Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

High-Throughput Transfection and Analysis of Soybean (Glycine max ) Protoplasts.

With the advent of plant synthetic biology, there is an urgent need to develop plant-based systems that are able to effectively enhance the speed of design-build-test cycles to screen large numbers of synthetic constructs. Thus far, protoplasts have served to fill this need, with cell suspension cultures serving as the primary source tissue to enable high-throughput protoplast experimentation. The possibility to use low-cost food-grade enzymes for cell wall digestion along with polyethylene glycol (PEG)-mediated transfection makes protoplasts particularly suited to automation and high-throughput screening. In other systems for which synthetic biology is well established (model bacteria and yeast), libraries of components, i.e., promoters, 5' untranslated regions, 3' untranslated regions, terminators, and transcription factors, serve as the basis for the design of complex genetic circuits. In order for synthetic biology to make similar strides in plant biology, well-characterized libraries of functional genetic parts for plants are required, necessitating the need for high-throughput protoplast assays.In this chapter, we describe an optimized method for the preparation of soybean (Glycine max ) dark-grown cell suspension cultures, followed by protoplast isolation, automated transfection , and subsequent screening.

The Q-System as a Synthetic Transcriptional Regulator in Plants.

A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.

Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop.

The droplet interface bilayer (DIB) method offers simple control over initial leaflet compositions in model membranes, enabling an experimental path to filling gaps in our knowledge about the interplay between compositional lipid asymmetry, membrane properties, and the behaviors of membrane-active species. Yet, the stability of lipid leaflet asymmetry in DIBs has received very little attention, particularly in the presence of peptides and ion channels that are often studied in DIBs. Herein, we demonstrate for the first time parallel, capacitance-based measurements of intramembrane potential with arrays of asymmetric DIBs assembled in a microfluidic device to characterize the stability of leaflet asymmetry over many hours in the presence and absence of membrane-active peptides. DIBs assembled from opposing monolayers of the ester (DPhPC) and ether (DOPhPC) forms of diphytanoyl-phosphatidylcholine yielded asymmetric bilayers with leaflet compositions that were stable for at least 18 h as indicated by a stable |137 mV| intramembrane potential. In contrast, the addition of surface-bound alamethicin peptides caused a gradual, concentration-dependent decrease in the magnitude of the dipole potential difference. Intermittent current-voltage measurements revealed that alamethicin in asymmetric DIBs also shifts the threshold voltage required to drive peptide insertion and ion channel formation. These outcomes take place over the course of 1 to 5 h after membrane formation, and suggest that alamethicin peptides promote lipid flip-flop, even in the un-inserted, surface-bound state, by disordering lipids in the monolayer to which they bind. Moreover, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.

Hydrodynamic trapping for rapid assembly and in situ electrical characterization of droplet interface bilayer arrays.

The droplet interface bilayer (DIB) is a modular technique for assembling planar lipid membranes between water droplets in oil. The DIB method thus provides a unique capability for developing digital, droplet-based membrane platforms for rapid membrane characterization, drug screening and ion channel recordings. This paper demonstrates a new, low-volume microfluidic system that automates droplet generation, sorting, and sequential trapping in designated locations to enable the rapid assembly of arrays of DIBs. The channel layout of the device is guided by an equivalent circuit model, which predicts that a serial arrangement of hydrodynamic DIB traps enables sequential droplet placement and minimizes the hydrodynamic pressure developed across filled traps to prevent squeeze-through of trapped droplets. Furthermore, the incorporation of thin-film electrodes fabricated via evaporation metal deposition onto the glass substrate beneath the channels allows for the first time in situ, simultaneous electrical interrogation of multiple DIBs within a sealed device. Combining electrical measurements with imaging enables measurements of membrane capacitance and resistance and bilayer area, and our data show that DIBs formed in different trap locations within the device exhibit similar sizes and transport properties. Simultaneous, single channel recordings of ion channel gating in multiple membranes are obtained when alamethicin peptides are incorporated into the captured droplets, qualifying the thin-film electrodes as a means for measuring stimuli-responsive functions of membrane-bound biomolecules. This novel microfluidic-electrophysiology platform provides a reproducible, high throughput method for performing electrical measurements to study transmembrane proteins and biomembranes in low-volume, droplet-based membranes.