switchboard sensor schematic. (B) Fold changes in levels of the four reporters for each induction condition. All values were normalized by OFF state (expression of all reporters repressed). Graphs depict the triplicate mean ± SEM.

Fig. 4.

Metabolism switchboard. (A) Metabolism switchboard schematic. (B) Overview of glucose-utilization pathways controlled by our metabolism switchboard (27). Glucose imported into the cell is converted quickly to glucose-6-phosphate (G6P). Phosphoglucose isomerase (Pgi; green), regulated here by Mg²⁺-sensitive pMgrB, converts G6P to fructose-6-phosphate (F6P), which is the first step in the EMP. Glucose-6-phosphate dehydrogenase (Zwf; red), regulated here by aTc-sensitive P_LtetO-1, converts G6P to 6-phosphogluconolactone, which then is converted to 6-phosphogluconate (6PG) by 6-phosphogluconolactonase (Pgl; not shown). Phosphogluconate dehydratase (Edd; blue), regulated here by IPTG-sensitive P_LlacO-1, converts 6PG to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is the first of only two steps in the EDP. 6-Phosphogluconate dehydrogenase (Gnd; purple), regulated here by AHL-sensitive pLuxI, converts 6PG to ribulose-5-phosphate (Ru5P), which is the distinguishing step in the PPP. (C) Metabolism switchboard performance at the RNA scale. Relative mRNA concentrations for the target metabolic genes, presented as the percentage of the total of all switchboard-regulated mRNA for each metabolic state. (D) Exponential growth rates per hour. The graph in C and table in D depict the triplicate mean ± SEM.

Fig. 5.

Metabolism switchboard performance. (A) Protein scale. Activities of Zwf, the EDP, and Gnd in nanomolars per minute per milligram. Graphs depict the triplicate mean ± SEM. (B–D) Metabolome scale. Using mass spectrometry, raw area counts for identified biochemicals were normalized by the total protein concentration and then rescaled by setting the median value equal to 1.0. Missing values were imputed with the minimum value. Graphs depict the sextuplicate mean ± SEM. (B) Levels of EMP intermediates. (C) Levels of methylglyoxal detoxification intermediates. (D) Levels of PPP intermediates.