Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.DOI: http://dx.doi.org/10.7554/eLife.10606.001

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

BackgroundThere is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable.Methodology/Principal FindingsWe describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant.Conclusions/SignificanceOur system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.

No abstract